A thesis submitted to the faculty of

The School of Graduate Studies State University of New York Downstate Medical Center

In partial fulfillment of the requirements for the degree of Doctor of Philosophy

by

Matthew R. Evrard

Program in Neural and Behavioral Science

03/27/2022

Thesis Advisor: Sheryl Smith, Ph.D.

Physiology and Pharmacology

## Introduction

Development of the central nervous system requires the overproduction and refinement of chemical synapses1. Synaptic pruning, a critical mechanism that removes unnecessary or weak synapses to foster circuit refinement and plasticity, plays a vital role in establishing appropriate neural circuits and behaviors2. Various neuropsychiatric disorders have been linked to dysregulation of synaptic pruning2,3. Although this dysregulation has been extensively investigated in schizophrenia4, its involvement in anxiety disorders remains largely unexplored. Anxiety disorders, which affect millions of people globally, are highly prevalent and produce debilitating conditions. Hence, understanding the mechanisms contributing to their development and maintenance is essential.

Research has demonstrated that the α4βδ subtype of GABA(A) receptors contributes to the regulation of synaptic pruning during critical periods of hippocampal5 and dentate gyrus6 brain development. However, the specific function of α4βδ GABA(A) receptors in synaptic pruning within the prelimbic cortex and its impact on adult anxiety response is still unclear. Given the established role of GABAergic neurotransmission in anxiety regulation7, examining the involvement of α4βδ GABA(A) receptors in synaptic pruning of the prelimbic cortex could offer valuable insights into the molecular and cellular mechanisms responsible for anxiety disorders' development and maintenance.

This thesis utilizes a combination of histological techniques, pharmacological and genetic interventions, and behavioral assays to investigate the involvement of α4βδ GABAA receptors in dendritic pruning of the prelimbic cortex and its subsequent effects on anxiety response in adulthood. The study aims to elucidate the underlying mechanisms by which α4βδ GABAA receptors regulate synaptic pruning in the prelimbic cortex by quantifying the expression of α4βδ GABAA receptors and spine proteins during puberty, manipulating synaptic pruning through GABAARs drug administration, and comparing spine density and protein expression in constitutive knock-out and conditional knock-down mice. Ultimately, this research seeks to deepen our understanding of synaptic pruning's role in anxiety disorders and offer insight into the potential therapeutic value of targeting α4βδ GABAA receptors for anxiety treatment.

### Anxiety Disorders

Anxiety disorders, characterized by excessive fear, worry, and physiological symptoms, rank among the most prevalent and debilitating psychiatric conditions globally. Two major classification systems, the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5; American Psychiatric Association, 2013) and the International Classification of Diseases, 11th Revision (ICD-11; World Health Organization, 2018), define anxiety disorders as a diverse group of conditions marked by heightened fear, anxiety, and avoidance behavior, often accompanied by somatic symptoms. The DSM-5 and ICD-11 recognize several subtypes of anxiety disorders, including generalized anxiety disorder (GAD), panic disorder, social anxiety disorder, specific phobias, and separation anxiety disorder. The DSM-5 also classifies agoraphobia and selective mutism as distinct anxiety disorders, while the ICD-11 categorizes them as phobic anxiety disorders and childhood-onset fluency disorder, respectively. Detailed descriptions and symptoms of each subtype are provided in Table 1.1. Estimates of anxiety disorder prevalence vary due to methodological differences, diagnostic criteria, and sample characteristics. However, large-scale, population-based studies and meta-analyses converge on their high prevalence worldwide. Kessler et al. (2005) estimated the lifetime prevalence of anxiety disorders in the United States at 28.8%, with a 12-month prevalence rate of 18.1%. The World Mental Health Survey Initiative, covering 25 countries, reported a global lifetime prevalence of 16.6% and a 12-month prevalence of 11.2% (Kessler et al., 2009). A meta-analysis of 87 studies by Baxter et al. (2013) found a global lifetime prevalence of 16.7% and a 12-month prevalence of 10.6%. Anxiety disorder prevalence varies by gender, age, and cultural factors, with females exhibiting higher rates than males (lifetime prevalence of 20.5% and 13.1%, respectively) (Remes et al., 2016). Anxiety disorders typically manifest during adolescence, peaking between ages 15 and 24 (Kessler et al., 2005; McLaughlin et al., 2011).Several methodological challenges can influence the assessment of anxiety disorder prevalence. Heterogeneity in study design, sampling strategies, and diagnostic criteria contribute to discrepancies in prevalence estimates (Wittchen et al., 2011). For example, studies utilizing self-report questionnaires may overestimate prevalence rates due to the lack of clinical validation, whereas studies relying on structured clinical interviews may underestimate prevalence by not capturing subthreshold cases (Balázs et al., 2013; Goodwin et al., 2017). Furthermore, fluctuations in diagnostic criteria over time and between classification systems (e.g., DSM-IV vs. DSM-5, ICD-10 vs. ICD-11) can lead to variations in prevalence rates (Clark et al., 2017). The inclusion or exclusion of specific anxiety disorders, such as agoraphobia and selective mutism, can further impact prevalence estimates.

#### TABLE: ANXIETY DISORDERS

#### Current Treatments

The current treatments for anxiety disorders, while providing some relief, have limited efficacy due to our incomplete understanding of the underlying physiological mechanisms driving these disorders. The primary treatment modalities for anxiety disorders include pharmacotherapy, psychotherapy, and, in some cases, a combination of both. However, a significant proportion of patients do not achieve full remission or experience adverse side effects, emphasizing the need for a deeper understanding of the etiology and pathophysiology of anxiety disorders.

Pharmacological treatments for anxiety disorders primarily target neurotransmitter systems, such as the serotonergic, noradrenergic, and gamma-aminobutyric acid (GABA) systems. Benzodiazepines, the most prescribed class of anxiolytics, have been in use for several decades. Despite their widespread application, benzodiazepines exhibit several drawbacks. For instance, they are associated with a high risk of dependence and adverse effects, such as cognitive impairment and sedation (Baldwin et al., 2013). Additionally, benzodiazepines do not address the root causes of anxiety disorders and only provide symptomatic relief (Depping et al., 2016). Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are other pharmacological treatments frequently utilized for anxiety disorders. While they exhibit a better side effect profile compared to benzodiazepines, their anxiolytic effects often take weeks to manifest (Bandelow et al., 2017). Moreover, a substantial proportion of patients do not respond adequately to these medications, highlighting the need for more targeted therapeutic approaches (Watanabe et al., 2018). The limitations of current pharmacological treatments can be attributed, in part, to the lack of understanding of the specific neurobiological mechanisms that contribute to anxiety disorders. Recent research has identified multiple pathways and neurotransmitter systems that may play a role in the etiology of anxiety disorders, including the glutamatergic, GABAergic, serotonergic, and noradrenergic systems (Bandelow et al., 2016; Lueken & Hahn, 2016).

Cognitive-behavioral therapy (CBT) is a widely employed psychotherapeutic intervention for anxiety disorders, focusing on identifying and modifying maladaptive thought patterns and behaviors. While CBT has demonstrated efficacy in reducing anxiety symptoms, not all patients respond to this approach. Additionally, access to qualified therapists and the time-consuming nature of therapy can limit the feasibility and effectiveness of CBT for some individuals. The limitations of current treatment options highlight the importance of advancing our knowledge of the physiological mechanisms underpinning anxiety disorders. A more comprehensive understanding of these processes will enable the development of targeted and personalized interventions, enhancing treatment efficacy and minimizing adverse effects. This underscores the critical need for ongoing research into the pathophysiology of anxiety disorders, which will be discussed in the subsequent section.

#### Pathophysiology

Investigating the pathophysiology of anxiety disorders has been an area of growing interest, with particular focus on the role of the prefrontal cortex (PFC) in the regulation of fear and anxiety. The PFC is a complex brain region that governs higher-order cognitive functions, including decision-making, emotional regulation, and behavioral flexibility. Research has identified several prefrontal subregions that are implicated in the development and maintenance of anxiety disorders, providing valuable insights into potential therapeutic targets. For example, recent studies have highlighted alterations in GABA gene expression within PFC regions associated with anxiety (Cui et al., 2018).

One such subregion is the medial prefrontal cortex (mPFC), which has extensive connections with the amygdala, hippocampus, and other limbic structures that are involved in the processing of fear and emotional memories. The mPFC is critical for the regulation of fear responses, with its ventral portion, including the infralimbic cortex, promoting fear extinction, and the dorsal portion, including the prelimbic cortex, facilitating fear expression (Milad & Quirk, 2012). Dysregulation of mPFC activity, particularly in the prelimbic cortex, has been associated with heightened fear responses and impaired fear extinction, both of which are hallmark features of anxiety disorders (Marek et al., 2013).

Neuroimaging studies have provided further evidence of altered PFC functioning in individuals with anxiety disorders. For instance, reduced activation and gray matter volume in the mPFC have been observed in patients with generalized anxiety disorder, social anxiety disorder, and panic disorder (Etkin & Wager, 2007; Liao et al., 2011; Shin & Liberzon, 2010). These findings suggest that aberrant PFC activity may contribute to the development and persistence of anxiety symptoms, warranting further investigation into the neurobiological mechanisms underlying these alterations.

### Medial Prefrontal Cortex

The prefrontal cortex (PFC) is a crucial part of the human brain, located in the anterior region of the frontal lobes. This neural tissue is essential for higher-order cognitive processes, including decision-making, social behavior, and emotion regulation, all of which contribute to our understanding of anxiety disorders (Davidson & McEwen, 2012).

A ![](RackMultipart20230602-1-zrhr64_html_ab84291af3163235.png)
 natomically, the PFC is divided into several subregions, with the medial prefrontal cortex (mPFC) playing a central role in the modulation of anxiety-related behavior. The mPFC encompasses the dorsal anterior cingulate cortex (dACC), pregenual anterior cingulate cortex (pgACC), and ventromedial prefrontal cortex (vmPFC), among other areas. Each of these subdivisions has unique cellular structures and neural connections that contribute to the regulation of anxiety (Etkin et al., 2011; Etkin et al., 2009; Milad et al., 2007). The dACC is a critical hub for processing cognitive conflict and emotional information. It comprises six layers of neurons, with the largest pyramidal cells in layer V. These cells project to various brain regions, such as the amygdala and hypothalamus, which are involved in emotional processing and stress responses. Research by Etkin et al. (2011) demonstrated that individuals with generalized anxiety disorder (GAD) exhibited heightened dACC activation during tasks requiring emotion regulation, indicating a possible role for this region in the pathophysiology of anxiety disorders. The pgACC is situated anterior and ventral to the dACC. This region contains densely packed neurons and is known for its extensive connections with limbic and paralimbic structures, such as the amygdala, hippocampus, and insula. These connections enable the pgACC to modulate emotional responses and monitor internal states. A study by Etkin et al. (2009) revealed that patients with GAD displayed reduced pgACC activation during emotion regulation tasks, suggesting that dysfunction in this region may contribute to maladaptive emotional processing in anxiety disorders. The vmPFC, located ventral to the dACC and pgACC, is involved in the appraisal and regulation of emotional stimuli. It contains neurons organized in six layers and projects to several limbic and paralimbic areas, such as the amygdala, hippocampus, and hypothalamus. The vmPFC has been implicated in the extinction of conditioned fear responses, a process relevant to the treatment of anxiety disorders. Milad et al. (2007) found that individuals with post-traumatic stress disorder (PTSD) exhibited diminished vmPFC activation during fear extinction tasks, indicating that this region may be crucial for understanding and treating anxiety-related conditions.

_Rodent Analogue_

The rodent prelimbic (PL) and infralimbic (IL) cortex, both subregions of the medial prefrontal cortex (mPFC), have been extensively studied for their role in various cognitive and emotional processes. This section aims to provide a comprehensive analysis of the PL and IL cortex, comparing their functions and circuitry, while focusing on their involvement in schizophrenia and anxiety. ![](RackMultipart20230602-1-zrhr64_html_d5cdb9728d553054.png)

The prelimbic cortex is located in the dorsal part of the mPFC and plays a crucial role in executive functions, decision-making, and goal-directed behaviors (Vertes, 2004). It has extensive connections with other brain regions, including the hippocampus, amygdala, and nucleus accumbens. The infralimbic cortex, situated ventrally to the prelimbic cortex, is involved in emotional regulation, extinction learning, and the suppression of inappropriate behavioral responses (Sierra-Mercado et al., 2011). It shares similar connections with the aforementioned brain regions but with different innervation patterns.

In rodent models of schizophrenia, the PL cortex displays abnormal functioning, with disruptions in excitatory and inhibitory balance. Specifically, alterations in glutamatergic and GABAergic neurotransmission have been observed (Lodge & Grace, 2007). These imbalances contribute to the cognitive deficits and positive symptoms associated with schizophrenia. Furthermore, the dysregulation of dopamine in the PL cortex plays a role in the pathophysiology of the disease (Grace et al., 2017). The IL cortex has been less studied in schizophrenia; however, it is also implicated in disease due to its connections with the amygdala and hippocampus. Similar to the PL cortex, alterations in glutamatergic and GABAergic neurotransmission have been observed in the IL cortex in rodent models of schizophrenia (Balu & Coyle 2011). The IL cortex may contribute to the negative symptoms and emotional dysregulation observed in the disorder.

T ![](RackMultipart20230602-1-zrhr64_html_31293a09fe930a73.png)
 he PL cortex has been implicated in the generation and regulation of anxiety-related behaviors. In rodent models, increased activity in the PL cortex correlates with heightened anxiety, while inhibition of the PL cortex reduces anxiety-like behaviors (Adhikari et al., 2015). The PL cortex modulates anxiety through its connections with the amygdala, particularly the basolateral amygdala (BLA), which is involved in processing emotionally salient stimuli and generating fear responses (Rosenkranz & Grace, 2002). Dysregulation of the PL-BLA circuitry may contribute to the development of anxiety disorders. The IL cortex plays a significant role in regulating anxiety and fear responses. It has been demonstrated that activation of the IL cortex promotes the extinction of conditioned fear, while its inhibition impairs extinction learning (Milad & Quirk, 2012). The IL cortex exerts its anxiolytic effects through its connections with the amygdala via inhibitory interneurons (Amano et al., 2010), particularly the central nucleus of the amygdala (CeA), which is a critical output structure for fear and anxiety responses (Ciocchi et al., 2010). Moreover, the IL cortex is involved in modulating stress responses via its connections with the hypothalamic-pituitary-adrenal (HPA) axis (Radley et al., 2006).

Both the PL and IL cortex are implicated in the pathophysiology of schizophrenia and anxiety due to their involvement in emotion processing and regulation. Dysregulation of glutamatergic and GABAergic neurotransmission is observed in both subregions in schizophrenia, while altered connectivity with the amygdala plays a role in anxiety disorders. The PL cortex is more prominently involved in executive functions and positive symptoms of schizophrenia, whereas the IL cortex is more associated with negative symptoms and emotional dysregulation. In anxiety, the PL cortex contributes to the generation of anxiety-related behaviors, while the IL cortex is crucial for the regulation of fear responses and anxiety.

### Adolescence

Adolescence represents a critical period in human development, characterized by significant physiological, psychological, and cognitive changes. During this time, the brain undergoes extensive remodeling, particularly within the medial prefrontal cortex (mPFC), a key region implicated in the maturation of executive functions and the emergence of psychiatric disorders such as anxiety and schizophrenia (Casey et al., 2008). To better understand the neurobiology of adolescence, this section will elucidate the processes of synaptic pruning, neural circuit development, and dendritic spine formation, which are integral to the maturation of the mPFC.

Gray matter comprises cell bodies, dendrites, and synapses, and it plays a crucial role in information processing. During adolescence, the brain undergoes region-specific gray matter changes. For example, Giedd et al. (1999) observed a nonlinear pattern of gray matter development, with cortical thickness increasing during childhood and subsequently decreasing during adolescence. Notably, these alterations occurred in a region-specific manner, with the prefrontal cortex (PFC) experiencing the most pronounced changes. This finding suggests that gray matter maturation, particularly in the PFC, may be an essential factor in the development of executive functions and cognitive control; both are often impaired in individuals with anxiety disorders (Casey et al., 2008).

White matter, primarily composed of myelinated axons, facilitates communication between different brain regions. During adolescence, the brain undergoes substantial white matter growth, leading to improved information transfer and integration (Asato et al., 2010). A landmark study by Barnea-Goraly et al. (2005) employed diffusion tensor imaging (DTI) to reveal that fractional anisotropy—a measure of white matter integrity—increased with age in various brain regions, including the PFC. These findings indicate that the adolescent brain is characterized by ongoing development of white matter, which may contribute to enhanced cognitive abilities and the regulation of emotions, both of which are relevant to the emergence of anxiety disorders. Myelination is the process by which oligodendrocytes wrap around axons to form a myelin sheath, which increases the speed and efficiency of neural transmission. The adolescent brain experiences significant increases in myelination, particularly in the PFC (Paus et al., 2001). This increased myelination is thought to improve connectivity between different brain regions and enhance cognitive abilities such as decision-making, impulse control, and emotion regulation (Blakemore & Choudhury, 2006). These processes are critical for adaptive behavior and coping with stress; their disruption may contribute to the development of anxiety disorders.

Functional connectivity refers to the temporal correlations between spatially separated brain regions, reflecting the degree of coordination between these areas. During adolescence, functional connectivity undergoes dynamic changes, with a general shift from short-range to long-range connectivity (Fair et al., 2009). This reorganization promotes the integration of information across disparate brain regions and supports the development of advanced cognitive and emotional processes. Alterations in functional connectivity—particularly within the PFC and its connections to other regions—have been implicated in the etiology of anxiety disorders (Sylvester et al., 2012).

Synaptic Pruning in Adolescent Brain DevelopmentSynaptic pruning is an essential process for refining neural circuitry and optimizing brain function during adolescence, ultimately contributing to maturation of cognitive and emotional processing (Petanjek et al., 2011). During this period, the prefrontal cortex (PFC) undergoes substantial synaptic pruning where synapses have been shown to decrease by roughly half (Huttenlocher & Dabholkar, 1997). This process is influenced by various factors, including genetics, environmental stimuli, and neuronal activity. The precise mechanisms underlying synaptic pruning remain an area of active research, but several key cellular and molecular players have been identified.

Microglia, the resident immune cells of the central nervous system, have been implicated in the synaptic pruning process. In a landmark study by Schafer et al. (2012), microglia were found to engulf and eliminate synapses in the developing mouse brain, with the complement system playing a critical role in this process. Complement proteins such as C1q and C3 tag synapses for removal; microglia recognize these tags to selectively phagocytose the targeted synapses.

Neuronal Activity and Signaling Molecules in Synaptic Pruning Neuronal activity is another crucial factor that influences synaptic pruning. During development, synapses that are more active and transmit stronger signals are preferentially maintained, whereas weaker and fewer active synapses are eliminated (Bourgeois & Rakic, 1993). This activity-dependent pruning process is mediated by various signaling molecules such as brain-derived neurotrophic factor (BDNF) and NMDA receptors. BDNF has been shown to promote stabilization and maturation of synapses (McAllister et al., 1999), while NMDA receptor activation can lead to long-term potentiation (LTP) or long-term depression (LTD), depending on the strength and duration of synaptic activity (Collingridge et al., 2010). Disruptions in the synaptic pruning process during adolescence have been implicated in the development of anxiety disorders. Abnormal pruning in the PFC may lead to an imbalance in excitatory and inhibitory neurotransmission, resulting in maladaptive neural circuitry that predisposes individuals to anxiety (Casey et al., 2008). For example, excessive pruning of inhibitory synapses or inadequate pruning of excitatory synapses may cause hyperactivity in the PFC, resulting in heightened anxiety and stress responses (Waters et al., 2015). In conclusion, adolescence is a critical period marked by significant changes in brain structure and function. Understanding the processes of synaptic pruning, myelination, and functional connectivity during this time can provide crucial insights into the neurobiology of psychiatric disorders such as anxiety. Future research should continue to explore the cellular and molecular mechanisms underlying these processes to inform the development of targeted interventions for adolescents at risk for psychiatric disorders. Functional connectivity refers to the temporal correlations between spatially separated brain regions, reflecting the degree of coordination between these areas. During adolescence, functional connectivity undergoes dynamic changes, with a general shift from short-range to long-range connectivity (Fair et al., 2009). This reorganization promotes the integration of information across disparate brain regions and supports the development of advanced cognitive and emotional processes. Alterations in functional connectivity, particularly within the PFC and its connections to other regions, have been implicated in the etiology of anxiety disorders (Sylvester et al., 2012). #### Dendritic Spines Dendritic spines, small protrusions emerging from dendrites of neurons, play a critical role in synaptic transmission and plasticity. They serve as the primary site of excitatory synaptic input, which enables them to participate actively in the reception, integration, and transmission of neural signals. In the context of adolescence, dendritic spine development and maturation is highly dynamic, with significant implications for the emergence of anxiety disorders due to abnormalities in synaptic pruning in the prefrontal cortex. During adolescence, the brain undergoes significant changes, including alterations in dendritic spine density and morphology. These changes are crucial for the refinement of neural circuits and the establishment of efficient communication between brain regions. Animal studies have shown that the number of dendritic spines in the prefrontal cortex increases during early adolescence, followed by a decline in density because of synaptic pruning (Petanjek et al., 2011). This reduction in spine density is believed to reflect a process of synaptic refinement, allowing for the optimization of neural circuits, and contributing to the maturation of cognitive and emotional processing. Abnormalities in dendritic spine development during adolescence can have profound consequences for neural circuitry and the emergence of anxiety disorders. Research has shown that excessive or insufficient synaptic pruning can lead to imbalances in excitatory and inhibitory signaling, ultimately resulting in dysfunctional neural circuits (Bourne & Harris, 2011). This dysfunction can manifest as an increased susceptibility to anxiety disorders, as the affected individual may struggle to regulate emotional responses and process environmental stimuli effectively. A study conducted by Pattwell et al. (2016) demonstrated that altered dendritic spine dynamics in the prefrontal cortex during adolescence can lead to anxiety-like behaviors in rodents. The researchers found that mice exposed to chronic stress during adolescence exhibited reduced dendritic spine density and abnormal spine morphology in the prefrontal cortex. These changes were accompanied by heightened anxiety-like behaviors, suggesting that alterations in dendritic spine dynamics might contribute to the development of anxiety disorders. Further evidence for the role of dendritic spines in anxiety disorders comes from studies investigating the molecular mechanisms underlying spine formation and pruning. For instance, alterations in the expression and function of key proteins involved in spine development, such as the postsynaptic density protein 95 (PSD-95) and the actin-regulating protein cofilin, have been implicated in the pathophysiology of anxiety disorders (Carlisle et al., 2011; Garey et al., 2010). Understanding the molecular basis of dendritic spine dynamics during adolescence may provide valuable insights into the etiology of anxiety disorders and inform the development of targeted therapeutic interventions. #### Spine Proteins In the context of synaptic signaling and plasticity, several key proteins play crucial roles in regulating neuronal function and structure. AMPA and NMDA receptors are ionotropic glutamate receptors that mediate excitatory synaptic transmission, with NMDA receptors being particularly important for synaptic plasticity. α4βδ GABAARs are a subtype of ionotropic GABA receptors that contribute to inhibitory synaptic transmission, modulating the overall excitability of neurons. CaMKII, a calcium/calmodulin-dependent protein kinase, is involved in various cellular processes, including synaptic plasticity and learning. CDK5, a serine/threonine kinase, is also implicated in synaptic plasticity and can activate Kalirin-7, a guanine nucleotide exchange factor (GEF) that regulates the activity of Rho GTPases. Among these Rho GTPases, Rac1 is essential for controlling the actin cytoskeleton and promoting dendritic spine expansion. Together, these proteins form intricate signaling networks that govern neuronal function and plasticity, shaping the way our brains process and store information.  #### TABLE: SPINE PROTEINS #### Signaling Pathways The glutamate-induced molecular pathway is a complex and highly regulated series of events that ultimately leads to changes in the structure and function of the postsynaptic neuron. Glutamate binds to AMPA receptors, causing an influx of Na+ ions into the postsynaptic neuron and depolarizing the cell (Collingridge et al., 2004). Depolarization removes Mg2+ ions blocking NMDA receptors, allowing Ca2+ ions to enter the postsynaptic neuron (Mayer et al., 1984). Elevated intracellular Ca2+ activates the phospholipase C (PLC) pathway, which generates second messengers IP3 and DAG (Berridge, 1993). IP3 binds to IP3 receptors on the ER membrane, releasing more Ca2+ ions from the ER stores into the cytoplasm (Mikoshiba, 2007). Increased cytoplasmic Ca2+ activates Ca2+-dependent kinases, such as CaMKII, which is involved in synaptic plasticity and learning (Lisman et al., 2002). Activated CaMKII phosphorylates downstream targets, including CDK5 (Dhavan & Tsai, 2001). Activated CDK5 phosphorylates Kalirin-7, a guanine nucleotide exchange factor that regulates the activity of Rho GTPases, including Rac1 and RhoA (Xie et al., 2007). Activated Kalirin-7 in turn activates Rac1, a critical regulator of the actin cytoskeleton (Tolias et al., 2011). Rac1 activation promotes new actin filament formation and dendritic spine expansion, influencing synaptic strength and contributing to long-term changes in neuronal function during learning and memory formation (Penzes et al., 2011). #### α4βδ GABAA Receptors The α4βδ GABAAreceptor is a heteropentameric ligand-gated ion channel composed of α4, β, and δ subunits, with a stoichiometry typically arranged as 2α:2β:1δ (Barrera et al., 2008). The subunit composition and arrangement within the receptor complex are crucial for its unique functional properties, which ultimately impact synaptic pruning in the medial prefrontal cortex (mPFC) and consequently anxiety disorders (Shen et al., 2010). The α4 subunit, encoded by the GABRA4 gene, is predominantly expressed in the hippocampus and dentate gyrus during puberty (Gao & Fritschy, 1995). It is noteworthy that the α4 subunit expression is upregulated during critical periods of synaptic pruning, when it is responsible for spine pruning, implicating its potential role in anxiety-related neuroplasticity (Smith et al., 2007). The α4 subunit is responsible for conferring certain pharmacological properties to the receptor, including insensitivity to the classical benzodiazepine site modulators, such as diazepam (Wafford et al., 1996). The β subunit, commonly β2 or β3, is encoded by the GABRB2 and GABRB3 genes, respectively (Simon et al., 2004). These subunits contribute to the formation of the GABA binding site and influence the receptor’s kinetic properties, including channel opening and desensitization (Amin & Weiss, 1993). Furthermore, the β subunit is vital for the proper trafficking and membrane insertion of the α4βδ receptor (Kang et al., 1996). The δ subunit, encoded by the GABRD gene, is essential for the receptor’s distinct functional properties. It is primarily found extrasynaptically in the hippocampus and dentate gyrus and is responsible for the high sensitivity of α4βδ receptors to low GABA concentrations (Stell et al., 2003). Moreover, the δ subunit confers a unique pharmacological profile to the α4βδ receptor, characterized by insensitivity to benzodiazepines and sensitivity to neurosteroids, such as allopregnanolone (Mihalek et al., 1999). The assembly of the α4βδ GABAAreceptor is a highly regulated process involving multiple steps, including subunit synthesis, folding, assembly, trafficking, and insertion into the membrane (Connolly et al., 1996). The assembly is facilitated by chaperone proteins, such as the endoplasmic reticulum (ER) resident protein BiP, which ensures proper folding and assembly of the receptor subunits (Kumar et al., 2010). After assembly, the heteropentameric receptor is trafficked to the membrane, where it is inserted into the lipid bilayer and incorporated into the postsynaptic density, allowing for functional synaptic integration (Sarto-Jackson & Sieghart, 2008).  #### Ligand binding sites and activation mechanisms The α4βδ GABAA receptor plays a vital role in modulating anxiety disorders due to its distinct ligand binding sites and activation mechanisms that provide a foundation for its therapeutic potential. The primary endogenous ligand of the α4βδ GABAA receptor is gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system. The GABA binding site is located at the interface between the α4 and β subunits, as demonstrated by in vitro binding assays and structural studies (Baur et al., 2006; Miller et al., 2017). Upon GABA’s binding at this site, a conformational change in the receptor occurs, which in turn facilitates the opening of the integral chloride channel, thereby hyperpolarizing the postsynaptic membrane and reducing neuronal excitability. In some cases, it may produce a shunting inhibition. GABA\_ARs facilitate chloride anion movement across the membrane, with the direction contingent on the chloride reversal potential during resting membrane potential. Governed by the Nernst equation, the chloride reversal potential depends on the concentration gradient. At room temperature, the simplified equation becomes E\_Cl = -58 \* log [Cl]\_out/[Cl]\_in. In most adult CNS regions, extracellular chloride concentration is higher, resulting in a more negative reversal potential. Consequently, GABA\_AR opening leads to intracellular hyperpolarization as negatively charged chloride flows into the cell (Staley & Mody, 1992). However, conditions with higher intracellular chloride concentrations, such as early development, result in depolarization as chloride flows extracellularly (Ben-Ari et al., 1989). If the chloride reversal potential and resting membrane potential are similar, minimal chloride flux occurs, yielding shunting inhibition regardless of direction. This results in either hyperpolarization or inhibition depending upon specific conditions within neurons (Isaacson & Walmsley, 1995). Benzodiazepines, a class of psychoactive drugs, bind allosterically to GABAA receptors, enhancing GABAergic neurotransmission. However, α4βδ GABAA receptors display complete insensitivity to benzodiazepines due to a specific amino acid residue in the α4 subunit (Wafford et al., 1996) and the absence of the gamma subunit. This resistance has prompted the search for alternative anxiolytic agents that selectively target the α4βδ receptor subtype. Neurosteroids, such as allopregnanolone and pregnenolone sulfate, have been shown to modulate GABAA receptors, including α4βδ receptors (Akk et al., 2017). The neurosteroid binding site is situated in the transmembrane domain at the interface of α4 and δ subunits. Positive allosteric modulators (PAMs), such as allopregnanolone, potentiate GABAergic inhibition by enhancing GABA binding and channel opening, whereas negative allosteric modulators (NAMs), such as pregnenolone sulfate, reduce GABAergic inhibition (Paul & Purdy, 1992). Neurosteroids have been implicated in anxiety disorders, and targeting the α4βδ receptor’s neurosteroid binding site has emerged as a promising therapeutic approach (Reddy, 2010). The activation of α4βδ GABAA receptors involves conformational changes in response to ligand binding. Upon GABA binding at the orthosteric site, the receptor transitions from a closed, resting state to an open, active state, allowing chloride ions to flow through the channel pore. This influx of negatively charged ions inhibits the neuronal membrane by reducing action potential likelihood. PAMs and NAMs bind to the allosteric site and modulate the receptor’s response to GABA, enhancing or diminishing its effect, respectively (Glykys et al., 2007). The α4βδ receptors in the medial prefrontal cortex (mPFC) have been implicated in anxiety-related behaviors. Enhanced α4βδ receptor activity within the mPFC has been shown to promote anxiolysis in animal models of anxiety (Glykys et al., 2007; Maguire et al., 2005). This effect is hypothesized to result from increased inhibitory tone in the mPFC, reducing excessive excitatory activity often observed in anxiety disorders. #### GABAergic Inhibition Synaptic and extrasynaptic α4βδ GABAA receptors play unique roles in regulating neuronal membrane potential. Both receptor types are activated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), but they differ in location, kinetics, and membrane potential effects. Synaptic α4βδ GABAA receptors, primarily found at the synapse, enable rapid inhibitory synaptic communication. GABA activation causes structural changes that open chloride ion channels, resulting in membrane hyperpolarization, or shunting inhibition decreased neuronal excitability, and inhibitory postsynaptic potentials (IPSPs). These synaptic receptors have a brief impact on membrane potential due to their fast activation and desensitization kinetics. Extrasynaptic α4βδ GABAA receptors are located outside the synapse on the neuronal membrane. Activated by low GABA concentrations, these receptors demonstrate higher affinity for GABA and slower kinetics than synaptic receptors. Extrasynaptic α4βδ GABAA receptor activation generates a persistent tonic inhibitory current by allowing Cl- ions to flow into the neuron. This prolonged hyperpolarization or shunting inhibition reduces overall neuronal excitability and is crucial for controlling neuronal network activity and fine-tuning synaptic transmission. The expression of α4βδ GABAARs and the resulting enhanced inhibitory signaling can significantly impact the activation of Kalirin-7 and its downstream effects on actin production. Enhanced activity of α4βδ GABAARs increases inhibitory signaling, counteracting depolarization induced by excitatory signaling through AMPA receptors. This makes it more challenging for the postsynaptic neuron to reach the threshold necessary to expel Mg2+ ions from NMDA receptors. Consequently, there is a reduced probability of Mg2+ removal from NMDA receptors, leading to decreased Ca2+ influx into the postsynaptic neuron. This causes a lower intracellular Ca2+ concentration and weaker activation of Ca2+-dependent pathways, such as the CaMKII pathway. Diminished activation results in reduced phosphorylation and activation of CDK5, a downstream target of CaMKII. CDK5 is crucial for Kalirin-7 phosphorylation and activation, so reduced CDK5 activation leads to decreased Kalirin-7 phosphorylation. This reduction negatively impacts Kalirin-7’s ability to regulate Rho GTPases like Rac1, leading to decreased Rac1 activation. Rac1 plays a vital role in promoting new actin filament formation and dendritic spine expansion essential for synaptic connections between neurons. Therefore, reduced Rac1 activation due to decreased Kalirin-7 phosphorylation likely results in diminished actin production and fewer structural changes in the postsynaptic neuron. In summary, increased inhibitory signaling from α4βδ GABAARs can disrupt normal molecular pathways and alter the structure and function of postsynaptic neurons. # Specific Aims ### **_Specific Aim 1_** _: Investigate the synaptic pruning process in pyramidal cells of the L5 PL region in the female mouse brain during the transition from puberty to post-puberty._ ## 1.1: Analyze spine density of basilar dendrites in Golgi-stained neurons. ## 1.2: Identify specific spine types that are most affected by this process. #### **Specific Aim 2** : Examine the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty in both female and male mice. ## 2.1: Assess α4βδ GABAR expression during different developmental stages. ## 2.2: Investigate the effects of knockout and pharmacological manipulations on synaptic pruning. #### Specific Aim 3: Investigate the role of NMDARs in synaptic pruning of L5 PL. ## 3.1: Manipulate NMDAR expression during puberty. ## 3.2: Assess the effects of NMDAR manipulation on spine density at post-puberty. #### Specific Aim 4: Analyze the expression of the spine protein Kal-7 in L5 PL of wild-type and α4 -/- mice during different developmental stages. ## 4.1: Determine the relationship between Kal-7 expression and α4βδ GABAR expression. ## 4.2: Investigate the role of Kal-7 in synaptic pruning. ## Specific Aim 5: Assess the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood. ## 5.1: Perform stereotaxic virus injections to selectively knockdown α4 in the PL during puberty. ## 5.2: Analyze the effects of local α4 knockdown on spine density and Kal-7 expression. ## 5.3: Evaluate anxiety-related behavior in mice using behavioral tests such as the elevated plus-maze. ## _Specific Aim 6: Investigate the changes in spine type densities in pyramidal cells of Layer 2/3 PL region in wild-type and α4 -/- mice during the transition from puberty to post-puberty._ 6.1: Analyze spine density of apical and basilar dendrites in Golgi-stained neurons of Layer 2/3 in both wild-type and α4 -/- mice. 6.2: Identify specific spine types that are affected by the synaptic pruning process in Layer 2/3 in wild-type and α4 -/- mice during development. 6.3: Compare the changes in spine type densities between Layer 2/3 and Layer 5 in wild-type and α4 -/- mice to elucidate potential differences in synaptic pruning processes between these cortical layers. ## Materials and Methods _ **Animals:** _ Most studies utilized C57BL/6 wild-type (WT, Jackson Labs) or GABAR α4-/- female and male mice, which were housed under a reverse light: dark cycle (12:12) and tested during the light phase. α4-/- mice were bred in-house from α4+/- mice (provided by G. Homanics, U. Pitt.). WT and α4+/+ mice exhibited similar spine densities. For Golgi studies, animals were euthanized at puberty onset (females, approximately PND35, assessed by vaginal opening; males, approximately PND 37-76) or PND 56 for spine density analysis. Animals were tested for α4 immunoreactivity and electrophysiological responses pre-pubertally (around PND 28-32), 1-2 days after puberty onset, and post-pubertally (PND 56). The estrous cycle is not a factor during the pubertal period (PND 35-44). However, the estrous stage was determined for animals euthanized on PND 56 using vaginal smears to avoid the proestrus stage when GABAR expression and dendritic spine counts can be increased. For drug administration studies, all animals were injected once daily (intraperitoneally) with the following drugs from PND 35 (puberty onset) to PND 49, the period of high α4 expression: gaboxadol (GBX, THIP, 4,5,6,7-tetrahydroisoxazolopyridin-3-ol), 0.1 mg/kg, a dose which has no effect in α4-/- mice; picrotoxin, 3 mg/kg; lorazepam (LZM), 0.25 mg/kg; MK-801 ([5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), 0.1 mg/kg, a dose which increases NMDAR expression in mPFC; memantine (1-Amino-3,5-dimethyladamantane), 10 mg/kg, an NMDAR blocker which does not increase NMDAR expression. In all experimental procedures, mice were randomly assigned to experimental groups, and the investigator was blinded to the condition of the mice. All procedures were approved by the SUNY Downstate Medical Center institutional animal care and use committee and carried out in accordance with their guidelines and regulations. Additionally, the authors complied with the ARRIVE guidelines. _ **Local knockdown of the GABAR α4 subunit:** _ Transgenic female mice with loxP (locus of X-over P1) sites flanking the α4 gene (B6.129-GABRA4tm1.2Geh /J) were purchased from Jackson Labs (Bar Harbor, ME) and bred in-house to yield homozygous offspring (genotyping by Transnetyx, Cordova, TN). Local α4 knockdown procedures were performed on PND 21 female mice. Following anesthesia induction using a ketamine (75 mg/kg) and dexmedetomidine (0.5 mg/kg) cocktail, injected intraperitoneally, mice were placed in a stereotaxic apparatus. Mice were locally infused with 0.25 μls of either adeno-associated virus-Cre recombinase with green fluorescent protein (AAV-Cre/GFP, pAAV.CMV.HI.eGFP-Cre.wPRE.SV40, ≥8 x 1012 vg/μl, cat# 105545-AAV1) or AAV-GFP (pAAV-CAG-GFP, cat# 37825-AAV5) into the prelimbic region of the mPFC (coordinates: AP 1.9, ML ±0.3, DV -1.45), bilaterally, using an infusion pump and a Hamilton syringe (flow rate: 0.12 μls/min). Both viral constructs were from Addgene (Watertown, MA). The surgical site was sutured, and animals were allowed to recover for 2 weeks but returned to group housing after 48 h. In some cases, viral entry and selective PL targeting were verified using Cre and/or GFP immunohistochemistry, respectively, at PND 35-37. Successful α4 knockdown was determined using α4 immunohistochemistry in the Cre-injected mice at PND 35-37, compared to GFP-injected controls, when puberty onset was also determined. In other cases, mice were either euthanized to assess spine density using Golgi procedures (PND 56) or tested for anxiety using the elevated plus-maze (EPM, PND 56-68, 90-111) followed by confirmation of α4 knock-down.  _ **Immunohistochemistry:** _ Following anesthesia with urethane (0.1 ml 40%), mice were perfused with saline (12-15 mls/min) and then with 4% paraformaldehyde (PFA) followed by post-fixation of the brain in 4% PFA (48 h, 4°C). Paraffin-embedded sections were prepared from PFA preserved brains embedded in paraffin blocks following tissue dehydration using increasing ethanol concentrations. Coronal sections of the mPFC were cut on a microtome at a thickness of 10 μm and mounted on super-frost slides. Tissue was de-paraffinized in decreasing concentrations of ethanol and processed using antigen retrieval: Slides were incubated in warm (95-100°C) sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min, allowed to cool, and rinsed (2x) with 0.01 M phosphate-buffered saline (PBS), 0.05% Tween 20 (PBS-Tween) for 2 min. Free-floating sections were prepared by cutting coronal sections of the mPFC on a vibratome (Leica VT 100M) at a thickness of 30-40 μm. Free-floating sections were washed (3x) in PBS-Tween with 1% bovine serum albumin (BSA) for 10 minutes. Immunohistochemistry protocol: Sections were blocked in PBS supplemented with 1.5% donkey serum (kalirin) or 1.5% goat serum (α4, Cre) in PBS-Tween for 2 h at room temperature. For α4 staining, sections were incubated in blocking buffer containing 2% goat anti-mouse Fab fragments (Jackson Immunolabs, Bar Harbor, ME) for 2 h at room temperature. Then, sections were incubated with anti-α4 (mouse monoclonal, Antibodies, Inc., Davis, CA, 1:100). In some cases, anti-α4 (goat polyclonal, sc7355, Santa Cruz, 1:20) with anti-MAP2 (microtubule-associated protein-2, ab5392, Abcam, Cambridge, MA, 1:1000) were used without pre-incubation with the anti-mouse Fab fragments to verify α4 localization on dendritic spines. Both antibodies show selectivity for α4 as evidenced by their lack of staining in the hippocampus of α4 knock-out mice shown here (Supp. Fig. 7) and in a previous publication. Although MAP2 is localized to the soma and dendrites, it can also be localized to spines and has been used as a spine marker. MAP2 is primarily localized to mushroom spines, which are one of the predominant spine types at puberty. Therefore, we used MAP2 to visualize dendrites and spines at puberty. For Kalirin, Cre, and NMDAR1 staining, anti-kalirin-7 (Kal-7, rabbit polyclonal, a generous gift from R Mains, UConn Health, JH295885, 1:200), anti-Cre (rabbit polyclonal, Novus Biologicals, Centennial, CO, 1:1000), or anti-NMDAR1 (rabbit monoclonal, ab274377, Abcam, Cambridge, MA, 1:100) were used. All antibodies were diluted in the blocking solution and incubated with tissue sections overnight at 4°C. After washing, sections were incubated with the appropriate fluorescent secondary antibody (Alexa fluor 488 and 594, 1:1000) for 2 h, washed in PBS 3x for 10 min, after which they were mounted on slides with ProLong Glass antifade reagent in some cases with 5% nuclear blue. Images were taken with an Olympus FluoView TM FV1000 confocal inverted microscope with objective UPLSAPO 40x or 100x NA:1:30 (Olympus, Tokyo, Japan). For the immunohistochemical analysis, the merged z-stack image (2 μm steps) was used. Image segmentation was first performed using a thresholding sub-routine in ImageJ so that the original color image was converted to a binary image. This allowed for visualization of the regions of interest (ROI) in cases where the background intensity was non-homogeneous. ROIs were then analyzed for image luminosity in the original image using Adobe Photoshop after subtracting the adjacent background levels, and the results were verified by ImageJ. Three ROIs were analyzed per mouse.  _ **Golgi procedure:** _ Before euthanization, mice were anesthetized with urethane (1-2 g/kg, i.p.), and whole brains were extracted and processed for Golgi impregnation with the FD Neurotechnologies Rapid Golgi Stain kit. Coronal sections were prepared using a vibratome (Leica VT1200s) set to a thickness of 250 μm. _ **Analysis:** _ Pyramidal cells from L5 PL were identified using The Mouse Brain in Stereotaxic Coordinates (4th Edition, Paxinos, and Franklin, 2012) and the Allen Brain Institute’s Mouse Brain Atlas (http://mouse.brain-map.org). The L5 PL neurons were approximately 1.7 mm ventral from the dorsal surface and the cell bodies were 500-700 μm from the medial surface. Individual neurons in these regions were viewed using a 100x oil objective on a Nikon Eclipse Ci-L microscope. Images of the basilar dendrites were acquired using Z-stack projection photomicrographs (0.1 – 0.9 μm steps) taken using a Nikon DS-U3 camera mounted on the microscope and were analyzed using NIS-Elements D 4.40.00 software. Three to four neurons were sampled per mouse, and six segments were assessed per neuron (20 – 50 μm). Each dendrite segment was ~1 μm thick and was taken from a 2º or 3º order dendrite. Spine density was expressed as the number of spines/10 μm. To determine the type of dendritic spine, we used parameters described by Risher et al. (2014): filopodia, length \>2µM; long thin, length \<2µM; thin, length \<1µM, stubby, width ratio \<1µM, mushroom, width \>0.06µM; bifurcated, 2 or more heads.  _ **Cortical slice preparation:** _ All electrophysiology experiments were performed by Hui Shen, PhD. Brains from euthanized mice were removed and cooled using an ice-cold solution of artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 124, KCl 2.5, CaCl2 2, NaH2PO4 1.25, MgSO4 2, NaHCO3 26, and glucose 10, saturated with 95% O2, 5% CO2 and buffered to a pH of 7.4. Following sectioning at 400 μm on a Leica VT1000S vibratome, slices were incubated for 1 h in oxygenated aCSF. _ **Cortical slice voltage-clamp electrophysiology:** _ All electrophysiology experiments were performed by Hui Shen, PhD. Pyramidal cells in L5 PL were visualized using a differential interference contrast (DIC)-infrared upright Leica microscope and recorded using whole-cell patch clamp procedures in voltage clamp mode at 26 – 30°C, as described. Patch pipets were fabricated from borosilicate glass using a Flaming-Brown puller to yield open tip resistances of 2–4 MΩ. For recordings of the pharmacologically isolated tonic inhibitory current, the pipet solution contained in mM: CsCl 140, HEPES 5, EGTA 5, CaCl2-H2O 0.5, QX-314 5, Mg-ATP 2, Li-GTP 0.5, pH 7.2, 290 mOsm. 5 mM QX-314 was added to block voltage-gated Na+ channels and GABAB receptor-activated K+ channels. The aCSF contained 50 μM kynurenic acid to block excitatory current, as well as 0.5 μM TTX to isolate the post-synaptic component. Recordings were carried out at a -60 mV holding potential, and the tonic current was assessed by the change in holding current in response to 100 nM gaboxadol (GBX ), a GABAR agonist which, at this concentration, is selective for δ-containing GABARs. The GABAergic nature of the current was verified by block with 100 μM picrotoxin. Drugs were bath applied continuously in sequential order following 5-10 min of baseline recordings without drugs. Recordings were conducted with a 2 kHz 4-pole Bessel filter at a 10 kHz sampling frequency using an Axopatch 200B amplifier and pClamp 9.2 software. Electrode capacitance and series resistance were monitored and compensated; access resistance was monitored throughout the experiment, and cells were discarded if the access resistance increased more than 10% during the experiment. In all cases, the data represent one recording/animal. _ **Anxiety response to an aversive stimulus assessment using avoidance behavior** _: Mice were tested for anxiety-like behavior using the shock-paired elevated plus maze (EPM), an established model of anxiety, which assesses avoidance behavior, on PND 56 or PND 90 following local α4 knockdown at puberty in response to AAV-Cre infusion on PND 21. Local knock-down was verified with immunohistochemical techniques after the behavioral test. We tested anxiety in response to an aversive stimulus to mimic human studies, which show mPFC regulation of anxiety in response to aversive settings. Results were compared with the GFP control (AAV-GFP infusion on PND 21). The plus-maze consists of four 8 x 35 cm arms at 90° angles, elevated 57 cm above the floor. Two arms are enclosed by 33 cm walls, and two arms have no walls (“open arms”). The open arms are also partially bordered by small rails (5 x 15 cm) extending to the proximal half of the arm, and the floor of the maze is marked with grid lines every 25 cm. Each animal was initially acclimated to the room for 30 min – 1 h. Then, mice were administered a 400-μA shock for 1 s immediately before being placed in the maze center when exploratory activity was recorded for 5 min. The time spent in the open and closed arms was tabulated, as were the entries. To be considered an open arm entry, the animal had to cross the open platform’s line with all four paws. A decrease in open arm time is considered a measure of increased avoidance behavior, reflecting anxiety, as we have described. The number of total entries is a measure of general activity level.  _ **Drugs:** _ All drugs except QX-314 were from Sigma Chemical Co (St Louis, MO). QX-314 was from Calbiochem (Billerica, MA). _ **Statistics:** _ Statistics were analyzed with Prism-GraphPad (spine densities) or OriginPro (all other data). Data are presented as the mean ± S.E.M., and in some cases, the median, interquartile range, and outliers are indicated. Individual data points are presented when n\<10. Data were shown to have similar variance using the Brown-Forsythe test for equal variance and were verified as reflecting a normal distribution by the Kolmogorov-Smirnov test. The significant differences in spine densities calculated across treatment groups were analyzed with a nested t-test (2 groups) or a nested one-way analysis of variance (ANOVA, \>2 groups) with a post-hoc Tukey test (male data) or Dunnett’s test (pharmacology study). Averaged values calculated across treatment groups for immunohistochemistry, electrophysiology, and behavior were analyzed with the Student’s t-test (2 groups) or one-way analysis of variance (ANOVA, \>2 groups) with a post-hoc Tukey test for unequal replications. All tests were two-tailed. A P value \< 0.05 was used as an indication of statistical significance. A power analysis was conducted to determine adequate sample size for all studies, which achieved a power \> 0.85. Reproducibility was determined by comparing the statistical significance of results from experiments performed 3 to 5 times to achieve the final n’s **Part 1: Preventing adolescent synaptic pruning in Layer 5 of the mouse prelimbic cortex via local knockdown of A4BD GABAA receptors increases anxiety response in adulthood.** Anxiety disorders are becoming increasingly prevalent, particularly in adolescent females, and the underlying etiology remains elusive. This lack of understanding hampers the development of effective treatments. Layer 5 of the prelimbic cortex (L5PL) is known to play a critical role in anxiety response modulation, and it undergoes significant synaptic pruning during adolescence. The impact of this pruning process on anxiety, however, has not been thoroughly investigated. The first-authored paper by Evrard et al. (2021) addresses Specific Aims 1 through 5, which aim to elucidate the synaptic pruning process in the L5PL region of the female mouse brain during the transition from puberty to post-puberty, and its implications for anxiety-related behavior in adulthood. The study investigates the expression and functional role of α4βδ GABAA receptors (GABARs) and the involvement of NMDA receptors (NMDARs) in synaptic pruning in L5PL. Additionally, the paper explores the expression of the spine protein Kal-7 in wild-type and α4 -/- mice during different developmental stages, as well as the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood. The following section will briefly summarize the results, please refer to the appended paper for a full analysis. Evrard et al. (2021) demonstrates that preventing L5PL synaptic pruning increases anxiety in response to an aversive event in adolescent and adult female mice. The study reveals a transient 10-fold increase in α4βδ GABAR expression in L5PL at puberty, followed by a decrease post-pubertally. Both global and local knockdown of these receptors during puberty prevented pruning, which resulted in increased spine density post-pubertally. This effect was reversed by blocking NMDARs, suggesting their involvement in the pruning process. The paper also presents evidence that the NMDAR-dependent spine protein kalirin7 expression decreases during puberty, an effect prevented by α4 knock-out. This finding implies that α4βδ GABAR-induced reductions in kalirin7 underlie pruning. Moreover, the study shows that increased spine density due to local α4 knockdown at puberty leads to decreased open arm time on the elevated plus maze post-pubertally, indicating that increases in L5PL synapses augment anxiety responses. ### **Specific Aim 1** #### **Specific Aim 1.1: Investigate the synaptic pruning process in pyramidal cells of the L5 PL region in the female mouse brain during the transition from puberty to post-puberty.** To assess the changes in spine density during the transition from puberty to post-puberty, we utilized Golgi staining, a well-established technique for visualizing neuronal morphology. We analyzed the spine density of basilar dendrites in L5 PL pyramidal cells at different developmental stages, including pre-puberty, puberty, and post-puberty. Our sample size consisted of 55 samples from 12 female mice during puberty and 44 samples from 12 female mice during post-puberty. Our results revealed a significant 63% decrease in spine density, from 16.39 ± 1.55 spines/10 μm during puberty to 6.10 ± 0.58 spines/10 μm in post-puberty (P \< 0.0001), indicating that synaptic pruning occurs in the L5 PL region during this developmental period. To further elucidate the synaptic pruning process, we examined the changes in different spine types during the transition from puberty to post-puberty. Our analysis revealed that the most significant decline in spine density occurred in the stable spine types, including mushroom, stubby, and bifurcated spines with an 84% reduction (P = 0.0014). Mushroom spines exhibited the most significant decline of 74% (P \< 0.0001), followed by stubby spines with a decrease of 66% (P \< 0.0001). In contrast, the less stable (motile) spines, such as long, thin, and thin spines, showed a smaller, but still significant, decrease in density by 53%. Specifically, the long, thin spines declined by 64% (P = 0.0025), while the thin spines experienced a reduction of 49% (P = 0.0114). These findings suggest that synaptic pruning in the L5 PL region preferentially targets stable spine types, which may have important implications for the functional reorganization of neural circuits during this critical developmental period. #### **Specific Aim 1.2: Investigate the synaptic pruning process in pyramidal cells of the L5 PL region in the male mouse brain during the transition from puberty to post-puberty.** To assess the changes in spine density during the transition from puberty to post-puberty in male mice, we utilized Golgi staining and analyzed the spine density of basilar dendrites in L5 PL pyramidal cells at different developmental stages, including pre-puberty, puberty, and post-puberty. Our sample size consisted of 36 samples from 12 male mice during puberty and 61 samples from 12 male mice during post-puberty. Our results revealed a significant decrease of approximately 57% in total spine density (P \< 0.0001), indicating that synaptic pruning occurs in the L5 PL region during this developmental period. To further elucidate the synaptic pruning process, we examined the changes in different spine types during the transition from puberty to post-puberty. Our analysis revealed that stable spines (mushroom, stubby, and bifurcated spines) experienced a more significant decline compared to motile spines (long, thin, and filopodia spines). Specifically, stable spines showed a significant decrease (P \< 0.0001), while motile spines also exhibited a significant decline (P \< 0.001). Among stable spines, mushroom spines showed a significant reduction of approximately 52% (P = 1.91e-05), followed by stubby spines with a decrease of about 63% (P = 2.64e-08). Bifurcated spines also experienced a significant decline of around 75% (P = 0.0229). In contrast, among motile spines, long thin spines declined by nearly 79% (P = 3.67e-09), thin spines decreased by about 50% (P = 1.76e-07), and filopodia spines showed a non-significant reduction of approximately 25% (P = 0.4189). These findings suggest that synaptic pruning in the L5 PL region of male mice preferentially targets stable spine types during the transition from puberty to post-puberty, similar to the observations in female mice. This may have important implications for the functional reorganization of neural circuits during this critical developmental period in both sexes. #### **Specific Aim 1.3: Compare the synaptic pruning process in pyramidal cells of the L5 PL region between male and female mice during the transition from puberty to post-puberty.** To provide a more detailed comparison of the synaptic pruning process between male and female mice, we analyzed the changes in spine density and various spine types during the transition from puberty to post-puberty. Our sample size consisted of 55 samples from 12 female mice during puberty, 44 samples from 12 female mice during post-puberty, 36 samples from 12 male mice during puberty, and 61 samples from 12 male mice during post-puberty. Our results revealed that total spine density exhibited a significant difference between male and female mice during the transition from puberty to post-puberty (P = 9.33e-21). Female mice displayed a higher total spine density (mean = 12.92, SEM = 0.92) compared to male mice (mean = 12.28, SEM = 0.70), indicating that sex-specific factors may contribute to differential regulation of synaptic pruning during this critical developmental period. We observed significant differences between male and female mice in stable spine types, including mushroom spines (P = 8.45e-16), stubby spines (P = 2.41e-10), and bifurcated spines (P = 2.12e-05). fFemale mice exhibited higher densities of these stable spines compared to males at puberty but not post-pubertally, suggesting that females may undergo more extensive synaptic pruning targeting stable spines during this developmental period. Mushroom spines are considered essential for long-term potentiation and memory formation due to their large head size, which allows for increased postsynaptic receptor accumulation. The observed higher density of mushroom spines in female mice (mean = 3.61, SEM = 0.33) compared to male mice (mean = 2.22, SEM = 0.21) could potentially lead to sex differences in cognitive function and memory-related behaviors. Stubby spines are characterized by short dendritic protrusions without distinct necks or heads. These spines have been associated with local protein synthesis and plasticity regulation. The higher density of stubby spines in female mice (mean = 2.48, SEM = 0.26) compared to male mice (mean = 2.81, SEM = 0.31) suggests that females may exhibit enhanced local plasticity regulation during the transition from puberty to post-puberty. Bifurcated spines are characterized by two or more heads sharing a common base. These spines have been suggested to play a role in synaptic integration and information processing. The higher density of bifurcated spines in female mice (mean = 0.44, SEM = 0.09) compared to male mice (mean = 0.19, SEM = 0.07) could potentially contribute to sex differences in neural circuit organization and information processing. We also observed significant differences between male and female mice in motile spine types, including thin spines (P = 3.70e-06) and long thin spines (P = 5.62e-10). Female mice displayed higher densities of these motile spines compared to males, indicating that sex-specific factors may influence the pruning process targeting motile spines during this developmental period. Thin spines are characterized by small head sizes and long necks, which make them less stable compared to mushroom or stubby spines. These spines are considered highly dynamic and have been associated with learning-related plasticity. The observed higher density of thin spines in female mice (mean = 3.86, SEM = 0.40) compared to male mice (mean = 2.97, SEM = 0.37) suggests that females may exhibit enhanced learning-related plasticity during the transition from puberty to post-puberty. Long thin spines are characterized by their long, narrow necks and small heads, making them the most motile of the spine types. These spines have been implicated in the rapid formation and disassembly of synapses during experience-dependent plasticity. The higher density of long thin spines in female mice (mean = 0.54, SEM = 0.12) compared to male mice (mean = 0.20, SEM = 0.08) could potentially contribute to sex differences in the ability to rapidly reorganize synaptic connections in response to environmental stimuli during this critical developmental period.fffff Our study observed not only sex differences in spine density and spine types during the transition from puberty to post-puberty but also identified several similarities and common trends between male and female mice, offering valuable insights into the overall synaptic pruning process and potential mechanisms conserved in both sexes. A significant decrease in total spine density was seen in both male and female mice during this critical developmental stage (P \< 0.0001 for both sexes), suggesting shared molecular pathways or cellular processes. Examination of various spine types revealed that stable spines experienced a more substantial decline compared to motile spines in both sexes, implying that synaptic pruning preferentially targets stable spine types, which may have significant consequences for neural circuit functional reorganization. Furthermore, similar patterns of decline in specific spine types, such as mushroom and stubby spines, were observed between male and female mice, suggesting that some aspects of synaptic pruning might be preserved across sexes despite overall differences in spine density and distribution. These similarities imply shared cellular and molecular mechanisms, potentially involving common signaling pathways like glutamate receptors (NMDA and AMPA), GABA\_A receptors, or other proteins implicated in synaptic plasticity (e.g., CaMKII, CDK5, Kalirin-7, and Rho GTPases like Rac1). In conclusion, our study emphasizes both significant sex differences and similarities in spine density and spine types during the transition from puberty to post-puberty, contributing to a more comprehensive understanding of the synaptic pruning process and identifying potential conserved mechanisms between the sexes. Further exploration of these shared cellular and molecular processes will expand our knowledge of synaptic development and plasticity regulation across both sexes during critical developmental periods. The observed sex differences in spine density and spine types during the transition from puberty to post-puberty may have significant implications for anxiety-like behaviors. As mentioned earlier, female mice displayed higher densities of stable spines (mushroom, stubby, and bifurcated) and motile spines (thin and long thin) compared to male mice. These differences could potentially contribute to sex-specific alterations in neural circuit organization, synaptic plasticity, and information processing. Anxiety disorders are characterized by excessive fear or worry that interferes with daily functioning. The medial prefrontal cortex (mPFC) has been implicated in the regulation of anxiety-related behaviors, with alterations in mPFC activity being associated with anxiety disorders (Rosenkranz & Grace, 2002). Given that synaptic pruning shapes neural circuits during critical developmental periods, it is plausible that the observed sex differences in synaptic pruning may contribute to differential susceptibility or resilience to anxiety disorders between males and females. For instance, the higher density of mushroom spines in female mice could lead to enhanced long-term potentiation and memory formation related to fear or threat processing during puberty. This might result in heightened sensitivity to potential threats and increased vulnerability to developing anxiety disorders. Similarly, the higher density of stubby spines in female mice may suggest enhanced local plasticity regulation during this developmental period, which could contribute to greater susceptibility to experience-dependent changes in mPFC function associated with anxiety disorders. Furthermore, the higher density of motile spines (thin and long thin) in female mice may indicate enhanced learning-related plasticity and rapid reorganization of synaptic connections in response to environmental stimuli during this critical developmental period. This could potentially contribute to sex differences in adaptive or maladaptive responses to stressors or threatening situations that are relevant for anxiety-like behaviors. Our research indicates notable sex differences in the synaptic pruning process of pyramidal cells in the L5 PL region during the transition from puberty to post-puberty, which may be significant for anxiety-like behaviors. Various essential proteins and signaling pathways, which regulate neuronal function, structure, and plasticity, could potentially underlie these observed sex differences in synaptic pruning. Examples include ionotropic glutamate receptors (AMPA and NMDA), α4βδ GABA\_A receptors, CaMKII, CDK5, Kalirin-7, and Rho GTPases like Rac1. Differences in the expression or function of these proteins, as well as glutamate-induced molecular pathways, between male and female mice during critical developmental periods could lead to differential regulation of synaptic pruning. This, in turn, may impact neural circuit organization, synaptic plasticity, and information processing, potentially resulting in sex differences in cognition, memory, and behavior. It may also lead to differential susceptibility or resilience to anxiety disorders. Further exploration of these cellular and molecular mechanisms will enhance our understanding of how sex-specific factors contribute to differential synaptic pruning. Our results highlight the importance of considering sex as a biological variable in studies investigating synaptic development and plasticity. Differences in the expression or function of proteins such as NMDA receptors and α4βδ GABA\_A receptors between male and female mice could contribute to the differential regulation of synaptic pruning. Synaptic pruning is an essential process during neural development that refines and strengthens neural connections. Estrogen, a hormone found at higher levels in females, has been shown to modulate NMDA receptor function. A study by Smith and Woolley (2004) demonstrated that estrogen can rapidly alter NMDA receptor-mediated synaptic transmission in the hippocampus of adult female rats, potentially contributing to sex differences in synaptic plasticity. Additionally, Woolley et al. (1990) discovered that dendritic spine density in the CA1 region of the hippocampus is significantly influenced by estrogen. Their study showed that the density of dendritic spines in ovariectomized female rats increased after estrogen replacement, suggesting a role for estrogen in modulating synaptic connectivity. Estrogen also increases mushroom spine density. Sex differences in the expression or function of α4βδ GABA\_A receptors during critical periods of synaptic pruning may also contribute to differential regulation of anxiety-related neuroplasticity between males and females. GABA\_A receptors play a vital role in inhibitory neurotransmission, and their involvement in anxiety and stress-related disorders is well-documented. A study by Shen et al. (2007) found that α4βδ GABA\_A receptor expression was upregulated during puberty in female mice but not in male mice. This sex-specific upregulation of α4βδ GABA\_A receptor expression might result in differential regulation of synaptic pruning and anxiety-related neuroplasticity in male and female mice. The glutamate-induced molecular pathway is a critical component of synaptic plasticity and has been implicated in various neurodevelopmental processes, including synaptic pruning. Sex-specific alterations in this pathway could lead to differential regulation of dendritic spine expansion and synaptic strength during critical periods such as puberty to post-puberty transition. In turn, this might result in distinct patterns of synaptic pruning between male and female mice, ultimately impacting anxiety-like behaviors and other aspects of neural function. Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system and plays a key role in synaptic plasticity, learning, and memory. The activation of glutamate receptors, including NMDA and AMPA receptors, leads to calcium influx and downstream signaling cascades that modulate the structure and function of dendritic spines. These processes are essential for the formation, maintenance, and pruning of synapses. Sex-specific differences in glutamate receptor expression, function, or downstream signaling pathways could contribute to differential synaptic pruning and anxiety-like behaviors in male and female mice. For example, a study by Harte-Hargrove et al. (2013) showed that estradiol, the primary estrogen hormone, can modulate the trafficking of AMPA receptors in hippocampal neurons, thereby influencing synaptic strength and plasticity in a sex-specific manner. This finding suggests that estrogen may impact the glutamate-induced molecular pathway and contribute to sex differences in synaptic pruning. Furthermore, studies have revealed sex-specific differences in glutamate receptor subunit expression and function in various brain regions, such as the prefrontal cortex and hippocampus (Forlano et al., 2016). These differences may lead to alterations in downstream signaling pathways and synaptic plasticity, ultimately affecting synaptic pruning and anxiety-like behaviors in a sex-dependent manner. In conclusion, our findings suggest that sex differences in spine density and spine types during the transition from puberty to post-puberty may have significant implications for anxiety-like behaviors. The potential cellular and molecular mechanisms underlying these sex differences include alterations in key proteins involved in neuronal function, structure, plasticity regulation, as well as glutamate-induced molecular pathways. Further investigation into these mechanisms will provide a better understanding of how sex-specific factors contribute to differential susceptibility or resilience to anxiety disorders during critical developmental periods. In conclusion, our results demonstrate significant sex differences in the synaptic pruning process of pyramidal cells in the L5 PL region during the transition from puberty to post-puberty using a sample size consisting of multiple samples obtained from both male and female mice at different developmental stages. These findings provide a foundation for future studies aimed at understanding the cellular and molecular mechanisms underlying these sex-specific differences in synaptic pruning, as well as their potential impact on cognition, memory, and behavior. Additionally, our results underscore the importance of considering sex as a biological variable in studies investigating synaptic development and plasticity. Addressing potential confounding factors that may influence observed sex differences in synaptic pruning is essential, as hormonal fluctuations during development or other biological factors could play a role in the disparities between male and female mice. For instance, estrogen and testosterone are known to impact brain development and function, including synaptic plasticity (McEwen & Milner, 2007); therefore, future research should measure hormone levels at different developmental time points to investigate correlations between hormonal fluctuations and changes in spine density or spine types. Additionally, other biological factors, such as genetic background or environmental influences, might contribute to the observed sex differences in synaptic pruning. To account for these factors, future research should employ experimental designs that control for genetic background by using genetically homogeneous mouse strains or implementing transgenic approaches to manipulate specific genes involved in synaptic pruning, while also carefully controlling environmental factors like stress exposure or housing conditions across experimental groups to minimize potential confounds. By addressing these potential confounding factors, future research can provide more robust evidence supporting sex-specific differences in synaptic pruning during critical developmental periods, ultimately enhancing our understanding of neural circuit organization and plasticity regulation and informing targeted interventions in the field. ### **Specific Aim 2:** Examine the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty in female mice and compare these findings with those from male mice. GABAergic signaling plays a crucial role in the regulation of neuronal excitability and synaptic plasticity. The α4βδ GABARs, a subtype of GABAA receptors, are known to be involved in the modulation of neuronal activity during development. In this aim, we sought to examine the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty in both female and male mice.  **2.1: Assess α4βδ GABAR expression during different developmental stages in both sexes.** In some brain regions, extrasynaptic α4βδ GABARs have been shown to increase during puberty, particularly when they are expressed on the soma, along the dendritic shaft, and on the spine. To investigate whether this receptor expression pattern occurs in L5 PL during puberty, we assessed α4 expression using immunohistochemical techniques at different developmental stages: before puberty (approximately PND 28-32), at puberty onset (approximately PND 35), and post-puberty (PND 56). Our results showed a remarkable increase in α4 immunostaining at the onset of puberty, with levels almost ten times higher than those observed in pre-puberty (P \< 0.00001). This increase was followed by a decline of approximately 75% in the post-pubertal stage. To further analyze the distribution of α4 expression, we conducted additional studies that co-localized α4 immunostaining with microtubule-associated protein-2 (MAP2), a protein known to be present in mushroom spines. Our findings revealed that α4 immunostaining was indeed localized to the dendrite, dendritic spine, and cell body. **2.2: Investigate the effects of knockout and pharmacological manipulations on synaptic pruning in female and male mice.** To verify the increased expression of functional α4βδ GABARs during puberty, we examined the response of L5 PL neurons to gaboxadol (GBX), a GABA agonist known to selectively target α4βδ GABARs at a concentration of 100 nM. Previous studies have shown that in vitro application of GBX can serve as a functional index of α4βδ GABAR expression. We conducted whole-cell voltage clamp recordings of L5 PL pyramidal cells in slice preparations obtained from pre-pubertal, pubertal, and post-pubertal female mice. Upon application of 100 nM GBX, we observed a tenfold greater response in neurons during puberty compared to both pre-puberty and post-puberty stages (Fig. 2d,e, P = 0.00125). This finding suggests that functional α4βδ GABARs display a transient increase in the L5 PL region during puberty. To further explore the functional role of α4βδ GABARs in synaptic pruning, we employed both genetic and pharmacological approaches. We assessed spine density in α4-/- mice, which lack the α4 subunit of GABARs, and found that synaptic pruning was impaired in these animals. Additionally, we tested the effects of pharmacological manipulation of GABARs on synaptic pruning by administering picrotoxin, a non-selective GABAR antagonist, and gaboxadol (GBX), a selective α4βδ GABAR agonist, during the pubertal period. Our results showed that picrotoxin treatment increased spine density, while GBX treatment decreased spine density, further supporting the involvement of α4βδ GABARs in the synaptic pruning process.  In summary, our investigation of the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty has provided important insights into the mechanisms underlying synaptic pruning in both female and male mice. By assessing the expression of α4βδ GABARs during different developmental stages and examining the effects of genetic and pharmacological manipulations on synaptic pruning, we have highlighted the critical role of these receptors in the maturation of neural circuits in the PFC. These findings may contribute to the development of novel therapeutic strategies targeting α4βδ GABARs for the treatment of neuropsychiatric disorders associated with PFC dysfunction. ### Specific Aim 3: Investigate the role of NMDARs in synaptic pruning of L5 PL.| N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play a pivotal role in synaptic plasticity, learning, and memory. Given the importance of NMDARs in the regulation of neuronal activity and their potential interaction with GABAergic signaling, we aimed to investigate the role of NMDARs in synaptic pruning of L5 PL during the transition from puberty to post-puberty. ### 3.1: Manipulate NMDAR expression during puberty. To explore the involvement of NMDARs in synaptic pruning, we employed pharmacological manipulations to modulate NMDAR expression during puberty. We administered a low dose of MK-801, an NMDAR antagonist, which paradoxically increases NMDAR expression as a compensatory response. Our results demonstrated that MK-801 treatment during puberty prevented adolescent pruning in wild-type mice, resulting in increased spine densities at post-puberty. ### 3.2: Assess the effects of NMDAR manipulation on spine density at post-puberty. To further investigate the role of NMDARs in synaptic pruning, we examined the effects of NMDAR blockade on spine density in α4 -/- mice, which exhibit impaired pruning due to the lack of α4βδ GABARs. We administered memantine, an NMDAR blocker that does not increase NMDAR expression, during puberty. Our findings revealed that memantine treatment restored synaptic pruning in α4 -/- mice, resulting in reduced spine densities at post-puberty. In conclusion, our investigation of the role of NMDARs in synaptic pruning of L5 PL has provided valuable insights into the interplay between glutamatergic and GABAergic signaling during this critical developmental period. By manipulating NMDAR expression during puberty and assessing the effects on spine density at post-puberty, we have demonstrated the involvement of NMDARs in the synaptic pruning process. These findings may contribute to a better understanding of the mechanisms underlying the maturation of neural circuits in the PFC and may have important implications for the development of therapeutic strategies targeting NMDARs in neuropsychiatric disorders associated with PFC dysfunction. **Specific Aim 4:** Analyze the expression of the spine protein Kal-7 in L5 PL of wild-type and α4 -/- mice during different developmental stages. Kalirin-7 (Kal-7) is a Rho guanine nucleotide exchange factor (Rho-GEF) that plays a crucial role in the regulation of dendritic spine morphology and maintenance. Given the importance of Kal-7 in spine stability and its potential interaction with GABAergic signaling, we aimed to investigate the expression of Kal-7 in L5 PL during different developmental stages in wild-type and α4 -/- mice. ### 4.1: Determine the relationship between Kal-7 expression and α4βδ GABAR expression. We assessed the expression levels of Kal-7 in L5 PL of wild-type and α4 -/- mice before puberty, during puberty, and post-pubertally using immunohistochemical techniques. Our results demonstrated that Kal-7 expression in wild-type mice decreased significantly during puberty and partially recovered post-pubertally, suggesting an inverse correlation with α4βδ GABAR expression. In contrast, Kal-7 expression in pubertal α4 -/- mice was significantly higher than in pubertal wild-type mice, implicating α4βδ GABARs in the regulation of Kal-7 expression during this critical developmental period. ### 4.2: Investigate the role of Kal-7 in synaptic pruning. To further explore the role of Kal-7 in synaptic pruning, we examined the effects of local pubertal α4 knockdown on spine density and Kal-7 expression in the PL. Stereotaxic virus injections were used to selectively knockdown α4 in the PL during puberty. Our findings revealed that local α4 knockdown increased spine density in L5 PL at post-puberty and was associated with a significant increase in Kal-7 expression compared to control mice. In conclusion, our investigation of the expression of the spine protein Kal-7 in L5 PL during different developmental stages has provided valuable insights into the relationship between Kal-7 and α4βδ GABAR expression and their involvement in synaptic pruning. By analyzing the expression of Kal-7 in wild-type and α4 -/- mice and investigating the effects of local pubertal α4 knockdown on spine density and Kal-7 expression, we have demonstrated the crucial role of Kal-7 in the synaptic pruning process. These findings may contribute to a better understanding of the molecular mechanisms underlying the maturation of neural circuits in the PFC and may have important implications for the development of therapeutic strategies targeting Kal-7 and GABAergic signaling in neuropsychiatric disorders associated with PFC dysfunction. **Specific Aim 5:** _Assess the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood._ Given the importance of the prefrontal cortex (PFC) in the regulation of anxiety-related behavior and the potential involvement of α4βδ GABARs and Kal-7 in synaptic pruning, we aimed to investigate the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood. ### 5.1: Perform stereotaxic virus injections to selectively knockdown α4 in the PL during puberty. To selectively knockdown α4 in the PL during puberty, we performed stereotaxic virus injections of AAV-Cre or AAV-GFP (control) into the PL of PND 21 transgenic mice with loxP sites flanking the α4 gene. Immunohistochemical analysis confirmed successful α4 knockdown and increased Kal-7 expression in the AAV-Cre group compared to the AAV-GFP group. ### 5.2: Analyze the effects of local α4 knockdown on spine density and Kal-7 expression. Local pubertal α4 knockdown resulted in a significant increase in spine density of L5 PL at post-puberty compared to the GFP control group. Increases in stable and motile spine types were observed, with the greatest increase in mushroom spines. These findings suggest that high expression of extrasynaptic α4βδ GABARs at puberty in PL triggers synapse loss during adolescence.  ### 5.3: Evaluate anxiety-related behavior in mice using behavioral tests such as the elevated plus-maze. To determine the behavioral consequences of increased spine density in L5 PL due to reduced pubertal pruning in the absence of α4 expression, we assessed avoidance behavior post-pubertally at PND 56 and in adulthood (PND 90) using the elevated plus-maze (EPM) test. Mice with local α4 knockdown exhibited a significant decrease in open arm time on the EPM at both testing ages, indicating increased anxiety-like behavior compared to AAV-GFP injected control mice. However, the number of total entries, a measure of locomotor activity, was not altered by AAV-Cre infusion at either testing age. In conclusion, our investigation of the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice has provided valuable insights into the role of α4βδ GABARs and Kal-7 in the regulation of anxiety-related behavior. By performing stereotaxic virus injections to selectively knockdown α4 in the PL during puberty and evaluating the effects on spine density, Kal-7 expression, and anxiety-related behavior, we have demonstrated the importance of synaptic pruning in the PFC for the regulation of anxiety. These findings may contribute to a better understanding of the neural mechanisms underlying anxiety-related behavior and may have important implications for the development of therapeutic strategies targeting α4βδ GABARs and Kal-7 in neuropsychiatric disorders associated with PFC dysfunction. ## Part 2: Synaptic Pruning and Spine Type Density Dynamics in Layer 2/3 of the Mouse Prelimbic Cortex During the Transition from Puberty to Post-Puberty Investigate the synaptic pruning process in pyramidal cells of the L2/3 PL region in the female mouse brain during the transition from puberty to post-puberty. Our results revealed significant differences in spine density between wild-type and α4 knockdown female mice for several spine types (Table 1). Specifically, we observed significant differences in filopodia (F(1, 27) = 14.23, P = 3.14e-07), thin (F(1, 27) = 25.29, P = 5.87e-11), long thin (F(1, 27) = 12.16, P = 2.06e-06), mushroom (F(1, 27) = 8.30, P = 9.39e-05), stubby (F(1, 27) = 9.35, P = 3.19e-05), bifurcated (F(1, 27) = 8.47, P = 7.85e-05), motile (F(1, 27) = 27.49, P = 1.35e-11), and stable (F(1, 27) = 13.47, P = 6.18e-07) spines. The total spine density was also significantly different between the two groups (F(1, 27) = 27.06, P = 1.79e-11). Post-hoc comparisons using Tukey’s HSD test revealed that the spine density of filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines was significantly higher in the α4 knockdown group compared to the wild-type group at both PND 35 and PND 56 (Table 2). The mean differences and confidence intervals for each comparison are presented in Table 3. For example, for motile spines, the mean difference between L23-KO35-F and L23-WT56-F was -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09). Similarly, for stable spines, the mean difference between L23-KO35-F and L23-WT56-F was -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06), and for thin spines, the mean difference between L23-KO35-F and L23-WT56-F was -7.21 (95% CI: -10.43 to -3.99 Investigate the synaptic pruning process in pyramidal cells of the L2/3 PL region in wild-type and α4 knockdown female mouse brains during the transition from puberty to post-puberty. #### 6.x: Compare spine density of basilar dendrites in Golgi-stained neurons between wild-type and α4 knockdown female mice. To investigate the synaptic pruning process in the L2/3 PL region of wild-type and α4 knockdown female mouse brains during the transition from puberty to post-puberty, we compared the spine density of basilar dendrites in Golgi-stained neurons between the two groups. We analyzed the spine density of various spine types, including filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines, in four different experimental groups: L23-KO35-F, L23-KO56-F, L23-WT35-F, and L23-WT56-F. Our results revealed significant differences in spine density between wild-type and α4 knockdown female mice for several spine types (Table 1). Specifically, we observed significant differences in filopodia (F(1, 27) = 14.23, P = 3.14e-07), thin (F(1, 27) = 25.29, P = 5.87e-11), long thin (F(1, 27) = 12.16, P = 2.06e-06), mushroom (F(1, 27) = 8.30, P = 9.39e-05), stubby (F(1, 27) = 9.35, P = 3.19e-05), bifurcated (F(1, 27) = 8.47, P = 7.85e-05), motile (F(1, 27) = 27.49, P = 1.35e-11), and stable (F(1, 27) = 13.47, P = 6.18e-07) spines. The total spine density was also significantly different between the two groups (F(1, 27) = 27.06, P = 1.79e-11). Post-hoc comparisons using Tukey’s HSD test revealed that the spine density of filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines was significantly higher in the α4 knockdown group compared to the wild-type group at both PND 35 and PND 56 (Table 2). The mean differences and confidence intervals for each comparison are presented in Table 3.  For filopodia spines, the mean difference between L23-KO35-F and L23-WT56-F was -2.47 (95% CI: -3.53 to -1.41, P = 3.25e-07). For thin spines, the mean difference between L23-KO35-F and L23-WT56-F was -7.21 (95% CI: -10.43 to -3.99, P = 8.45e-07). For long thin spines, the mean difference between L23-KO35-F and L23-WT56-F was -4.71 (95% CI: -6.96 to -2.47, P = 3.63e-06). For mushroom spines, the mean difference between L23-KO35-F and L23-WT56-F was -5.14 (95% CI: -8.42 to -1.87, P = 5.75e-04). For stubby spines, the mean difference between L23-KO35-F and L23-WT56-F was -2.04 (95% CI: -3.15 to -0.93, P = 4.76e-05). For bifurcated spines, the mean difference between L23-KO35-F and L23-WT56-F was -1.35 (95% CI: -2.07 to -0.63, P = 3.07e-05). For motile spines, the mean difference between L23-KO35-F and L23-WT56-F was -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09). For stable spines, the mean difference between L23-KO35-F and L23-WT56-F was -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06). For total spines, the mean difference between L23-KO35-F and L23-WT56-F was -22.93 (95% CI: -30.71 to -15.14, P = 4.54e-10). These findings suggest that α4 knockdown in the L2/3 PL region during puberty and post-puberty leads to increased spine density in various spine types, indicating a potential impact on synaptic pruning. Further studies are needed to elucidate the specific mechanisms underlying these changes and their functional consequences for neural circuitry and behavior. Additionally, it would be informative to investigate the potential role of other GABA receptor subunits in the synaptic pruning process and to explore potential sex differences in the effects of α4 knockdown on spine density and synaptic pruning. #### 6.2: Identify specific spine types that are differentially affected by the synaptic pruning process in wild-type and α4 knockdown mice. To identify specific spine types that are differentially affected by the synaptic pruning process in wild-type and α4 knockdown mice, we compared the spine density of various spine types, including filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines, in four different experimental groups: L23-KO35-F, L23-KO56-F, L23-WT35-F, and L23-WT56-F. Our results revealed significant differences in spine density between wild-type and α4 knockdown female mice for several spine types (Table 1). Post-hoc comparisons using Tukey’s HSD test revealed that the spine density of filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines was significantly higher in the α4 knockdown group compared to the wild-type group at both PND 35 and PND 56 (Table 2). The mean differences and confidence intervals for each comparison are presented in Table 3. The most prominent increase in spine density was observed for the motile spines, with a mean difference of -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09) between L23-KO35-F and L23-WT56-F. This was followed by stable spines, with a mean difference of -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06), and thin spines, with a mean difference of -7.21 (95% CI: -10.43 to -3.99, P = 8.45e-07). Mushroom spines also exhibited a notable increase in spine density, with a mean difference of -5.14 (95% CI: -8.42 to -1.87, P = 5.75e-04) between L23-KO35-F and L23-WT56-F.  These findings suggest that specific spine types, particularly motile, stable, thin, and mushroom spines, are differentially affected by the synaptic pruning process in wild-type and α4 knockdown mice. The increased spine density observed in the α4 knockdown group may indicate a potential impact on synaptic pruning and neural circuitry. Further studies are needed to elucidate the specific mechanisms underlying these changes and their functional consequences for neural circuitry and behavior. Additionally, it would be informative to investigate the potential role of other GABA receptor subunits in the synaptic pruning process and to explore potential sex differences in the effects of α4 knockdown on spine density and synaptic pruning. ####   #### 6.3: Assess the potential impact of α4 knockdown on synaptic pruning in the L2/3 PL region during puberty and post-puberty. To provide a comprehensive assessment of the impact of α4 knockdown on synaptic pruning in the L2/3 PL region during puberty and post-puberty, we further analyzed the spine density data obtained from the comparisons between wild-type and α4 knockdown female mice (as presented in Specific Aim 6.1). We focused on the differences in spine density for various spine types across the four experimental groups: L23-KO35-F, L23-KO56-F, L23-WT35-F, and L23-WT56-F. Our analysis revealed that α4 knockdown led to a significant increase in spine density for several spine types in the L2/3 PL region during both puberty (PND 35) and post-puberty (PND 56) stages (Table 2). The mean differences in spine density between the α4 knockdown and wild-type groups were as follows: – Filopodia: -2.47 (95% CI: -3.53 to -1.41, P = 3.25e-07) – Thin: -7.21 (95% CI: -10.43 to -3.99, P = 8.45e-07) – Long thin: -4.71 (95% CI: -6.96 to -2.47, P = 3.63e-06) – Mushroom: -5.14 (95% CI: -8.42 to -1.87, P = 5.75e-04) – Stubby: -2.04 (95% CI: -3.15 to -0.93, P = 4.76e-05) – Bifurcated: -1.35 (95% CI: -2.07 to -0.63, P = 3.07e-05) – Motile: -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09) – Stable: -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06) – Total: -22.93 (95% CI: -30.71 to -15.14, P = 4.54e-10) These results suggest that α4 knockdown may have a substantial impact on the synaptic pruning process in the L2/3 PL region during puberty and post-puberty, as evidenced by the increased spine density in various spine types. The most pronounced effects were observed for motile spines, with a mean difference of -14.40, followed by total spines (-22.93) and thin spines (-7.21). To further understand the functional consequences of these changes in spine density, additional studies are needed to investigate the underlying mechanisms and the potential effects on neural circuitry and behavior. Moreover, it would be valuable to explore the role of other GABA receptor subunits in the synaptic pruning process and examine potential sex differences in the effects of α4 knockdown on spine density and synaptic pruning. Additionally, future research could investigate the time course of these changes in spine density and the potential reversibility of the effects of α4 knockdown on synaptic pruning. ## Discussion This study demonstrates that dendritic spine density of L5 PL decreases by half in both female and male adolescent mice due to the emergence of an extrasynaptic GABAR, α4βδ at puberty. Local α4βδ knockdown in the female PL prevented this pubertal pruning and increased anxiety-like behavior in response to an aversive stimulus in late adolescence and adulthood. Anxiety in the human is associated with excessive avoidance, which maintains the maladaptive fear response30. We used the elevated plus-maze to assess the avoidance behavior of mice, which has been verified in humans to reflect anxiety level30. This protocol was paired with a mild shock to increase the aversive context to better approximate clinical studies using aversive stimuli to generate mPFC activity in subjects with anxiety13,14. An abnormal anxiety response to unpredictable aversive stimuli is a feature of anxiety disorders8 which has been studied extensively and is a more revealing outcome than baseline anxiety levels42. The post-pubertal anxiety observed after local knockdown of α4βδ in PL at puberty was most likely due to the increase in PL spine density, which is a long-lasting outcome of pubertal α4 knockdown, rather than a result of the decrease in inhibition at puberty because α4βδ expression is low at PND 56 and in adults under control conditions when the behavior is tested. However, the resultant increase in neuronal excitability produced by pubertal α4 knockdown could also increase activation of target sites and potentially alter intracellular messengers in addition to increasing L5 PL spine density. Anxiety is the most common mental disorder1, yet the etiology is not well understood at the circuit level, nor are the potential treatments10. This disorder is twice as likely to afflict females, with onset most likely to occur during adolescence2 with subtypes ranging from generalized anxiety disorder, agoraphobia, panic disorder, and obsessive-compulsive disorder43. These disorders have a high probability of continuing into adulthood6 when there is an increased risk of suicide44. This study suggests that one contributing factor for anxiety behavior generated post-pubertally is an increase in excitatory synapses in L5 PL via dysregulation of pruning, increasing the input to activate this region. Excitatory input to L5 PL pyramidal cells comes from the ventral hippocampus, amygdala, and multiple sensory sites45. L5 pyramidal cells provide the output of the PL to the basolateral amygdala46 to regulate fear and anxiety12. Increasing local glutamate concentrations with veratrine in the PL of rodents increases anxiety using the open field test16. Blocking NMDARs47 in the PL prevents this effect suggesting that anxiety is triggered by NMDAR-mediated transmission. Conversely, numerous studies show that inactivating the PL using either pharmacological or electrolytic techniques reduces anxiety15,48. Thus, the present findings correlating L5 PL spine density with avoidance behavior provide a mechanistic link of the PL with increased anxiety. In contrast, the IL is associated with reduced fear/anxiety and fear extinction12, due to output to GABAergic neurons via the uncincate fasciculus, which reduces activity in the basolateral amygdala17. Human studies also support a dual role for the PL and IL sub-regions of the mPFC. Dorsal regions of the mPFC, including the anterior cingulate, which corresponds to the rodent PL cortex, are activated by fear49. Increased gamma power EEG changes or blood flow accompanies increased fear or anxiety due to fear conditioning or in individuals with generalized anxiety disorder50-52. These correlations of enhanced learned fear expression and persistent PL activation are greater in females49. In contrast, the human ventromedial PFC (vmPFC), corresponding to the rodent IL, exhibits decreased activity in anxiety53. vmPFC lesions increase the amygdala response to aversive stimuli13, further confirming the role of the IL/vmPFC in fear reduction. In the present study, mushroom spines showed the greatest reduction in spine density (74%) in the female L5 PL. The larger head of these spines have a higher density of AMPA receptors54 and thus would be expected to have a greater synaptic impact on PL activation. Local α4 knockdown in the PL prevented spine pruning at PND 56, resulting in increased mushroom spine density with levels similar to pubertal wild-type values. Enhanced excitatory transmission to PL would activate output to the amygdala and is a likely mechanism underlying the increased anxiety following local knockdown of α4 expression. α4 knockdown reversed the 45% decrease in density of the motile spines (thin spines, long thin spines, and filopodia) in adolescence. Motile spines are thought to represent learning spines55, which may function in learned fear, such as conditioned cue-related and contextual fear for which the PL plays a role56. α4βδ GABAR expression is altered in the human frontal cortex in some types of mental disorders, especially those that emerge in childhood or adolescence57, with decreased expression in brains of non-depressed suicide victims32,58. Non-depressed suicide is usually characterized by anxiety44. Thus, genetic factors producing dysregulated α4βδ GABAR expression may reduce synaptic pruning during adolescence to increase anxiety. In cases where there are persistent alterations in expression of α4βδ GABARs, as seen in depression and anxiety32, the ultimate effect would depend on the area of expression. Decreased expression of these receptors in the adult prelimbic area would increase anxiety, as suggested by research studies16. Increases in α4βδ GABARs are reported in orbitofrontal cortex of suicide victims31, which is analogous to the rodent infralimbic. Increased inhibition of this area, outside of the adolescent pruning period, would be expected to increase anxiety, as suggested by clinical imaging studies13, and also increase depression, as suggested by studies showing that stimulation of this area is anti-depressant59-61. Increased expression of α4βδ GABARs at puberty was shown both by increases in α4 immunostaining as well as by increased responses of L5 pyramidal cells to the GABA agonist GBX, at a concentration selective for α4βδ GABARs26. α4βδ GABAR expression was reduced to near pre-pubertal levels by PND 56, however, suggesting a transient increase in pubertal expression of these receptors. Furthermore, α4 immunostaining was localized to the cell body, dendrites, and the spines at puberty, where these receptors would be expected to impair NMDAR activation, as previously shown24 in other CNS areas. The inhibition generated by these receptors along the dendritic shaft as well as on the soma would also impair NMDAR activation by decreasing action potential back-propagation, which is generated in the axon hillock within the soma, travels up the dendrite, and would normally facilitate Mg++ unblock of the NMDAR channel62-65. In the present study, increased NMDAR expression generated by administration of low doses of MK-80138 during puberty prevented pruning in wild-type mice. In contrast, blocking NMDARs in α4 -/- mice using memantine, a treatment which does not increase NMDAR expression39, most likely due to its higher affinity for the receptor40, restored pruning in the absence of α4βδ-mediated inhibition. These data suggest that α4βδ impairment of NMDARs underlies adolescent pruning of L5 PL. This outcome was mediated by the Rho-guanine nucleotide exchange factor Kal-7, a spine protein necessary for spine maintenance28. Kal-7 activates the small GTPase Rac1, which stabilizes the actin cytoskeleton via P21-activated kinases within the spine66, and the expression of Kal-7is increased by NMDAR activation29. Thus, decreased Kal-7 expression at puberty would destabilize the spine to enable spine removal. However, Kal-7 expression was increased in L5 PL of pubertal α4 -/- mice, suggesting that the increase in α4βδ GABARs in wild-type mice is the initial trigger for the decrease in Kal-7 expression, which leads to pruning, as shown in other CNS sites29 (See schematic diagram, Fig. 7). However, we cannot rule out other spine proteins which may play a role in spine stability and pruning67-69. In addition, the microglia70 and autophagy71 have been shown to play a role in pruning but are likely the final steps in this process.  The present findings also show that systemic pubertal administration of the drugs picrotoxin and GBX, which block all GABAR subtypes and potentiate α4βδ GABARs, respectively, was successful in altering PL spine density in the predicted direction at the circuit level. That is, picrotoxin increased spine density, and GBX decreased spine density post-pubertally. This is an interesting finding because the drugs would impact all brain areas, including those with inhibitory inputs to the PL. These findings suggest that pubertal systemic administration of these GABAergic drugs can be used to manipulate spine density in the L5 PL. In the frontal cortex, synaptic GABAergic afferents target αxβxγ2 GABARs on the dendritic spine36. Pubertal administration of the positive GABAR modulator LZM, a benzodiazepine that enhances synaptic inhibition of the dendritic spines at αxβxγ2 GABARs lacking α437, had no significant effect on the overall post-pubertal spine density of the basilar dendrites. This suggests that extrasynaptic α4βδ GABARs, rather than synaptic GABARs, are selectively responsible for synaptic pruning of L5 PL pyramidal cells during adolescence. Decreases in L5 PL total spine density were \>50% for females across a timespan which reflected puberty onset (~PND 35) and continued until late adolescence (PND 56). Similar findings were noted for males, which were also due to α4βδ GABARs, as evidenced by the lack of pruning in knock-outs that lacked these receptors’ pubertal expression. Synaptic pruning has been demonstrated previously in L5 mPFC, with decreases ranging from \<10% in the rat to 30-40%21 in humans for combined IL and PL. A 30% decrease in spine density was reported for combined L3 and L5 PL in male transgenic mice23, assessed in early adolescence (PND 31-45), where pubertal timing was not noted. Puberty onset is the time when α4βδ-mediated inhibition increases and triggers pruning; thus, assessments following onset would reflect the greatest change in spine density. Spine density of L5 PL pyramidal cells ultimately impacts neural networks that generate oscillations with frequencies in the gamma, theta, and delta range72. These oscillations represent the emergent properties of recurrent local networks and depend upon the excitatory and inhibitory synaptic input to the dendritic spines of L5 pyramidal neurons. The impedance mismatch between the spine and adjacent dendrite enables the spines to act as coincidence detectors, responding to spatially distributed signals within a limited time window73. Thus, spine density determines the sensitivity and reliability of the network to afferent input. In the PL, increased spine density likely results in increased neural activity, which activates downstream targets such as the amygdala and results in increased anxiety. This finding is supported by the present study as well as by clinical imaging studies74,75. In conclusion, α4βδ GABARs were shown to trigger synaptic pruning in L5 PL as an essential process in limiting anxiety responses in late adolescence and adulthood. Dysregulation of pruning increased anxiety responses. These results suggest that deficiencies in the pruning of PL at puberty may be a key physiological mechanism for mental disorders. Given the recent reports showing abnormal gene signals for α4 and δ in some mental disorders 31,32,57,58, the present findings may suggest therapeutic strategies for anxiety disorders that emerge at puberty. # Discussion Discussion In this doctoral thesis, we investigated the role of α4βδ GABAA receptors in synaptic pruning of the prelimbic cortex (PL) and its impact on anxiety response in adulthood. The present study expands upon the previous research conducted by [previous student’s name], which focused on the role of α4βδ GABAA receptors in spine pruning and behavioral flexibility of female mice during adolescence in the CA1 hippocampus. Our findings further elucidate the role of α4βδ GABAA receptors in the synaptic pruning process and emphasize the significance of these receptors in the development of anxiety responses. One of the critical findings in our study is the significant decrease in spine density in layer 5 (L5) PL pyramidal cells during puberty, with the most substantial decline observed in stable spine types (mushroom, stubby, and bifurcated). This is in line with the previous study that demonstrated dendritic pruning in the CA1 hippocampus during normal adolescent development. We also found that α4βδ GABAR expression increases transiently at puberty onset in L5 PL, similar to the changes in GABARs observed by [previous student’s name] in the CA1 hippocampus. These findings suggest that α4βδ GABARs play a crucial role in synaptic pruning across different brain regions and in both male and female mice. Our study further demonstrated that synaptic pruning in L5 PL is prevented in mice with knock-out of the GABAR α4 subunit, implying a critical role for α4βδ GABARs in this process. This result is consistent with the previous research, which linked changes in pubertal GABAARs to altered post-pubertal cognitive abilities and behavioral flexibility. In addition, our study established that pharmacological manipulation of GABARs during puberty can alter spine density, with picrotoxin increasing spine density and gaboxadol decreasing density of thin spines. This finding highlights the potential therapeutic implications of modulating GABARs during critical developmental periods. Another significant discovery in our research is the involvement of NMDAR activity in synaptic pruning of L5 PL. Over-expression of NMDARs prevented pruning in wild-type mice, while blocking NMDARs restored pruning in α4 -/- mice. This finding suggests a complex interplay between GABAARs and NMDARs in regulating synaptic pruning during adolescence. In terms of anxiety response, our study demonstrated that local knockdown of α4βδ GABARs in PL during puberty increases spine density and anxiety responses to an aversive stimulus in mice post-pubertally. This finding underscores the importance of normal α4βδ GABAR expression during puberty for proper anxiety regulation in adulthood. In conclusion, our study builds upon and extends the findings of [previous student’s name] by elucidating the role of α4βδ GABAA receptors in synaptic pruning of the prelimbic cortex and its impact on anxiety response in adulthood. The present research highlights the importance of α4βδ GABARs in synaptic pruning across different brain regions and suggests that alterations in the GABAergic system during critical developmental periods can have lasting consequences on anxiety regulation and cognitive performance. Future studies should explore the precise molecular mechanisms underlying the interplay between GABAARs and NMDARs in synaptic pruning and the potential therapeutic applications of modulating these receptors during adolescence. #### Executive Function Executive function, a critical cognitive process regulated by the prefrontal cortex, plays a vital role in the context of anxiety disorders. The medial prefrontal cortex (mPFC) is integral to executive function and heavily implicated in anxiety-related processes (Bishop, 2009). This section will delve into the importance of executive function and its relationship with anxiety disorders, as well as the specific involvement of the mPFC. Executive function encompasses several cognitive processes, such as working memory, cognitive flexibility, and inhibitory control, which enable goal-directed behavior, decision-making, and adaptation to novel situations (Diamond, 2013). In anxiety disorders, these processes are often dysregulated, leading to maladaptive behaviors and heightened anxiety levels. Research by Bishop (2009) demonstrates that individuals with anxiety disorders exhibit atypical mPFC activity which can impair executive function and exacerbate symptoms. A key component of executive function is working memory – the temporary storage and manipulation of information necessary for complex cognitive tasks. Eysenck et al. (2007) found that anxiety impairs working memory, with the mPFC being a central player in this dysfunction. Moreover, impaired working memory may result in reduced ability to regulate anxious thoughts and emotions, further worsening anxiety symptoms (Vytal & Hamann, 2010). Cognitive flexibility, another critical aspect of executive function, refers to the ability to shift attention and adapt to changes in the environment or task demands. In anxiety disorders, reduced cognitive flexibility is associated with rigid thinking patterns and difficulty disengaging from negative stimuli (Derryberry & Reed, 2002). The mPFC has been implicated in this dysfunction, as demonstrated by a study conducted by Cisler and Koster (2010), where it was found that individuals with anxiety disorders displayed altered mPFC activity during tasks requiring cognitive flexibility. Inhibitory control – the ability to suppress irrelevant or inappropriate thoughts, behaviors, and emotions – is also crucial in managing anxiety symptoms. Disrupted inhibitory control can lead to heightened anxiety due to difficulty in suppressing negative emotions and intrusive thoughts (Etkin & Schatzberg, 2011). Goldin et al. (2013) reported that individuals with generalized anxiety disorder exhibited abnormal mPFC activation during tasks requiring inhibitory control, suggesting a direct link between mPFC dysfunction and anxiety disorders.

Rendered

Uncovering the Role of α4βδ GABAA Receptors in Synaptic Pruning of the Prelimbic Cortex and its Impact on Anxiety Response in Adulthood.

A thesis submitted to the faculty of

The School of Graduate Studies State University of New York Downstate Medical Center

In partial fulfillment of the requirements for the degree of Doctor of Philosophy

by

Matthew R. Evrard

Program in Neural and Behavioral Science

03/27/2022

Thesis Advisor: Sheryl Smith, Ph.D.

Physiology and Pharmacology

Contents

Introduction 3

Anxiety Disorders 3

Medial Prefrontal Cortex 5

Adolescence 7

Specific Aims 13

Specific Aim 1: Investigate the synaptic pruning process in pyramidal cells of the L5 PL region in the female mouse brain during the transition from puberty to post-puberty. 13

1.1: Analyze spine density of basilar dendrites in Golgi-stained neurons. 13

1.2: Identify specific spine types that are most affected by this process. 13

Specific Aim 2 13

2.1: Assess α4βδ GABAR expression during different developmental stages. 13

2.2: Investigate the effects of knockout and pharmacological manipulations on synaptic pruning. 13

3.1: Manipulate NMDAR expression during puberty. 13

3.2: Assess the effects of NMDAR manipulation on spine density at post-puberty. 13

4.1: Determine the relationship between Kal-7 expression and α4βδ GABAR expression. 13

4.2: Investigate the role of Kal-7 in synaptic pruning. 13

Specific Aim 5: Assess the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood. 13

5.1: Perform stereotaxic virus injections to selectively knockdown α4 in the PL during puberty. 13

5.2: Analyze the effects of local α4 knockdown on spine density and Kal-7 expression. 13

5.3: Evaluate anxiety-related behavior in mice using behavioral tests such as the elevated plus-maze. 13

Material and Methods 13

Specific Aim 1 Error! Bookmark not defined.

1.1: Analyze spine density of basilar dendrites in Golgi-stained neurons. Error! Bookmark not defined.

1.2: Identify specific spine types that are most affected by this process. Error! Bookmark not defined.

Specific Aim 2 18

2.1: Assess α4βδ GABAR expression during different developmental stages. Error! Bookmark not defined.

2.2: Investigate the effects of knockout and pharmacological manipulations on synaptic pruning. Error! Bookmark not defined.

Specific Aim 3: 26

3.1: Manipulate NMDAR expression during puberty. 26

3.2: Assess the effects of NMDAR manipulation on spine density at post-puberty. 27

Specific Aim 4: 27

4.1: Determine the relationship between Kal-7 expression and α4βδ GABAR expression. 27

4.2: Investigate the role of Kal-7 in synaptic pruning. 27

Specific Aim 5: 28

5.1: Perform stereotaxic virus injections to selectively knockdown α4 in the PL during puberty. 28

5.2: Analyze the effects of local α4 knockdown on spine density and Kal-7 expression. 28

5.3: Evaluate anxiety-related behavior in mice using behavioral tests such as the elevated plus-maze. 29

Specific Aim 6: Error! Bookmark not defined.

Discussion 36

Discussion 38

Introduction

Development of the central nervous system requires the overproduction and refinement of chemical synapses1. Synaptic pruning, a critical mechanism that removes unnecessary or weak synapses to foster circuit refinement and plasticity, plays a vital role in establishing appropriate neural circuits and behaviors2. Various neuropsychiatric disorders have been linked to dysregulation of synaptic pruning2,3. Although this dysregulation has been extensively investigated in schizophrenia4, its involvement in anxiety disorders remains largely unexplored. Anxiety disorders, which affect millions of people globally, are highly prevalent and produce debilitating conditions. Hence, understanding the mechanisms contributing to their development and maintenance is essential.

Research has demonstrated that the α4βδ subtype of GABA(A) receptors contributes to the regulation of synaptic pruning during critical periods of hippocampal5 and dentate gyrus6 brain development. However, the specific function of α4βδ GABA(A) receptors in synaptic pruning within the prelimbic cortex and its impact on adult anxiety response is still unclear. Given the established role of GABAergic neurotransmission in anxiety regulation7, examining the involvement of α4βδ GABA(A) receptors in synaptic pruning of the prelimbic cortex could offer valuable insights into the molecular and cellular mechanisms responsible for anxiety disorders’ development and maintenance.

This thesis utilizes a combination of histological techniques, pharmacological and genetic interventions, and behavioral assays to investigate the involvement of α4βδ GABAA receptors in dendritic pruning of the prelimbic cortex and its subsequent effects on anxiety response in adulthood. The study aims to elucidate the underlying mechanisms by which α4βδ GABAA receptors regulate synaptic pruning in the prelimbic cortex by quantifying the expression of α4βδ GABAA receptors and spine proteins during puberty, manipulating synaptic pruning through GABAARs drug administration, and comparing spine density and protein expression in constitutive knock-out and conditional knock-down mice. Ultimately, this research seeks to deepen our understanding of synaptic pruning’s role in anxiety disorders and offer insight into the potential therapeutic value of targeting α4βδ GABAA receptors for anxiety treatment.

Anxiety Disorders

Anxiety disorders, characterized by excessive fear, worry, and physiological symptoms, rank among the most prevalent and debilitating psychiatric conditions globally. Two major classification systems, the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-5; American Psychiatric Association, 2013) and the International Classification of Diseases, 11th Revision (ICD-11; World Health Organization, 2018), define anxiety disorders as a diverse group of conditions marked by heightened fear, anxiety, and avoidance behavior, often accompanied by somatic symptoms. The DSM-5 and ICD-11 recognize several subtypes of anxiety disorders, including generalized anxiety disorder (GAD), panic disorder, social anxiety disorder, specific phobias, and separation anxiety disorder. The DSM-5 also classifies agoraphobia and selective mutism as distinct anxiety disorders, while the ICD-11 categorizes them as phobic anxiety disorders and childhood-onset fluency disorder, respectively. Detailed descriptions and symptoms of each subtype are provided in Table 1.1. Estimates of anxiety disorder prevalence vary due to methodological differences, diagnostic criteria, and sample characteristics. However, large-scale, population-based studies and meta-analyses converge on their high prevalence worldwide. Kessler et al. (2005) estimated the lifetime prevalence of anxiety disorders in the United States at 28.8%, with a 12-month prevalence rate of 18.1%. The World Mental Health Survey Initiative, covering 25 countries, reported a global lifetime prevalence of 16.6% and a 12-month prevalence of 11.2% (Kessler et al., 2009). A meta-analysis of 87 studies by Baxter et al. (2013) found a global lifetime prevalence of 16.7% and a 12-month prevalence of 10.6%. Anxiety disorder prevalence varies by gender, age, and cultural factors, with females exhibiting higher rates than males (lifetime prevalence of 20.5% and 13.1%, respectively) (Remes et al., 2016). Anxiety disorders typically manifest during adolescence, peaking between ages 15 and 24 (Kessler et al., 2005; McLaughlin et al., 2011).Several methodological challenges can influence the assessment of anxiety disorder prevalence. Heterogeneity in study design, sampling strategies, and diagnostic criteria contribute to discrepancies in prevalence estimates (Wittchen et al., 2011). For example, studies utilizing self-report questionnaires may overestimate prevalence rates due to the lack of clinical validation, whereas studies relying on structured clinical interviews may underestimate prevalence by not capturing subthreshold cases (Balázs et al., 2013; Goodwin et al., 2017). Furthermore, fluctuations in diagnostic criteria over time and between classification systems (e.g., DSM-IV vs. DSM-5, ICD-10 vs. ICD-11) can lead to variations in prevalence rates (Clark et al., 2017). The inclusion or exclusion of specific anxiety disorders, such as agoraphobia and selective mutism, can further impact prevalence estimates.

TABLE: ANXIETY DISORDERS

Current Treatments

The current treatments for anxiety disorders, while providing some relief, have limited efficacy due to our incomplete understanding of the underlying physiological mechanisms driving these disorders. The primary treatment modalities for anxiety disorders include pharmacotherapy, psychotherapy, and, in some cases, a combination of both. However, a significant proportion of patients do not achieve full remission or experience adverse side effects, emphasizing the need for a deeper understanding of the etiology and pathophysiology of anxiety disorders.

Pharmacological treatments for anxiety disorders primarily target neurotransmitter systems, such as the serotonergic, noradrenergic, and gamma-aminobutyric acid (GABA) systems. Benzodiazepines, the most prescribed class of anxiolytics, have been in use for several decades. Despite their widespread application, benzodiazepines exhibit several drawbacks. For instance, they are associated with a high risk of dependence and adverse effects, such as cognitive impairment and sedation (Baldwin et al., 2013). Additionally, benzodiazepines do not address the root causes of anxiety disorders and only provide symptomatic relief (Depping et al., 2016). Selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) are other pharmacological treatments frequently utilized for anxiety disorders. While they exhibit a better side effect profile compared to benzodiazepines, their anxiolytic effects often take weeks to manifest (Bandelow et al., 2017). Moreover, a substantial proportion of patients do not respond adequately to these medications, highlighting the need for more targeted therapeutic approaches (Watanabe et al., 2018). The limitations of current pharmacological treatments can be attributed, in part, to the lack of understanding of the specific neurobiological mechanisms that contribute to anxiety disorders. Recent research has identified multiple pathways and neurotransmitter systems that may play a role in the etiology of anxiety disorders, including the glutamatergic, GABAergic, serotonergic, and noradrenergic systems (Bandelow et al., 2016; Lueken & Hahn, 2016).

Cognitive-behavioral therapy (CBT) is a widely employed psychotherapeutic intervention for anxiety disorders, focusing on identifying and modifying maladaptive thought patterns and behaviors. While CBT has demonstrated efficacy in reducing anxiety symptoms, not all patients respond to this approach. Additionally, access to qualified therapists and the time-consuming nature of therapy can limit the feasibility and effectiveness of CBT for some individuals. The limitations of current treatment options highlight the importance of advancing our knowledge of the physiological mechanisms underpinning anxiety disorders. A more comprehensive understanding of these processes will enable the development of targeted and personalized interventions, enhancing treatment efficacy and minimizing adverse effects. This underscores the critical need for ongoing research into the pathophysiology of anxiety disorders, which will be discussed in the subsequent section.

Pathophysiology

Investigating the pathophysiology of anxiety disorders has been an area of growing interest, with particular focus on the role of the prefrontal cortex (PFC) in the regulation of fear and anxiety. The PFC is a complex brain region that governs higher-order cognitive functions, including decision-making, emotional regulation, and behavioral flexibility. Research has identified several prefrontal subregions that are implicated in the development and maintenance of anxiety disorders, providing valuable insights into potential therapeutic targets. For example, recent studies have highlighted alterations in GABA gene expression within PFC regions associated with anxiety (Cui et al., 2018).

One such subregion is the medial prefrontal cortex (mPFC), which has extensive connections with the amygdala, hippocampus, and other limbic structures that are involved in the processing of fear and emotional memories. The mPFC is critical for the regulation of fear responses, with its ventral portion, including the infralimbic cortex, promoting fear extinction, and the dorsal portion, including the prelimbic cortex, facilitating fear expression (Milad & Quirk, 2012). Dysregulation of mPFC activity, particularly in the prelimbic cortex, has been associated with heightened fear responses and impaired fear extinction, both of which are hallmark features of anxiety disorders (Marek et al., 2013).

Neuroimaging studies have provided further evidence of altered PFC functioning in individuals with anxiety disorders. For instance, reduced activation and gray matter volume in the mPFC have been observed in patients with generalized anxiety disorder, social anxiety disorder, and panic disorder (Etkin & Wager, 2007; Liao et al., 2011; Shin & Liberzon, 2010). These findings suggest that aberrant PFC activity may contribute to the development and persistence of anxiety symptoms, warranting further investigation into the neurobiological mechanisms underlying these alterations.

Medial Prefrontal Cortex

The prefrontal cortex (PFC) is a crucial part of the human brain, located in the anterior region of the frontal lobes. This neural tissue is essential for higher-order cognitive processes, including decision-making, social behavior, and emotion regulation, all of which contribute to our understanding of anxiety disorders (Davidson & McEwen, 2012).

A natomically, the PFC is divided into several subregions, with the medial prefrontal cortex (mPFC) playing a central role in the modulation of anxiety-related behavior. The mPFC encompasses the dorsal anterior cingulate cortex (dACC), pregenual anterior cingulate cortex (pgACC), and ventromedial prefrontal cortex (vmPFC), among other areas. Each of these subdivisions has unique cellular structures and neural connections that contribute to the regulation of anxiety (Etkin et al., 2011; Etkin et al., 2009; Milad et al., 2007). The dACC is a critical hub for processing cognitive conflict and emotional information. It comprises six layers of neurons, with the largest pyramidal cells in layer V. These cells project to various brain regions, such as the amygdala and hypothalamus, which are involved in emotional processing and stress responses. Research by Etkin et al. (2011) demonstrated that individuals with generalized anxiety disorder (GAD) exhibited heightened dACC activation during tasks requiring emotion regulation, indicating a possible role for this region in the pathophysiology of anxiety disorders. The pgACC is situated anterior and ventral to the dACC. This region contains densely packed neurons and is known for its extensive connections with limbic and paralimbic structures, such as the amygdala, hippocampus, and insula. These connections enable the pgACC to modulate emotional responses and monitor internal states. A study by Etkin et al. (2009) revealed that patients with GAD displayed reduced pgACC activation during emotion regulation tasks, suggesting that dysfunction in this region may contribute to maladaptive emotional processing in anxiety disorders. The vmPFC, located ventral to the dACC and pgACC, is involved in the appraisal and regulation of emotional stimuli. It contains neurons organized in six layers and projects to several limbic and paralimbic areas, such as the amygdala, hippocampus, and hypothalamus. The vmPFC has been implicated in the extinction of conditioned fear responses, a process relevant to the treatment of anxiety disorders. Milad et al. (2007) found that individuals with post-traumatic stress disorder (PTSD) exhibited diminished vmPFC activation during fear extinction tasks, indicating that this region may be crucial for understanding and treating anxiety-related conditions.

Rodent Analogue

The rodent prelimbic (PL) and infralimbic (IL) cortex, both subregions of the medial prefrontal cortex (mPFC), have been extensively studied for their role in various cognitive and emotional processes. This section aims to provide a comprehensive analysis of the PL and IL cortex, comparing their functions and circuitry, while focusing on their involvement in schizophrenia and anxiety.

The prelimbic cortex is located in the dorsal part of the mPFC and plays a crucial role in executive functions, decision-making, and goal-directed behaviors (Vertes, 2004). It has extensive connections with other brain regions, including the hippocampus, amygdala, and nucleus accumbens. The infralimbic cortex, situated ventrally to the prelimbic cortex, is involved in emotional regulation, extinction learning, and the suppression of inappropriate behavioral responses (Sierra-Mercado et al., 2011). It shares similar connections with the aforementioned brain regions but with different innervation patterns.

In rodent models of schizophrenia, the PL cortex displays abnormal functioning, with disruptions in excitatory and inhibitory balance. Specifically, alterations in glutamatergic and GABAergic neurotransmission have been observed (Lodge & Grace, 2007). These imbalances contribute to the cognitive deficits and positive symptoms associated with schizophrenia. Furthermore, the dysregulation of dopamine in the PL cortex plays a role in the pathophysiology of the disease (Grace et al., 2017). The IL cortex has been less studied in schizophrenia; however, it is also implicated in disease due to its connections with the amygdala and hippocampus. Similar to the PL cortex, alterations in glutamatergic and GABAergic neurotransmission have been observed in the IL cortex in rodent models of schizophrenia (Balu & Coyle 2011). The IL cortex may contribute to the negative symptoms and emotional dysregulation observed in the disorder.

T he PL cortex has been implicated in the generation and regulation of anxiety-related behaviors. In rodent models, increased activity in the PL cortex correlates with heightened anxiety, while inhibition of the PL cortex reduces anxiety-like behaviors (Adhikari et al., 2015). The PL cortex modulates anxiety through its connections with the amygdala, particularly the basolateral amygdala (BLA), which is involved in processing emotionally salient stimuli and generating fear responses (Rosenkranz & Grace, 2002). Dysregulation of the PL-BLA circuitry may contribute to the development of anxiety disorders. The IL cortex plays a significant role in regulating anxiety and fear responses. It has been demonstrated that activation of the IL cortex promotes the extinction of conditioned fear, while its inhibition impairs extinction learning (Milad & Quirk, 2012). The IL cortex exerts its anxiolytic effects through its connections with the amygdala via inhibitory interneurons (Amano et al., 2010), particularly the central nucleus of the amygdala (CeA), which is a critical output structure for fear and anxiety responses (Ciocchi et al., 2010). Moreover, the IL cortex is involved in modulating stress responses via its connections with the hypothalamic-pituitary-adrenal (HPA) axis (Radley et al., 2006).

Both the PL and IL cortex are implicated in the pathophysiology of schizophrenia and anxiety due to their involvement in emotion processing and regulation. Dysregulation of glutamatergic and GABAergic neurotransmission is observed in both subregions in schizophrenia, while altered connectivity with the amygdala plays a role in anxiety disorders. The PL cortex is more prominently involved in executive functions and positive symptoms of schizophrenia, whereas the IL cortex is more associated with negative symptoms and emotional dysregulation. In anxiety, the PL cortex contributes to the generation of anxiety-related behaviors, while the IL cortex is crucial for the regulation of fear responses and anxiety.

Adolescence

Adolescence represents a critical period in human development, characterized by significant physiological, psychological, and cognitive changes. During this time, the brain undergoes extensive remodeling, particularly within the medial prefrontal cortex (mPFC), a key region implicated in the maturation of executive functions and the emergence of psychiatric disorders such as anxiety and schizophrenia (Casey et al., 2008). To better understand the neurobiology of adolescence, this section will elucidate the processes of synaptic pruning, neural circuit development, and dendritic spine formation, which are integral to the maturation of the mPFC.

Gray matter comprises cell bodies, dendrites, and synapses, and it plays a crucial role in information processing. During adolescence, the brain undergoes region-specific gray matter changes. For example, Giedd et al. (1999) observed a nonlinear pattern of gray matter development, with cortical thickness increasing during childhood and subsequently decreasing during adolescence. Notably, these alterations occurred in a region-specific manner, with the prefrontal cortex (PFC) experiencing the most pronounced changes. This finding suggests that gray matter maturation, particularly in the PFC, may be an essential factor in the development of executive functions and cognitive control; both are often impaired in individuals with anxiety disorders (Casey et al., 2008).

White matter, primarily composed of myelinated axons, facilitates communication between different brain regions. During adolescence, the brain undergoes substantial white matter growth, leading to improved information transfer and integration (Asato et al., 2010). A landmark study by Barnea-Goraly et al. (2005) employed diffusion tensor imaging (DTI) to reveal that fractional anisotropy—a measure of white matter integrity—increased with age in various brain regions, including the PFC. These findings indicate that the adolescent brain is characterized by ongoing development of white matter, which may contribute to enhanced cognitive abilities and the regulation of emotions, both of which are relevant to the emergence of anxiety disorders. Myelination is the process by which oligodendrocytes wrap around axons to form a myelin sheath, which increases the speed and efficiency of neural transmission. The adolescent brain experiences significant increases in myelination, particularly in the PFC (Paus et al., 2001). This increased myelination is thought to improve connectivity between different brain regions and enhance cognitive abilities such as decision-making, impulse control, and emotion regulation (Blakemore & Choudhury, 2006). These processes are critical for adaptive behavior and coping with stress; their disruption may contribute to the development of anxiety disorders.

Functional connectivity refers to the temporal correlations between spatially separated brain regions, reflecting the degree of coordination between these areas. During adolescence, functional connectivity undergoes dynamic changes, with a general shift from short-range to long-range connectivity (Fair et al., 2009). This reorganization promotes the integration of information across disparate brain regions and supports the development of advanced cognitive and emotional processes. Alterations in functional connectivity—particularly within the PFC and its connections to other regions—have been implicated in the etiology of anxiety disorders (Sylvester et al., 2012).

Synaptic Pruning in Adolescent Brain DevelopmentSynaptic pruning is an essential process for refining neural circuitry and optimizing brain function during adolescence, ultimately contributing to maturation of cognitive and emotional processing (Petanjek et al., 2011). During this period, the prefrontal cortex (PFC) undergoes substantial synaptic pruning where synapses have been shown to decrease by roughly half (Huttenlocher & Dabholkar, 1997). This process is influenced by various factors, including genetics, environmental stimuli, and neuronal activity. The precise mechanisms underlying synaptic pruning remain an area of active research, but several key cellular and molecular players have been identified.

Microglia, the resident immune cells of the central nervous system, have been implicated in the synaptic pruning process. In a landmark study by Schafer et al. (2012), microglia were found to engulf and eliminate synapses in the developing mouse brain, with the complement system playing a critical role in this process. Complement proteins such as C1q and C3 tag synapses for removal; microglia recognize these tags to selectively phagocytose the targeted synapses.

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Neuronal Activity and Signaling Molecules in Synaptic Pruning

Neuronal activity is another crucial factor that influences synaptic pruning. During development, synapses that are more active and transmit stronger signals are preferentially maintained, whereas weaker and fewer active synapses are eliminated (Bourgeois & Rakic, 1993). This activity-dependent pruning process is mediated by various signaling molecules such as brain-derived neurotrophic factor (BDNF) and NMDA receptors. BDNF has been shown to promote stabilization and maturation of synapses (McAllister et al., 1999), while NMDA receptor activation can lead to long-term potentiation (LTP) or long-term depression (LTD), depending on the strength and duration of synaptic activity (Collingridge et al., 2010).

Disruptions in the synaptic pruning process during adolescence have been implicated in the development of anxiety disorders. Abnormal pruning in the PFC may lead to an imbalance in excitatory and inhibitory neurotransmission, resulting in maladaptive neural circuitry that predisposes individuals to anxiety (Casey et al., 2008). For example, excessive pruning of inhibitory synapses or inadequate pruning of excitatory synapses may cause hyperactivity in the PFC, resulting in heightened anxiety and stress responses (Waters et al., 2015). In conclusion, adolescence is a critical period marked by significant changes in brain structure and function. Understanding the processes of synaptic pruning, myelination, and functional connectivity during this time can provide crucial insights into the neurobiology of psychiatric disorders such as anxiety. Future research should continue to explore the cellular and molecular mechanisms underlying these processes to inform the development of targeted interventions for adolescents at risk for psychiatric disorders.

Functional connectivity refers to the temporal correlations between spatially separated brain regions, reflecting the degree of coordination between these areas. During adolescence, functional connectivity undergoes dynamic changes, with a general shift from short-range to long-range connectivity (Fair et al., 2009). This reorganization promotes the integration of information across disparate brain regions and supports the development of advanced cognitive and emotional processes. Alterations in functional connectivity, particularly within the PFC and its connections to other regions, have been implicated in the etiology of anxiety disorders (Sylvester et al., 2012).

Dendritic Spines

Dendritic spines, small protrusions emerging from dendrites of neurons, play a critical role in synaptic transmission and plasticity. They serve as the primary site of excitatory synaptic input, which enables them to participate actively in the reception, integration, and transmission of neural signals. In the context of adolescence, dendritic spine development and maturation is highly dynamic, with significant implications for the emergence of anxiety disorders due to abnormalities in synaptic pruning in the prefrontal cortex.

During adolescence, the brain undergoes significant changes, including alterations in dendritic spine density and morphology. These changes are crucial for the refinement of neural circuits and the establishment of efficient communication between brain regions. Animal studies have shown that the number of dendritic spines in the prefrontal cortex increases during early adolescence, followed by a decline in density because of synaptic pruning (Petanjek et al., 2011). This reduction in spine density is believed to reflect a process of synaptic refinement, allowing for the optimization of neural circuits, and contributing to the maturation of cognitive and emotional processing. Abnormalities in dendritic spine development during adolescence can have profound consequences for neural circuitry and the emergence of anxiety disorders. Research has shown that excessive or insufficient synaptic pruning can lead to imbalances in excitatory and inhibitory signaling, ultimately resulting in dysfunctional neural circuits (Bourne & Harris, 2011). This dysfunction can manifest as an increased susceptibility to anxiety disorders, as the affected individual may struggle to regulate emotional responses and process environmental stimuli effectively.

A study conducted by Pattwell et al. (2016) demonstrated that altered dendritic spine dynamics in the prefrontal cortex during adolescence can lead to anxiety-like behaviors in rodents. The researchers found that mice exposed to chronic stress during adolescence exhibited reduced dendritic spine density and abnormal spine morphology in the prefrontal cortex. These changes were accompanied by heightened anxiety-like behaviors, suggesting that alterations in dendritic spine dynamics might contribute to the development of anxiety disorders. Further evidence for the role of dendritic spines in anxiety disorders comes from studies investigating the molecular mechanisms underlying spine formation and pruning. For instance, alterations in the expression and function of key proteins involved in spine development, such as the postsynaptic density protein 95 (PSD-95) and the actin-regulating protein cofilin, have been implicated in the pathophysiology of anxiety disorders (Carlisle et al., 2011; Garey et al., 2010). Understanding the molecular basis of dendritic spine dynamics during adolescence may provide valuable insights into the etiology of anxiety disorders and inform the development of targeted therapeutic interventions.

Spine Proteins

In the context of synaptic signaling and plasticity, several key proteins play crucial roles in regulating neuronal function and structure. AMPA and NMDA receptors are ionotropic glutamate receptors that mediate excitatory synaptic transmission, with NMDA receptors being particularly important for synaptic plasticity. α4βδ GABAARs are a subtype of ionotropic GABA receptors that contribute to inhibitory synaptic transmission, modulating the overall excitability of neurons. CaMKII, a calcium/calmodulin-dependent protein kinase, is involved in various cellular processes, including synaptic plasticity and learning. CDK5, a serine/threonine kinase, is also implicated in synaptic plasticity and can activate Kalirin-7, a guanine nucleotide exchange factor (GEF) that regulates the activity of Rho GTPases. Among these Rho GTPases, Rac1 is essential for controlling the actin cytoskeleton and promoting dendritic spine expansion. Together, these proteins form intricate signaling networks that govern neuronal function and plasticity, shaping the way our brains process and store information.

TABLE: SPINE PROTEINS

Signaling Pathways

The glutamate-induced molecular pathway is a complex and highly regulated series of events that ultimately leads to changes in the structure and function of the postsynaptic neuron. Glutamate binds to AMPA receptors, causing an influx of Na+ ions into the postsynaptic neuron and depolarizing the cell (Collingridge et al., 2004). Depolarization removes Mg2+ ions blocking NMDA receptors, allowing Ca2+ ions to enter the postsynaptic neuron (Mayer et al., 1984). Elevated intracellular Ca2+ activates the phospholipase C (PLC) pathway, which generates second messengers IP3 and DAG (Berridge, 1993). IP3 binds to IP3 receptors on the ER membrane, releasing more Ca2+ ions from the ER stores into the cytoplasm (Mikoshiba, 2007). Increased cytoplasmic Ca2+ activates Ca2+-dependent kinases, such as CaMKII, which is involved in synaptic plasticity and learning (Lisman et al., 2002). Activated CaMKII phosphorylates downstream targets, including CDK5 (Dhavan & Tsai, 2001). Activated CDK5 phosphorylates Kalirin-7, a guanine nucleotide exchange factor that regulates the activity of Rho GTPases, including Rac1 and RhoA (Xie et al., 2007). Activated Kalirin-7 in turn activates Rac1, a critical regulator of the actin cytoskeleton (Tolias et al., 2011). Rac1 activation promotes new actin filament formation and dendritic spine expansion, influencing synaptic strength and contributing to long-term changes in neuronal function during learning and memory formation (Penzes et al., 2011).

α4βδ GABAA Receptors

The α4βδ GABAAreceptor is a heteropentameric ligand-gated ion channel composed of α4, β, and δ subunits, with a stoichiometry typically arranged as 2α:2β:1δ (Barrera et al., 2008). The subunit composition and arrangement within the receptor complex are crucial for its unique functional properties, which ultimately impact synaptic pruning in the medial prefrontal cortex (mPFC) and consequently anxiety disorders (Shen et al., 2010).

The α4 subunit, encoded by the GABRA4 gene, is predominantly expressed in the hippocampus and dentate gyrus during puberty (Gao & Fritschy, 1995). It is noteworthy that the α4 subunit expression is upregulated during critical periods of synaptic pruning, when it is responsible for spine pruning, implicating its potential role in anxiety-related neuroplasticity (Smith et al., 2007). The α4 subunit is responsible for conferring certain pharmacological properties to the receptor, including insensitivity to the classical benzodiazepine site modulators, such as diazepam (Wafford et al., 1996). The β subunit, commonly β2 or β3, is encoded by the GABRB2 and GABRB3 genes, respectively (Simon et al., 2004). These subunits contribute to the formation of the GABA binding site and influence the receptor’s kinetic properties, including channel opening and desensitization (Amin & Weiss, 1993). Furthermore, the β subunit is vital for the proper trafficking and membrane insertion of the α4βδ receptor (Kang et al., 1996). The δ subunit, encoded by the GABRD gene, is essential for the receptor’s distinct functional properties. It is primarily found extrasynaptically in the hippocampus and dentate gyrus and is responsible for the high sensitivity of α4βδ receptors to low GABA concentrations (Stell et al., 2003). Moreover, the δ subunit confers a unique pharmacological profile to the α4βδ receptor, characterized by insensitivity to benzodiazepines and sensitivity to neurosteroids, such as allopregnanolone (Mihalek et al., 1999).

The assembly of the α4βδ GABAAreceptor is a highly regulated process involving multiple steps, including subunit synthesis, folding, assembly, trafficking, and insertion into the membrane (Connolly et al., 1996). The assembly is facilitated by chaperone proteins, such as the endoplasmic reticulum (ER) resident protein BiP, which ensures proper folding and assembly of the receptor subunits (Kumar et al., 2010). After assembly, the heteropentameric receptor is trafficked to the membrane, where it is inserted into the lipid bilayer and incorporated into the postsynaptic density, allowing for functional synaptic integration (Sarto-Jackson & Sieghart, 2008).

Ligand binding sites and activation mechanisms

The α4βδ GABAA receptor plays a vital role in modulating anxiety disorders due to its distinct ligand binding sites and activation mechanisms that provide a foundation for its therapeutic potential. The primary endogenous ligand of the α4βδ GABAA receptor is gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the central nervous system. The GABA binding site is located at the interface between the α4 and β subunits, as demonstrated by in vitro binding assays and structural studies (Baur et al., 2006; Miller et al., 2017). Upon GABA’s binding at this site, a conformational change in the receptor occurs, which in turn facilitates the opening of the integral chloride channel, thereby hyperpolarizing the postsynaptic membrane and reducing neuronal excitability. In some cases, it may produce a shunting inhibition.

GABA_ARs facilitate chloride anion movement across the membrane, with the direction contingent on the chloride reversal potential during resting membrane potential. Governed by the Nernst equation, the chloride reversal potential depends on the concentration gradient. At room temperature, the simplified equation becomes E_Cl = -58 * log [Cl]_out/[Cl]_in. In most adult CNS regions, extracellular chloride concentration is higher, resulting in a more negative reversal potential. Consequently, GABA_AR opening leads to intracellular hyperpolarization as negatively charged chloride flows into the cell (Staley & Mody, 1992). However, conditions with higher intracellular chloride concentrations, such as early development, result in depolarization as chloride flows extracellularly (Ben-Ari et al., 1989). If the chloride reversal potential and resting membrane potential are similar, minimal chloride flux occurs, yielding shunting inhibition regardless of direction. This results in either hyperpolarization or inhibition depending upon specific conditions within neurons (Isaacson & Walmsley, 1995).

Benzodiazepines, a class of psychoactive drugs, bind allosterically to GABAA receptors, enhancing GABAergic neurotransmission. However, α4βδ GABAA receptors display complete insensitivity to benzodiazepines due to a specific amino acid residue in the α4 subunit (Wafford et al., 1996) and the absence of the gamma subunit. This resistance has prompted the search for alternative anxiolytic agents that selectively target the α4βδ receptor subtype.

Neurosteroids, such as allopregnanolone and pregnenolone sulfate, have been shown to modulate GABAA receptors, including α4βδ receptors (Akk et al., 2017). The neurosteroid binding site is situated in the transmembrane domain at the interface of α4 and δ subunits. Positive allosteric modulators (PAMs), such as allopregnanolone, potentiate GABAergic inhibition by enhancing GABA binding and channel opening, whereas negative allosteric modulators (NAMs), such as pregnenolone sulfate, reduce GABAergic inhibition (Paul & Purdy, 1992). Neurosteroids have been implicated in anxiety disorders, and targeting the α4βδ receptor’s neurosteroid binding site has emerged as a promising therapeutic approach (Reddy, 2010).

The activation of α4βδ GABAA receptors involves conformational changes in response to ligand binding. Upon GABA binding at the orthosteric site, the receptor transitions from a closed, resting state to an open, active state, allowing chloride ions to flow through the channel pore. This influx of negatively charged ions inhibits the neuronal membrane by reducing action potential likelihood. PAMs and NAMs bind to the allosteric site and modulate the receptor’s response to GABA, enhancing or diminishing its effect, respectively (Glykys et al., 2007). The α4βδ receptors in the medial prefrontal cortex (mPFC) have been implicated in anxiety-related behaviors. Enhanced α4βδ receptor activity within the mPFC has been shown to promote anxiolysis in animal models of anxiety (Glykys et al., 2007; Maguire et al., 2005). This effect is hypothesized to result from increased inhibitory tone in the mPFC, reducing excessive excitatory activity often observed in anxiety disorders.

GABAergic Inhibition

Synaptic and extrasynaptic α4βδ GABAA receptors play unique roles in regulating neuronal membrane potential. Both receptor types are activated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), but they differ in location, kinetics, and membrane potential effects. Synaptic α4βδ GABAA receptors, primarily found at the synapse, enable rapid inhibitory synaptic communication. GABA activation causes structural changes that open chloride ion channels, resulting in membrane hyperpolarization, or shunting inhibition decreased neuronal excitability, and inhibitory postsynaptic potentials (IPSPs). These synaptic receptors have a brief impact on membrane potential due to their fast activation and desensitization kinetics. Extrasynaptic α4βδ GABAA receptors are located outside the synapse on the neuronal membrane. Activated by low GABA concentrations, these receptors demonstrate higher affinity for GABA and slower kinetics than synaptic receptors. Extrasynaptic α4βδ GABAA receptor activation generates a persistent tonic inhibitory current by allowing Cl- ions to flow into the neuron. This prolonged hyperpolarization or shunting inhibition reduces overall neuronal excitability and is crucial for controlling neuronal network activity and fine-tuning synaptic transmission.

The expression of α4βδ GABAARs and the resulting enhanced inhibitory signaling can significantly impact the activation of Kalirin-7 and its downstream effects on actin production. Enhanced activity of α4βδ GABAARs increases inhibitory signaling, counteracting depolarization induced by excitatory signaling through AMPA receptors. This makes it more challenging for the postsynaptic neuron to reach the threshold necessary to expel Mg2+ ions from NMDA receptors. Consequently, there is a reduced probability of Mg2+ removal from NMDA receptors, leading to decreased Ca2+ influx into the postsynaptic neuron. This causes a lower intracellular Ca2+ concentration and weaker activation of Ca2+-dependent pathways, such as the CaMKII pathway. Diminished activation results in reduced phosphorylation and activation of CDK5, a downstream target of CaMKII. CDK5 is crucial for Kalirin-7 phosphorylation and activation, so reduced CDK5 activation leads to decreased Kalirin-7 phosphorylation. This reduction negatively impacts Kalirin-7’s ability to regulate Rho GTPases like Rac1, leading to decreased Rac1 activation. Rac1 plays a vital role in promoting new actin filament formation and dendritic spine expansion essential for synaptic connections between neurons. Therefore, reduced Rac1 activation due to decreased Kalirin-7 phosphorylation likely results in diminished actin production and fewer structural changes in the postsynaptic neuron. In summary, increased inhibitory signaling from α4βδ GABAARs can disrupt normal molecular pathways and alter the structure and function of postsynaptic neurons.

Specific Aims

Specific Aim 1 : Investigate the synaptic pruning process in pyramidal cells of the L5 PL region in the female mouse brain during the transition from puberty to post-puberty.

1.1: Analyze spine density of basilar dendrites in Golgi-stained neurons.

1.2: Identify specific spine types that are most affected by this process.

Specific Aim 2 : Examine the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty in both female and male mice.

2.1: Assess α4βδ GABAR expression during different developmental stages.

2.2: Investigate the effects of knockout and pharmacological manipulations on synaptic pruning.

Specific Aim 3: Investigate the role of NMDARs in synaptic pruning of L5 PL.

3.1: Manipulate NMDAR expression during puberty.

3.2: Assess the effects of NMDAR manipulation on spine density at post-puberty.

Specific Aim 4: Analyze the expression of the spine protein Kal-7 in L5 PL of wild-type and α4 -/- mice during different developmental stages.

4.1: Determine the relationship between Kal-7 expression and α4βδ GABAR expression.

4.2: Investigate the role of Kal-7 in synaptic pruning.

Specific Aim 5: Assess the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood.

5.1: Perform stereotaxic virus injections to selectively knockdown α4 in the PL during puberty.

5.2: Analyze the effects of local α4 knockdown on spine density and Kal-7 expression.

5.3: Evaluate anxiety-related behavior in mice using behavioral tests such as the elevated plus-maze.

Specific Aim 6: Investigate the changes in spine type densities in pyramidal cells of Layer 2/3 PL region in wild-type and α4 -/- mice during the transition from puberty to post-puberty.

6.1: Analyze spine density of apical and basilar dendrites in Golgi-stained neurons of Layer 2/3 in both wild-type and α4 -/- mice.

6.2: Identify specific spine types that are affected by the synaptic pruning process in Layer 2/3 in wild-type and α4 -/- mice during development.

6.3: Compare the changes in spine type densities between Layer 2/3 and Layer 5 in wild-type and α4 -/- mice to elucidate potential differences in synaptic pruning processes between these cortical layers.

Materials and Methods

_ Animals: _ Most studies utilized C57BL/6 wild-type (WT, Jackson Labs) or GABAR α4-/- female and male mice, which were housed under a reverse light: dark cycle (12:12) and tested during the light phase. α4-/- mice were bred in-house from α4+/- mice (provided by G. Homanics, U. Pitt.). WT and α4+/+ mice exhibited similar spine densities. For Golgi studies, animals were euthanized at puberty onset (females, approximately PND35, assessed by vaginal opening; males, approximately PND 37-76) or PND 56 for spine density analysis. Animals were tested for α4 immunoreactivity and electrophysiological responses pre-pubertally (around PND 28-32), 1-2 days after puberty onset, and post-pubertally (PND 56). The estrous cycle is not a factor during the pubertal period (PND 35-44). However, the estrous stage was determined for animals euthanized on PND 56 using vaginal smears to avoid the proestrus stage when GABAR expression and dendritic spine counts can be increased. For drug administration studies, all animals were injected once daily (intraperitoneally) with the following drugs from PND 35 (puberty onset) to PND 49, the period of high α4 expression: gaboxadol (GBX, THIP, 4,5,6,7-tetrahydroisoxazolopyridin-3-ol), 0.1 mg/kg, a dose which has no effect in α4-/- mice; picrotoxin, 3 mg/kg; lorazepam (LZM), 0.25 mg/kg; MK-801 ([5R,10S]-[+]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine), 0.1 mg/kg, a dose which increases NMDAR expression in mPFC; memantine (1-Amino-3,5-dimethyladamantane), 10 mg/kg, an NMDAR blocker which does not increase NMDAR expression. In all experimental procedures, mice were randomly assigned to experimental groups, and the investigator was blinded to the condition of the mice. All procedures were approved by the SUNY Downstate Medical Center institutional animal care and use committee and carried out in accordance with their guidelines and regulations. Additionally, the authors complied with the ARRIVE guidelines.

_ Local knockdown of the GABAR α4 subunit: _ Transgenic female mice with loxP (locus of X-over P1) sites flanking the α4 gene (B6.129-GABRA4tm1.2Geh /J) were purchased from Jackson Labs (Bar Harbor, ME) and bred in-house to yield homozygous offspring (genotyping by Transnetyx, Cordova, TN). Local α4 knockdown procedures were performed on PND 21 female mice. Following anesthesia induction using a ketamine (75 mg/kg) and dexmedetomidine (0.5 mg/kg) cocktail, injected intraperitoneally, mice were placed in a stereotaxic apparatus. Mice were locally infused with 0.25 μls of either adeno-associated virus-Cre recombinase with green fluorescent protein (AAV-Cre/GFP, pAAV.CMV.HI.eGFP-Cre.wPRE.SV40, ≥8 x 1012 vg/μl, cat# 105545-AAV1) or AAV-GFP (pAAV-CAG-GFP, cat# 37825-AAV5) into the prelimbic region of the mPFC (coordinates: AP 1.9, ML ±0.3, DV -1.45), bilaterally, using an infusion pump and a Hamilton syringe (flow rate: 0.12 μls/min). Both viral constructs were from Addgene (Watertown, MA). The surgical site was sutured, and animals were allowed to recover for 2 weeks but returned to group housing after 48 h. In some cases, viral entry and selective PL targeting were verified using Cre and/or GFP immunohistochemistry, respectively, at PND 35-37. Successful α4 knockdown was determined using α4 immunohistochemistry in the Cre-injected mice at PND 35-37, compared to GFP-injected controls, when puberty onset was also determined. In other cases, mice were either euthanized to assess spine density using Golgi procedures (PND 56) or tested for anxiety using the elevated plus-maze (EPM, PND 56-68, 90-111) followed by confirmation of α4 knock-down.

_ Immunohistochemistry: _ Following anesthesia with urethane (0.1 ml 40%), mice were perfused with saline (12-15 mls/min) and then with 4% paraformaldehyde (PFA) followed by post-fixation of the brain in 4% PFA (48 h, 4°C). Paraffin-embedded sections were prepared from PFA preserved brains embedded in paraffin blocks following tissue dehydration using increasing ethanol concentrations. Coronal sections of the mPFC were cut on a microtome at a thickness of 10 μm and mounted on super-frost slides. Tissue was de-paraffinized in decreasing concentrations of ethanol and processed using antigen retrieval: Slides were incubated in warm (95-100°C) sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min, allowed to cool, and rinsed (2x) with 0.01 M phosphate-buffered saline (PBS), 0.05% Tween 20 (PBS-Tween) for 2 min. Free-floating sections were prepared by cutting coronal sections of the mPFC on a vibratome (Leica VT 100M) at a thickness of 30-40 μm. Free-floating sections were washed (3x) in PBS-Tween with 1% bovine serum albumin (BSA) for 10 minutes. Immunohistochemistry protocol: Sections were blocked in PBS supplemented with 1.5% donkey serum (kalirin) or 1.5% goat serum (α4, Cre) in PBS-Tween for 2 h at room temperature. For α4 staining, sections were incubated in blocking buffer containing 2% goat anti-mouse Fab fragments (Jackson Immunolabs, Bar Harbor, ME) for 2 h at room temperature. Then, sections were incubated with anti-α4 (mouse monoclonal, Antibodies, Inc., Davis, CA, 1:100). In some cases, anti-α4 (goat polyclonal, sc7355, Santa Cruz, 1:20) with anti-MAP2 (microtubule-associated protein-2, ab5392, Abcam, Cambridge, MA, 1:1000) were used without pre-incubation with the anti-mouse Fab fragments to verify α4 localization on dendritic spines. Both antibodies show selectivity for α4 as evidenced by their lack of staining in the hippocampus of α4 knock-out mice shown here (Supp. Fig. 7) and in a previous publication. Although MAP2 is localized to the soma and dendrites, it can also be localized to spines and has been used as a spine marker. MAP2 is primarily localized to mushroom spines, which are one of the predominant spine types at puberty. Therefore, we used MAP2 to visualize dendrites and spines at puberty.

For Kalirin, Cre, and NMDAR1 staining, anti-kalirin-7 (Kal-7, rabbit polyclonal, a generous gift from R Mains, UConn Health, JH295885, 1:200), anti-Cre (rabbit polyclonal, Novus Biologicals, Centennial, CO, 1:1000), or anti-NMDAR1 (rabbit monoclonal, ab274377, Abcam, Cambridge, MA, 1:100) were used. All antibodies were diluted in the blocking solution and incubated with tissue sections overnight at 4°C. After washing, sections were incubated with the appropriate fluorescent secondary antibody (Alexa fluor 488 and 594, 1:1000) for 2 h, washed in PBS 3x for 10 min, after which they were mounted on slides with ProLong Glass antifade reagent in some cases with 5% nuclear blue. Images were taken with an Olympus FluoView TM FV1000 confocal inverted microscope with objective UPLSAPO 40x or 100x NA:1:30 (Olympus, Tokyo, Japan). For the immunohistochemical analysis, the merged z-stack image (2 μm steps) was used. Image segmentation was first performed using a thresholding sub-routine in ImageJ so that the original color image was converted to a binary image. This allowed for visualization of the regions of interest (ROI) in cases where the background intensity was non-homogeneous. ROIs were then analyzed for image luminosity in the original image using Adobe Photoshop after subtracting the adjacent background levels, and the results were verified by ImageJ. Three ROIs were analyzed per mouse.

_ Golgi procedure: _ Before euthanization, mice were anesthetized with urethane (1-2 g/kg, i.p.), and whole brains were extracted and processed for Golgi impregnation with the FD Neurotechnologies Rapid Golgi Stain kit. Coronal sections were prepared using a vibratome (Leica VT1200s) set to a thickness of 250 μm.

_ Analysis: _ Pyramidal cells from L5 PL were identified using The Mouse Brain in Stereotaxic Coordinates (4th Edition, Paxinos, and Franklin, 2012) and the Allen Brain Institute’s Mouse Brain Atlas (http://mouse.brain-map.org). The L5 PL neurons were approximately 1.7 mm ventral from the dorsal surface and the cell bodies were 500-700 μm from the medial surface. Individual neurons in these regions were viewed using a 100x oil objective on a Nikon Eclipse Ci-L microscope. Images of the basilar dendrites were acquired using Z-stack projection photomicrographs (0.1 – 0.9 μm steps) taken using a Nikon DS-U3 camera mounted on the microscope and were analyzed using NIS-Elements D 4.40.00 software. Three to four neurons were sampled per mouse, and six segments were assessed per neuron (20 – 50 μm). Each dendrite segment was ~1 μm thick and was taken from a 2º or 3º order dendrite. Spine density was expressed as the number of spines/10 μm. To determine the type of dendritic spine, we used parameters described by Risher et al. (2014): filopodia, length >2µM; long thin, length <2µM; thin, length <1µM, stubby, width ratio <1µM, mushroom, width >0.06µM; bifurcated, 2 or more heads.

_ Cortical slice preparation: _ All electrophysiology experiments were performed by Hui Shen, PhD. Brains from euthanized mice were removed and cooled using an ice-cold solution of artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 124, KCl 2.5, CaCl2 2, NaH2PO4 1.25, MgSO4 2, NaHCO3 26, and glucose 10, saturated with 95% O2, 5% CO2 and buffered to a pH of 7.4. Following sectioning at 400 μm on a Leica VT1000S vibratome, slices were incubated for 1 h in oxygenated aCSF.

_ Cortical slice voltage-clamp electrophysiology: _ All electrophysiology experiments were performed by Hui Shen, PhD. Pyramidal cells in L5 PL were visualized using a differential interference contrast (DIC)-infrared upright Leica microscope and recorded using whole-cell patch clamp procedures in voltage clamp mode at 26 – 30°C, as described. Patch pipets were fabricated from borosilicate glass using a Flaming-Brown puller to yield open tip resistances of 2–4 MΩ. For recordings of the pharmacologically isolated tonic inhibitory current, the pipet solution contained in mM: CsCl 140, HEPES 5, EGTA 5, CaCl2-H2O 0.5, QX-314 5, Mg-ATP 2, Li-GTP 0.5, pH 7.2, 290 mOsm. 5 mM QX-314 was added to block voltage-gated Na+ channels and GABAB receptor-activated K+ channels. The aCSF contained 50 μM kynurenic acid to block excitatory current, as well as 0.5 μM TTX to isolate the post-synaptic component. Recordings were carried out at a -60 mV holding potential, and the tonic current was assessed by the change in holding current in response to 100 nM gaboxadol (GBX ), a GABAR agonist which, at this concentration, is selective for δ-containing GABARs. The GABAergic nature of the current was verified by block with 100 μM picrotoxin. Drugs were bath applied continuously in sequential order following 5-10 min of baseline recordings without drugs. Recordings were conducted with a 2 kHz 4-pole Bessel filter at a 10 kHz sampling frequency using an Axopatch 200B amplifier and pClamp 9.2 software. Electrode capacitance and series resistance were monitored and compensated; access resistance was monitored throughout the experiment, and cells were discarded if the access resistance increased more than 10% during the experiment. In all cases, the data represent one recording/animal.

_ Anxiety response to an aversive stimulus assessment using avoidance behavior _: Mice were tested for anxiety-like behavior using the shock-paired elevated plus maze (EPM), an established model of anxiety, which assesses avoidance behavior, on PND 56 or PND 90 following local α4 knockdown at puberty in response to AAV-Cre infusion on PND 21. Local knock-down was verified with immunohistochemical techniques after the behavioral test. We tested anxiety in response to an aversive stimulus to mimic human studies, which show mPFC regulation of anxiety in response to aversive settings. Results were compared with the GFP control (AAV-GFP infusion on PND 21). The plus-maze consists of four 8 x 35 cm arms at 90° angles, elevated 57 cm above the floor. Two arms are enclosed by 33 cm walls, and two arms have no walls (“open arms”). The open arms are also partially bordered by small rails (5 x 15 cm) extending to the proximal half of the arm, and the floor of the maze is marked with grid lines every 25 cm. Each animal was initially acclimated to the room for 30 min – 1 h. Then, mice were administered a 400-μA shock for 1 s immediately before being placed in the maze center when exploratory activity was recorded for 5 min. The time spent in the open and closed arms was tabulated, as were the entries. To be considered an open arm entry, the animal had to cross the open platform’s line with all four paws. A decrease in open arm time is considered a measure of increased avoidance behavior, reflecting anxiety, as we have described. The number of total entries is a measure of general activity level.

_ Drugs: _ All drugs except QX-314 were from Sigma Chemical Co (St Louis, MO). QX-314 was from Calbiochem (Billerica, MA).

_ Statistics: _ Statistics were analyzed with Prism-GraphPad (spine densities) or OriginPro (all other data). Data are presented as the mean ± S.E.M., and in some cases, the median, interquartile range, and outliers are indicated. Individual data points are presented when n<10. Data were shown to have similar variance using the Brown-Forsythe test for equal variance and were verified as reflecting a normal distribution by the Kolmogorov-Smirnov test. The significant differences in spine densities calculated across treatment groups were analyzed with a nested t-test (2 groups) or a nested one-way analysis of variance (ANOVA, >2 groups) with a post-hoc Tukey test (male data) or Dunnett’s test (pharmacology study). Averaged values calculated across treatment groups for immunohistochemistry, electrophysiology, and behavior were analyzed with the Student’s t-test (2 groups) or one-way analysis of variance (ANOVA, >2 groups) with a post-hoc Tukey test for unequal replications. All tests were two-tailed. A P value < 0.05 was used as an indication of statistical significance. A power analysis was conducted to determine adequate sample size for all studies, which achieved a power > 0.85. Reproducibility was determined by comparing the statistical significance of results from experiments performed 3 to 5 times to achieve the final n’s

Part 1: Preventing adolescent synaptic pruning in Layer 5 of the mouse prelimbic cortex via local knockdown of A4BD GABAA receptors increases anxiety response in adulthood.

Anxiety disorders are becoming increasingly prevalent, particularly in adolescent females, and the underlying etiology remains elusive. This lack of understanding hampers the development of effective treatments. Layer 5 of the prelimbic cortex (L5PL) is known to play a critical role in anxiety response modulation, and it undergoes significant synaptic pruning during adolescence. The impact of this pruning process on anxiety, however, has not been thoroughly investigated.

The first-authored paper by Evrard et al. (2021) addresses Specific Aims 1 through 5, which aim to elucidate the synaptic pruning process in the L5PL region of the female mouse brain during the transition from puberty to post-puberty, and its implications for anxiety-related behavior in adulthood. The study investigates the expression and functional role of α4βδ GABAA receptors (GABARs) and the involvement of NMDA receptors (NMDARs) in synaptic pruning in L5PL. Additionally, the paper explores the expression of the spine protein Kal-7 in wild-type and α4 -/- mice during different developmental stages, as well as the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood. The following section will briefly summarize the results, please refer to the appended paper for a full analysis.

Evrard et al. (2021) demonstrates that preventing L5PL synaptic pruning increases anxiety in response to an aversive event in adolescent and adult female mice. The study reveals a transient 10-fold increase in α4βδ GABAR expression in L5PL at puberty, followed by a decrease post-pubertally. Both global and local knockdown of these receptors during puberty prevented pruning, which resulted in increased spine density post-pubertally. This effect was reversed by blocking NMDARs, suggesting their involvement in the pruning process.

The paper also presents evidence that the NMDAR-dependent spine protein kalirin7 expression decreases during puberty, an effect prevented by α4 knock-out. This finding implies that α4βδ GABAR-induced reductions in kalirin7 underlie pruning. Moreover, the study shows that increased spine density due to local α4 knockdown at puberty leads to decreased open arm time on the elevated plus maze post-pubertally, indicating that increases in L5PL synapses augment anxiety responses.

Specific Aim 1

Specific Aim 1.1: Investigate the synaptic pruning process in pyramidal cells of the L5 PL region in the female mouse brain during the transition from puberty to post-puberty.

To assess the changes in spine density during the transition from puberty to post-puberty, we utilized Golgi staining, a well-established technique for visualizing neuronal morphology. We analyzed the spine density of basilar dendrites in L5 PL pyramidal cells at different developmental stages, including pre-puberty, puberty, and post-puberty. Our sample size consisted of 55 samples from 12 female mice during puberty and 44 samples from 12 female mice during post-puberty. Our results revealed a significant 63% decrease in spine density, from 16.39 ± 1.55 spines/10 μm during puberty to 6.10 ± 0.58 spines/10 μm in post-puberty (P < 0.0001), indicating that synaptic pruning occurs in the L5 PL region during this developmental period.

To further elucidate the synaptic pruning process, we examined the changes in different spine types during the transition from puberty to post-puberty. Our analysis revealed that the most significant decline in spine density occurred in the stable spine types, including mushroom, stubby, and bifurcated spines with an 84% reduction (P = 0.0014). Mushroom spines exhibited the most significant decline of 74% (P < 0.0001), followed by stubby spines with a decrease of 66% (P < 0.0001). In contrast, the less stable (motile) spines, such as long, thin, and thin spines, showed a smaller, but still significant, decrease in density by 53%. Specifically, the long, thin spines declined by 64% (P = 0.0025), while the thin spines experienced a reduction of 49% (P = 0.0114). These findings suggest that synaptic pruning in the L5 PL region preferentially targets stable spine types, which may have important implications for the functional reorganization of neural circuits during this critical developmental period.

Specific Aim 1.2: Investigate the synaptic pruning process in pyramidal cells of the L5 PL region in the male mouse brain during the transition from puberty to post-puberty.

To assess the changes in spine density during the transition from puberty to post-puberty in male mice, we utilized Golgi staining and analyzed the spine density of basilar dendrites in L5 PL pyramidal cells at different developmental stages, including pre-puberty, puberty, and post-puberty. Our sample size consisted of 36 samples from 12 male mice during puberty and 61 samples from 12 male mice during post-puberty. Our results revealed a significant decrease of approximately 57% in total spine density (P < 0.0001), indicating that synaptic pruning occurs in the L5 PL region during this developmental period.

To further elucidate the synaptic pruning process, we examined the changes in different spine types during the transition from puberty to post-puberty. Our analysis revealed that stable spines (mushroom, stubby, and bifurcated spines) experienced a more significant decline compared to motile spines (long, thin, and filopodia spines). Specifically, stable spines showed a significant decrease (P < 0.0001), while motile spines also exhibited a significant decline (P < 0.001). Among stable spines, mushroom spines showed a significant reduction of approximately 52% (P = 1.91e-05), followed by stubby spines with a decrease of about 63% (P = 2.64e-08). Bifurcated spines also experienced a significant decline of around 75% (P = 0.0229). In contrast, among motile spines, long thin spines declined by nearly 79% (P = 3.67e-09), thin spines decreased by about 50% (P = 1.76e-07), and filopodia spines showed a non-significant reduction of approximately 25% (P = 0.4189).

These findings suggest that synaptic pruning in the L5 PL region of male mice preferentially targets stable spine types during the transition from puberty to post-puberty, similar to the observations in female mice. This may have important implications for the functional reorganization of neural circuits during this critical developmental period in both sexes.

Specific Aim 1.3: Compare the synaptic pruning process in pyramidal cells of the L5 PL region between male and female mice during the transition from puberty to post-puberty.

To provide a more detailed comparison of the synaptic pruning process between male and female mice, we analyzed the changes in spine density and various spine types during the transition from puberty to post-puberty. Our sample size consisted of 55 samples from 12 female mice during puberty, 44 samples from 12 female mice during post-puberty, 36 samples from 12 male mice during puberty, and 61 samples from 12 male mice during post-puberty. Our results revealed that total spine density exhibited a significant difference between male and female mice during the transition from puberty to post-puberty (P = 9.33e-21). Female mice displayed a higher total spine density (mean = 12.92, SEM = 0.92) compared to male mice (mean = 12.28, SEM = 0.70), indicating that sex-specific factors may contribute to differential regulation of synaptic pruning during this critical developmental period.

We observed significant differences between male and female mice in stable spine types, including mushroom spines (P = 8.45e-16), stubby spines (P = 2.41e-10), and bifurcated spines (P = 2.12e-05). fFemale mice exhibited higher densities of these stable spines compared to males at puberty but not post-pubertally, suggesting that females may undergo more extensive synaptic pruning targeting stable spines during this developmental period. Mushroom spines are considered essential for long-term potentiation and memory formation due to their large head size, which allows for increased postsynaptic receptor accumulation. The observed higher density of mushroom spines in female mice (mean = 3.61, SEM = 0.33) compared to male mice (mean = 2.22, SEM = 0.21) could potentially lead to sex differences in cognitive function and memory-related behaviors. Stubby spines are characterized by short dendritic protrusions without distinct necks or heads. These spines have been associated with local protein synthesis and plasticity regulation. The higher density of stubby spines in female mice (mean = 2.48, SEM = 0.26) compared to male mice (mean = 2.81, SEM = 0.31) suggests that females may exhibit enhanced local plasticity regulation during the transition from puberty to post-puberty. Bifurcated spines are characterized by two or more heads sharing a common base. These spines have been suggested to play a role in synaptic integration and information processing. The higher density of bifurcated spines in female mice (mean = 0.44, SEM = 0.09) compared to male mice (mean = 0.19, SEM = 0.07) could potentially contribute to sex differences in neural circuit organization and information processing.

We also observed significant differences between male and female mice in motile spine types, including thin spines (P = 3.70e-06) and long thin spines (P = 5.62e-10). Female mice displayed higher densities of these motile spines compared to males, indicating that sex-specific factors may influence the pruning process targeting motile spines during this developmental period. Thin spines are characterized by small head sizes and long necks, which make them less stable compared to mushroom or stubby spines. These spines are considered highly dynamic and have been associated with learning-related plasticity. The observed higher density of thin spines in female mice (mean = 3.86, SEM = 0.40) compared to male mice (mean = 2.97, SEM = 0.37) suggests that females may exhibit enhanced learning-related plasticity during the transition from puberty to post-puberty. Long thin spines are characterized by their long, narrow necks and small heads, making them the most motile of the spine types. These spines have been implicated in the rapid formation and disassembly of synapses during experience-dependent plasticity. The higher density of long thin spines in female mice (mean = 0.54, SEM = 0.12) compared to male mice (mean = 0.20, SEM = 0.08) could potentially contribute to sex differences in the ability to rapidly reorganize synaptic connections in response to environmental stimuli during this critical developmental period.fffff

Our study observed not only sex differences in spine density and spine types during the transition from puberty to post-puberty but also identified several similarities and common trends between male and female mice, offering valuable insights into the overall synaptic pruning process and potential mechanisms conserved in both sexes. A significant decrease in total spine density was seen in both male and female mice during this critical developmental stage (P < 0.0001 for both sexes), suggesting shared molecular pathways or cellular processes. Examination of various spine types revealed that stable spines experienced a more substantial decline compared to motile spines in both sexes, implying that synaptic pruning preferentially targets stable spine types, which may have significant consequences for neural circuit functional reorganization. Furthermore, similar patterns of decline in specific spine types, such as mushroom and stubby spines, were observed between male and female mice, suggesting that some aspects of synaptic pruning might be preserved across sexes despite overall differences in spine density and distribution. These similarities imply shared cellular and molecular mechanisms, potentially involving common signaling pathways like glutamate receptors (NMDA and AMPA), GABA_A receptors, or other proteins implicated in synaptic plasticity (e.g., CaMKII, CDK5, Kalirin-7, and Rho GTPases like Rac1). In conclusion, our study emphasizes both significant sex differences and similarities in spine density and spine types during the transition from puberty to post-puberty, contributing to a more comprehensive understanding of the synaptic pruning process and identifying potential conserved mechanisms between the sexes. Further exploration of these shared cellular and molecular processes will expand our knowledge of synaptic development and plasticity regulation across both sexes during critical developmental periods.

The observed sex differences in spine density and spine types during the transition from puberty to post-puberty may have significant implications for anxiety-like behaviors. As mentioned earlier, female mice displayed higher densities of stable spines (mushroom, stubby, and bifurcated) and motile spines (thin and long thin) compared to male mice. These differences could potentially contribute to sex-specific alterations in neural circuit organization, synaptic plasticity, and information processing.

Anxiety disorders are characterized by excessive fear or worry that interferes with daily functioning. The medial prefrontal cortex (mPFC) has been implicated in the regulation of anxiety-related behaviors, with alterations in mPFC activity being associated with anxiety disorders (Rosenkranz & Grace, 2002). Given that synaptic pruning shapes neural circuits during critical developmental periods, it is plausible that the observed sex differences in synaptic pruning may contribute to differential susceptibility or resilience to anxiety disorders between males and females. For instance, the higher density of mushroom spines in female mice could lead to enhanced long-term potentiation and memory formation related to fear or threat processing during puberty. This might result in heightened sensitivity to potential threats and increased vulnerability to developing anxiety disorders. Similarly, the higher density of stubby spines in female mice may suggest enhanced local plasticity regulation during this developmental period, which could contribute to greater susceptibility to experience-dependent changes in mPFC function associated with anxiety disorders. Furthermore, the higher density of motile spines (thin and long thin) in female mice may indicate enhanced learning-related plasticity and rapid reorganization of synaptic connections in response to environmental stimuli during this critical developmental period. This could potentially contribute to sex differences in adaptive or maladaptive responses to stressors or threatening situations that are relevant for anxiety-like behaviors.

Our research indicates notable sex differences in the synaptic pruning process of pyramidal cells in the L5 PL region during the transition from puberty to post-puberty, which may be significant for anxiety-like behaviors. Various essential proteins and signaling pathways, which regulate neuronal function, structure, and plasticity, could potentially underlie these observed sex differences in synaptic pruning. Examples include ionotropic glutamate receptors (AMPA and NMDA), α4βδ GABA_A receptors, CaMKII, CDK5, Kalirin-7, and Rho GTPases like Rac1. Differences in the expression or function of these proteins, as well as glutamate-induced molecular pathways, between male and female mice during critical developmental periods could lead to differential regulation of synaptic pruning. This, in turn, may impact neural circuit organization, synaptic plasticity, and information processing, potentially resulting in sex differences in cognition, memory, and behavior. It may also lead to differential susceptibility or resilience to anxiety disorders. Further exploration of these cellular and molecular mechanisms will enhance our understanding of how sex-specific factors contribute to differential synaptic pruning. Our results highlight the importance of considering sex as a biological variable in studies investigating synaptic development and plasticity.

Differences in the expression or function of proteins such as NMDA receptors and α4βδ GABA_A receptors between male and female mice could contribute to the differential regulation of synaptic pruning. Synaptic pruning is an essential process during neural development that refines and strengthens neural connections. Estrogen, a hormone found at higher levels in females, has been shown to modulate NMDA receptor function. A study by Smith and Woolley (2004) demonstrated that estrogen can rapidly alter NMDA receptor-mediated synaptic transmission in the hippocampus of adult female rats, potentially contributing to sex differences in synaptic plasticity. Additionally, Woolley et al. (1990) discovered that dendritic spine density in the CA1 region of the hippocampus is significantly influenced by estrogen. Their study showed that the density of dendritic spines in ovariectomized female rats increased after estrogen replacement, suggesting a role for estrogen in modulating synaptic connectivity. Estrogen also increases mushroom spine density. Sex differences in the expression or function of α4βδ GABA_A receptors during critical periods of synaptic pruning may also contribute to differential regulation of anxiety-related neuroplasticity between males and females. GABA_A receptors play a vital role in inhibitory neurotransmission, and their involvement in anxiety and stress-related disorders is well-documented. A study by Shen et al. (2007) found that α4βδ GABA_A receptor expression was upregulated during puberty in female mice but not in male mice. This sex-specific upregulation of α4βδ GABA_A receptor expression might result in differential regulation of synaptic pruning and anxiety-related neuroplasticity in male and female mice.

The glutamate-induced molecular pathway is a critical component of synaptic plasticity and has been implicated in various neurodevelopmental processes, including synaptic pruning. Sex-specific alterations in this pathway could lead to differential regulation of dendritic spine expansion and synaptic strength during critical periods such as puberty to post-puberty transition. In turn, this might result in distinct patterns of synaptic pruning between male and female mice, ultimately impacting anxiety-like behaviors and other aspects of neural function.

Glutamate is the primary excitatory neurotransmitter in the mammalian central nervous system and plays a key role in synaptic plasticity, learning, and memory. The activation of glutamate receptors, including NMDA and AMPA receptors, leads to calcium influx and downstream signaling cascades that modulate the structure and function of dendritic spines. These processes are essential for the formation, maintenance, and pruning of synapses.

Sex-specific differences in glutamate receptor expression, function, or downstream signaling pathways could contribute to differential synaptic pruning and anxiety-like behaviors in male and female mice. For example, a study by Harte-Hargrove et al. (2013) showed that estradiol, the primary estrogen hormone, can modulate the trafficking of AMPA receptors in hippocampal neurons, thereby influencing synaptic strength and plasticity in a sex-specific manner. This finding suggests that estrogen may impact the glutamate-induced molecular pathway and contribute to sex differences in synaptic pruning. Furthermore, studies have revealed sex-specific differences in glutamate receptor subunit expression and function in various brain regions, such as the prefrontal cortex and hippocampus (Forlano et al., 2016). These differences may lead to alterations in downstream signaling pathways and synaptic plasticity, ultimately affecting synaptic pruning and anxiety-like behaviors in a sex-dependent manner.

In conclusion, our findings suggest that sex differences in spine density and spine types during the transition from puberty to post-puberty may have significant implications for anxiety-like behaviors. The potential cellular and molecular mechanisms underlying these sex differences include alterations in key proteins involved in neuronal function, structure, plasticity regulation, as well as glutamate-induced molecular pathways. Further investigation into these mechanisms will provide a better understanding of how sex-specific factors contribute to differential susceptibility or resilience to anxiety disorders during critical developmental periods.

In conclusion, our results demonstrate significant sex differences in the synaptic pruning process of pyramidal cells in the L5 PL region during the transition from puberty to post-puberty using a sample size consisting of multiple samples obtained from both male and female mice at different developmental stages. These findings provide a foundation for future studies aimed at understanding the cellular and molecular mechanisms underlying these sex-specific differences in synaptic pruning, as well as their potential impact on cognition, memory, and behavior. Additionally, our results underscore the importance of considering sex as a biological variable in studies investigating synaptic development and plasticity.

Addressing potential confounding factors that may influence observed sex differences in synaptic pruning is essential, as hormonal fluctuations during development or other biological factors could play a role in the disparities between male and female mice. For instance, estrogen and testosterone are known to impact brain development and function, including synaptic plasticity (McEwen & Milner, 2007); therefore, future research should measure hormone levels at different developmental time points to investigate correlations between hormonal fluctuations and changes in spine density or spine types. Additionally, other biological factors, such as genetic background or environmental influences, might contribute to the observed sex differences in synaptic pruning. To account for these factors, future research should employ experimental designs that control for genetic background by using genetically homogeneous mouse strains or implementing transgenic approaches to manipulate specific genes involved in synaptic pruning, while also carefully controlling environmental factors like stress exposure or housing conditions across experimental groups to minimize potential confounds. By addressing these potential confounding factors, future research can provide more robust evidence supporting sex-specific differences in synaptic pruning during critical developmental periods, ultimately enhancing our understanding of neural circuit organization and plasticity regulation and informing targeted interventions in the field.

Specific Aim 2: Examine the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty in female mice and compare these findings with those from male mice.

GABAergic signaling plays a crucial role in the regulation of neuronal excitability and synaptic plasticity. The α4βδ GABARs, a subtype of GABAA receptors, are known to be involved in the modulation of neuronal activity during development. In this aim, we sought to examine the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty in both female and male mice.

2.1: Assess α4βδ GABAR expression during different developmental stages in both sexes.

In some brain regions, extrasynaptic α4βδ GABARs have been shown to increase during puberty, particularly when they are expressed on the soma, along the dendritic shaft, and on the spine. To investigate whether this receptor expression pattern occurs in L5 PL during puberty, we assessed α4 expression using immunohistochemical techniques at different developmental stages: before puberty (approximately PND 28-32), at puberty onset (approximately PND 35), and post-puberty (PND 56).

Our results showed a remarkable increase in α4 immunostaining at the onset of puberty, with levels almost ten times higher than those observed in pre-puberty (P < 0.00001). This increase was followed by a decline of approximately 75% in the post-pubertal stage. To further analyze the distribution of α4 expression, we conducted additional studies that co-localized α4 immunostaining with microtubule-associated protein-2 (MAP2), a protein known to be present in mushroom spines. Our findings revealed that α4 immunostaining was indeed localized to the dendrite, dendritic spine, and cell body.

2.2: Investigate the effects of knockout and pharmacological manipulations on synaptic pruning in female and male mice.

To verify the increased expression of functional α4βδ GABARs during puberty, we examined the response of L5 PL neurons to gaboxadol (GBX), a GABA agonist known to selectively target α4βδ GABARs at a concentration of 100 nM. Previous studies have shown that in vitro application of GBX can serve as a functional index of α4βδ GABAR expression.

We conducted whole-cell voltage clamp recordings of L5 PL pyramidal cells in slice preparations obtained from pre-pubertal, pubertal, and post-pubertal female mice. Upon application of 100 nM GBX, we observed a tenfold greater response in neurons during puberty compared to both pre-puberty and post-puberty stages (Fig. 2d,e, P = 0.00125). This finding suggests that functional α4βδ GABARs display a transient increase in the L5 PL region during puberty.

To further explore the functional role of α4βδ GABARs in synaptic pruning, we employed both genetic and pharmacological approaches. We assessed spine density in α4-/- mice, which lack the α4 subunit of GABARs, and found that synaptic pruning was impaired in these animals. Additionally, we tested the effects of pharmacological manipulation of GABARs on synaptic pruning by administering picrotoxin, a non-selective GABAR antagonist, and gaboxadol (GBX), a selective α4βδ GABAR agonist, during the pubertal period. Our results showed that picrotoxin treatment increased spine density, while GBX treatment decreased spine density, further supporting the involvement of α4βδ GABARs in the synaptic pruning process.

In summary, our investigation of the expression and functional role of α4βδ GABARs in the L5 PL region during puberty and post-puberty has provided important insights into the mechanisms underlying synaptic pruning in both female and male mice. By assessing the expression of α4βδ GABARs during different developmental stages and examining the effects of genetic and pharmacological manipulations on synaptic pruning, we have highlighted the critical role of these receptors in the maturation of neural circuits in the PFC. These findings may contribute to the development of novel therapeutic strategies targeting α4βδ GABARs for the treatment of neuropsychiatric disorders associated with PFC dysfunction.

Specific Aim 3:

Investigate the role of NMDARs in synaptic pruning of L5 PL.|

N-methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels that play a pivotal role in synaptic plasticity, learning, and memory. Given the importance of NMDARs in the regulation of neuronal activity and their potential interaction with GABAergic signaling, we aimed to investigate the role of NMDARs in synaptic pruning of L5 PL during the transition from puberty to post-puberty.

3.1: Manipulate NMDAR expression during puberty.

To explore the involvement of NMDARs in synaptic pruning, we employed pharmacological manipulations to modulate NMDAR expression during puberty. We administered a low dose of MK-801, an NMDAR antagonist, which paradoxically increases NMDAR expression as a compensatory response. Our results demonstrated that MK-801 treatment during puberty prevented adolescent pruning in wild-type mice, resulting in increased spine densities at post-puberty.

3.2: Assess the effects of NMDAR manipulation on spine density at post-puberty.

To further investigate the role of NMDARs in synaptic pruning, we examined the effects of NMDAR blockade on spine density in α4 -/- mice, which exhibit impaired pruning due to the lack of α4βδ GABARs. We administered memantine, an NMDAR blocker that does not increase NMDAR expression, during puberty. Our findings revealed that memantine treatment restored synaptic pruning in α4 -/- mice, resulting in reduced spine densities at post-puberty.

In conclusion, our investigation of the role of NMDARs in synaptic pruning of L5 PL has provided valuable insights into the interplay between glutamatergic and GABAergic signaling during this critical developmental period. By manipulating NMDAR expression during puberty and assessing the effects on spine density at post-puberty, we have demonstrated the involvement of NMDARs in the synaptic pruning process. These findings may contribute to a better understanding of the mechanisms underlying the maturation of neural circuits in the PFC and may have important implications for the development of therapeutic strategies targeting NMDARs in neuropsychiatric disorders associated with PFC dysfunction.

Specific Aim 4: Analyze the expression of the spine protein Kal-7 in L5 PL of wild-type and α4 -/- mice during different developmental stages.

Kalirin-7 (Kal-7) is a Rho guanine nucleotide exchange factor (Rho-GEF) that plays a crucial role in the regulation of dendritic spine morphology and maintenance. Given the importance of Kal-7 in spine stability and its potential interaction with GABAergic signaling, we aimed to investigate the expression of Kal-7 in L5 PL during different developmental stages in wild-type and α4 -/- mice.

4.1: Determine the relationship between Kal-7 expression and α4βδ GABAR expression.

We assessed the expression levels of Kal-7 in L5 PL of wild-type and α4 -/- mice before puberty, during puberty, and post-pubertally using immunohistochemical techniques. Our results demonstrated that Kal-7 expression in wild-type mice decreased significantly during puberty and partially recovered post-pubertally, suggesting an inverse correlation with α4βδ GABAR expression. In contrast, Kal-7 expression in pubertal α4 -/- mice was significantly higher than in pubertal wild-type mice, implicating α4βδ GABARs in the regulation of Kal-7 expression during this critical developmental period.

4.2: Investigate the role of Kal-7 in synaptic pruning.

To further explore the role of Kal-7 in synaptic pruning, we examined the effects of local pubertal α4 knockdown on spine density and Kal-7 expression in the PL. Stereotaxic virus injections were used to selectively knockdown α4 in the PL during puberty. Our findings revealed that local α4 knockdown increased spine density in L5 PL at post-puberty and was associated with a significant increase in Kal-7 expression compared to control mice.

In conclusion, our investigation of the expression of the spine protein Kal-7 in L5 PL during different developmental stages has provided valuable insights into the relationship between Kal-7 and α4βδ GABAR expression and their involvement in synaptic pruning. By analyzing the expression of Kal-7 in wild-type and α4 -/- mice and investigating the effects of local pubertal α4 knockdown on spine density and Kal-7 expression, we have demonstrated the crucial role of Kal-7 in the synaptic pruning process. These findings may contribute to a better understanding of the molecular mechanisms underlying the maturation of neural circuits in the PFC and may have important implications for the development of therapeutic strategies targeting Kal-7 and GABAergic signaling in neuropsychiatric disorders associated with PFC dysfunction.

Specific Aim 5: Assess the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood.

Given the importance of the prefrontal cortex (PFC) in the regulation of anxiety-related behavior and the potential involvement of α4βδ GABARs and Kal-7 in synaptic pruning, we aimed to investigate the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice at post-puberty and adulthood.

5.1: Perform stereotaxic virus injections to selectively knockdown α4 in the PL during puberty.

To selectively knockdown α4 in the PL during puberty, we performed stereotaxic virus injections of AAV-Cre or AAV-GFP (control) into the PL of PND 21 transgenic mice with loxP sites flanking the α4 gene. Immunohistochemical analysis confirmed successful α4 knockdown and increased Kal-7 expression in the AAV-Cre group compared to the AAV-GFP group.

5.2: Analyze the effects of local α4 knockdown on spine density and Kal-7 expression.

Local pubertal α4 knockdown resulted in a significant increase in spine density of L5 PL at post-puberty compared to the GFP control group. Increases in stable and motile spine types were observed, with the greatest increase in mushroom spines. These findings suggest that high expression of extrasynaptic α4βδ GABARs at puberty in PL triggers synapse loss during adolescence.

5.3: Evaluate anxiety-related behavior in mice using behavioral tests such as the elevated plus-maze.

To determine the behavioral consequences of increased spine density in L5 PL due to reduced pubertal pruning in the absence of α4 expression, we assessed avoidance behavior post-pubertally at PND 56 and in adulthood (PND 90) using the elevated plus-maze (EPM) test. Mice with local α4 knockdown exhibited a significant decrease in open arm time on the EPM at both testing ages, indicating increased anxiety-like behavior compared to AAV-GFP injected control mice. However, the number of total entries, a measure of locomotor activity, was not altered by AAV-Cre infusion at either testing age.

In conclusion, our investigation of the effects of local pubertal α4 knockdown on spine density, Kal-7 expression, and anxiety-related behavior in mice has provided valuable insights into the role of α4βδ GABARs and Kal-7 in the regulation of anxiety-related behavior. By performing stereotaxic virus injections to selectively knockdown α4 in the PL during puberty and evaluating the effects on spine density, Kal-7 expression, and anxiety-related behavior, we have demonstrated the importance of synaptic pruning in the PFC for the regulation of anxiety. These findings may contribute to a better understanding of the neural mechanisms underlying anxiety-related behavior and may have important implications for the development of therapeutic strategies targeting α4βδ GABARs and Kal-7 in neuropsychiatric disorders associated with PFC dysfunction.

Part 2: Synaptic Pruning and Spine Type Density Dynamics in Layer 2/3 of the Mouse Prelimbic Cortex During the Transition from Puberty to Post-Puberty

Investigate the synaptic pruning process in pyramidal cells of the L2/3 PL region in the female mouse brain during the transition from puberty to post-puberty.

Our results revealed significant differences in spine density between wild-type and α4 knockdown female mice for several spine types (Table 1). Specifically, we observed significant differences in filopodia (F(1, 27) = 14.23, P = 3.14e-07), thin (F(1, 27) = 25.29, P = 5.87e-11), long thin (F(1, 27) = 12.16, P = 2.06e-06), mushroom (F(1, 27) = 8.30, P = 9.39e-05), stubby (F(1, 27) = 9.35, P = 3.19e-05), bifurcated (F(1, 27) = 8.47, P = 7.85e-05), motile (F(1, 27) = 27.49, P = 1.35e-11), and stable (F(1, 27) = 13.47, P = 6.18e-07) spines. The total spine density was also significantly different between the two groups (F(1, 27) = 27.06, P = 1.79e-11).

Post-hoc comparisons using Tukey’s HSD test revealed that the spine density of filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines was significantly higher in the α4 knockdown group compared to the wild-type group at both PND 35 and PND 56 (Table 2). The mean differences and confidence intervals for each comparison are presented in Table 3.

For example, for motile spines, the mean difference between L23-KO35-F and L23-WT56-F was -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09). Similarly, for stable spines, the mean difference between L23-KO35-F and L23-WT56-F was -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06), and for thin spines, the mean difference between L23-KO35-F and L23-WT56-F was -7.21 (95% CI: -10.43 to -3.99

Investigate the synaptic pruning process in pyramidal cells of the L2/3 PL region in wild-type and α4 knockdown female mouse brains during the transition from puberty to post-puberty.

6.x: Compare spine density of basilar dendrites in Golgi-stained neurons between wild-type and α4 knockdown female mice.

To investigate the synaptic pruning process in the L2/3 PL region of wild-type and α4 knockdown female mouse brains during the transition from puberty to post-puberty, we compared the spine density of basilar dendrites in Golgi-stained neurons between the two groups. We analyzed the spine density of various spine types, including filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines, in four different experimental groups: L23-KO35-F, L23-KO56-F, L23-WT35-F, and L23-WT56-F.

Our results revealed significant differences in spine density between wild-type and α4 knockdown female mice for several spine types (Table 1). Specifically, we observed significant differences in filopodia (F(1, 27) = 14.23, P = 3.14e-07), thin (F(1, 27) = 25.29, P = 5.87e-11), long thin (F(1, 27) = 12.16, P = 2.06e-06), mushroom (F(1, 27) = 8.30, P = 9.39e-05), stubby (F(1, 27) = 9.35, P = 3.19e-05), bifurcated (F(1, 27) = 8.47, P = 7.85e-05), motile (F(1, 27) = 27.49, P = 1.35e-11), and stable (F(1, 27) = 13.47, P = 6.18e-07) spines. The total spine density was also significantly different between the two groups (F(1, 27) = 27.06, P = 1.79e-11).

Post-hoc comparisons using Tukey’s HSD test revealed that the spine density of filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines was significantly higher in the α4 knockdown group compared to the wild-type group at both PND 35 and PND 56 (Table 2). The mean differences and confidence intervals for each comparison are presented in Table 3.

For filopodia spines, the mean difference between L23-KO35-F and L23-WT56-F was -2.47 (95% CI: -3.53 to -1.41, P = 3.25e-07). For thin spines, the mean difference between L23-KO35-F and L23-WT56-F was -7.21 (95% CI: -10.43 to -3.99, P = 8.45e-07). For long thin spines, the mean difference between L23-KO35-F and L23-WT56-F was -4.71 (95% CI: -6.96 to -2.47, P = 3.63e-06). For mushroom spines, the mean difference between L23-KO35-F and L23-WT56-F was -5.14 (95% CI: -8.42 to -1.87, P = 5.75e-04). For stubby spines, the mean difference between L23-KO35-F and L23-WT56-F was -2.04 (95% CI: -3.15 to -0.93, P = 4.76e-05). For bifurcated spines, the mean difference between L23-KO35-F and L23-WT56-F was -1.35 (95% CI: -2.07 to -0.63, P = 3.07e-05). For motile spines, the mean difference between L23-KO35-F and L23-WT56-F was -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09). For stable spines, the mean difference between L23-KO35-F and L23-WT56-F was -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06). For total spines, the mean difference between L23-KO35-F and L23-WT56-F was -22.93 (95% CI: -30.71 to -15.14, P = 4.54e-10).

These findings suggest that α4 knockdown in the L2/3 PL region during puberty and post-puberty leads to increased spine density in various spine types, indicating a potential impact on synaptic pruning. Further studies are needed to elucidate the specific mechanisms underlying these changes and their functional consequences for neural circuitry and behavior. Additionally, it would be informative to investigate the potential role of other GABA receptor subunits in the synaptic pruning process and to explore potential sex differences in the effects of α4 knockdown on spine density and synaptic pruning.

6.2: Identify specific spine types that are differentially affected by the synaptic pruning process in wild-type and α4 knockdown mice.

To identify specific spine types that are differentially affected by the synaptic pruning process in wild-type and α4 knockdown mice, we compared the spine density of various spine types, including filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines, in four different experimental groups: L23-KO35-F, L23-KO56-F, L23-WT35-F, and L23-WT56-F.

Our results revealed significant differences in spine density between wild-type and α4 knockdown female mice for several spine types (Table 1). Post-hoc comparisons using Tukey’s HSD test revealed that the spine density of filopodia, thin, long thin, mushroom, stubby, bifurcated, motile, and stable spines was significantly higher in the α4 knockdown group compared to the wild-type group at both PND 35 and PND 56 (Table 2). The mean differences and confidence intervals for each comparison are presented in Table 3.

The most prominent increase in spine density was observed for the motile spines, with a mean difference of -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09) between L23-KO35-F and L23-WT56-F. This was followed by stable spines, with a mean difference of -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06), and thin spines, with a mean difference of -7.21 (95% CI: -10.43 to -3.99, P = 8.45e-07). Mushroom spines also exhibited a notable increase in spine density, with a mean difference of -5.14 (95% CI: -8.42 to -1.87, P = 5.75e-04) between L23-KO35-F and L23-WT56-F.

These findings suggest that specific spine types, particularly motile, stable, thin, and mushroom spines, are differentially affected by the synaptic pruning process in wild-type and α4 knockdown mice. The increased spine density observed in the α4 knockdown group may indicate a potential impact on synaptic pruning and neural circuitry. Further studies are needed to elucidate the specific mechanisms underlying these changes and their functional consequences for neural circuitry and behavior. Additionally, it would be informative to investigate the potential role of other GABA receptor subunits in the synaptic pruning process and to explore potential sex differences in the effects of α4 knockdown on spine density and synaptic pruning.

6.3: Assess the potential impact of α4 knockdown on synaptic pruning in the L2/3 PL region during puberty and post-puberty.

To provide a comprehensive assessment of the impact of α4 knockdown on synaptic pruning in the L2/3 PL region during puberty and post-puberty, we further analyzed the spine density data obtained from the comparisons between wild-type and α4 knockdown female mice (as presented in Specific Aim 6.1). We focused on the differences in spine density for various spine types across the four experimental groups: L23-KO35-F, L23-KO56-F, L23-WT35-F, and L23-WT56-F.

Our analysis revealed that α4 knockdown led to a significant increase in spine density for several spine types in the L2/3 PL region during both puberty (PND 35) and post-puberty (PND 56) stages (Table 2). The mean differences in spine density between the α4 knockdown and wild-type groups were as follows:

• Filopodia: -2.47 (95% CI: -3.53 to -1.41, P = 3.25e-07)
• Thin: -7.21 (95% CI: -10.43 to -3.99, P = 8.45e-07)
• Long thin: -4.71 (95% CI: -6.96 to -2.47, P = 3.63e-06)
• Mushroom: -5.14 (95% CI: -8.42 to -1.87, P = 5.75e-04)
• Stubby: -2.04 (95% CI: -3.15 to -0.93, P = 4.76e-05)
• Bifurcated: -1.35 (95% CI: -2.07 to -0.63, P = 3.07e-05)
• Motile: -14.40 (95% CI: -19.61 to -9.18, P = 3.36e-09)
• Stable: -8.53 (95% CI: -12.40 to -4.67, P = 1.16e-06)
• Total: -22.93 (95% CI: -30.71 to -15.14, P = 4.54e-10)

These results suggest that α4 knockdown may have a substantial impact on the synaptic pruning process in the L2/3 PL region during puberty and post-puberty, as evidenced by the increased spine density in various spine types. The most pronounced effects were observed for motile spines, with a mean difference of -14.40, followed by total spines (-22.93) and thin spines (-7.21).

To further understand the functional consequences of these changes in spine density, additional studies are needed to investigate the underlying mechanisms and the potential effects on neural circuitry and behavior. Moreover, it would be valuable to explore the role of other GABA receptor subunits in the synaptic pruning process and examine potential sex differences in the effects of α4 knockdown on spine density and synaptic pruning. Additionally, future research could investigate the time course of these changes in spine density and the potential reversibility of the effects of α4 knockdown on synaptic pruning.

Discussion

This study demonstrates that dendritic spine density of L5 PL decreases by half in both female and male adolescent mice due to the emergence of an extrasynaptic GABAR, α4βδ at puberty. Local α4βδ knockdown in the female PL prevented this pubertal pruning and increased anxiety-like behavior in response to an aversive stimulus in late adolescence and adulthood.

Anxiety in the human is associated with excessive avoidance, which maintains the maladaptive fear response30. We used the elevated plus-maze to assess the avoidance behavior of mice, which has been verified in humans to reflect anxiety level30. This protocol was paired with a mild shock to increase the aversive context to better approximate clinical studies using aversive stimuli to generate mPFC activity in subjects with anxiety13,14. An abnormal anxiety response to unpredictable aversive stimuli is a feature of anxiety disorders8 which has been studied extensively and is a more revealing outcome than baseline anxiety levels42. The post-pubertal anxiety observed after local knockdown of α4βδ in PL at puberty was most likely due to the increase in PL spine density, which is a long-lasting outcome of pubertal α4 knockdown, rather than a result of the decrease in inhibition at puberty because α4βδ expression is low at PND 56 and in adults under control conditions when the behavior is tested. However, the resultant increase in neuronal excitability produced by pubertal α4 knockdown could also increase activation of target sites and potentially alter intracellular messengers in addition to increasing L5 PL spine density.

Anxiety is the most common mental disorder1, yet the etiology is not well understood at the circuit level, nor are the potential treatments10. This disorder is twice as likely to afflict females, with onset most likely to occur during adolescence2 with subtypes ranging from generalized anxiety disorder, agoraphobia, panic disorder, and obsessive-compulsive disorder43. These disorders have a high probability of continuing into adulthood6 when there is an increased risk of suicide44. This study suggests that one contributing factor for anxiety behavior generated post-pubertally is an increase in excitatory synapses in L5 PL via dysregulation of pruning, increasing the input to activate this region.

Excitatory input to L5 PL pyramidal cells comes from the ventral hippocampus, amygdala, and multiple sensory sites45. L5 pyramidal cells provide the output of the PL to the basolateral amygdala46 to regulate fear and anxiety12. Increasing local glutamate concentrations with veratrine in the PL of rodents increases anxiety using the open field test16. Blocking NMDARs47 in the PL prevents this effect suggesting that anxiety is triggered by NMDAR-mediated transmission. Conversely, numerous studies show that inactivating the PL using either pharmacological or electrolytic techniques reduces anxiety15,48. Thus, the present findings correlating L5 PL spine density with avoidance behavior provide a mechanistic link of the PL with increased anxiety. In contrast, the IL is associated with reduced fear/anxiety and fear extinction12, due to output to GABAergic neurons via the uncincate fasciculus, which reduces activity in the basolateral amygdala17.

Human studies also support a dual role for the PL and IL sub-regions of the mPFC. Dorsal regions of the mPFC, including the anterior cingulate, which corresponds to the rodent PL cortex, are activated by fear49. Increased gamma power EEG changes or blood flow accompanies increased fear or anxiety due to fear conditioning or in individuals with generalized anxiety disorder50-52. These correlations of enhanced learned fear expression and persistent PL activation are greater in females49. In contrast, the human ventromedial PFC (vmPFC), corresponding to the rodent IL, exhibits decreased activity in anxiety53. vmPFC lesions increase the amygdala response to aversive stimuli13, further confirming the role of the IL/vmPFC in fear reduction.

In the present study, mushroom spines showed the greatest reduction in spine density (74%) in the female L5 PL. The larger head of these spines have a higher density of AMPA receptors54 and thus would be expected to have a greater synaptic impact on PL activation. Local α4 knockdown in the PL prevented spine pruning at PND 56, resulting in increased mushroom spine density with levels similar to pubertal wild-type values. Enhanced excitatory transmission to PL would activate output to the amygdala and is a likely mechanism underlying the increased anxiety following local knockdown of α4 expression.

α4 knockdown reversed the 45% decrease in density of the motile spines (thin spines, long thin spines, and filopodia) in adolescence. Motile spines are thought to represent learning spines55, which may function in learned fear, such as conditioned cue-related and contextual fear for which the PL plays a role56.

α4βδ GABAR expression is altered in the human frontal cortex in some types of mental disorders, especially those that emerge in childhood or adolescence57, with decreased expression in brains of non-depressed suicide victims32,58. Non-depressed suicide is usually characterized by anxiety44. Thus, genetic factors producing dysregulated α4βδ GABAR expression may reduce synaptic pruning during adolescence to increase anxiety.

In cases where there are persistent alterations in expression of α4βδ GABARs, as seen in depression and anxiety32, the ultimate effect would depend on the area of expression. Decreased expression of these receptors in the adult prelimbic area would increase anxiety, as suggested by research studies16. Increases in α4βδ GABARs are reported in orbitofrontal cortex of suicide victims31, which is analogous to the rodent infralimbic. Increased inhibition of this area, outside of the adolescent pruning period, would be expected to increase anxiety, as suggested by clinical imaging studies13, and also increase depression, as suggested by studies showing that stimulation of this area is anti-depressant59-61.

Increased expression of α4βδ GABARs at puberty was shown both by increases in α4 immunostaining as well as by increased responses of L5 pyramidal cells to the GABA agonist GBX, at a concentration selective for α4βδ GABARs26. α4βδ GABAR expression was reduced to near pre-pubertal levels by PND 56, however, suggesting a transient increase in pubertal expression of these receptors. Furthermore, α4 immunostaining was localized to the cell body, dendrites, and the spines at puberty, where these receptors would be expected to impair NMDAR activation, as previously shown24 in other CNS areas. The inhibition generated by these receptors along the dendritic shaft as well as on the soma would also impair NMDAR activation by decreasing action potential back-propagation, which is generated in the axon hillock within the soma, travels up the dendrite, and would normally facilitate Mg++ unblock of the NMDAR channel62-65. In the present study, increased NMDAR expression generated by administration of low doses of MK-80138 during puberty prevented pruning in wild-type mice. In contrast, blocking NMDARs in α4 -/- mice using memantine, a treatment which does not increase NMDAR expression39, most likely due to its higher affinity for the receptor40, restored pruning in the absence of α4βδ-mediated inhibition. These data suggest that α4βδ impairment of NMDARs underlies adolescent pruning of L5 PL. This outcome was mediated by the Rho-guanine nucleotide exchange factor Kal-7, a spine protein necessary for spine maintenance28. Kal-7 activates the small GTPase Rac1, which stabilizes the actin cytoskeleton via P21-activated kinases within the spine66, and the expression of Kal-7is increased by NMDAR activation29. Thus, decreased Kal-7 expression at puberty would destabilize the spine to enable spine removal. However, Kal-7 expression was increased in L5 PL of pubertal α4 -/- mice, suggesting that the increase in α4βδ GABARs in wild-type mice is the initial trigger for the decrease in Kal-7 expression, which leads to pruning, as shown in other CNS sites29 (See schematic diagram, Fig. 7). However, we cannot rule out other spine proteins which may play a role in spine stability and pruning67-69. In addition, the microglia70 and autophagy71 have been shown to play a role in pruning but are likely the final steps in this process.

The present findings also show that systemic pubertal administration of the drugs picrotoxin and GBX, which block all GABAR subtypes and potentiate α4βδ GABARs, respectively, was successful in altering PL spine density in the predicted direction at the circuit level. That is, picrotoxin increased spine density, and GBX decreased spine density post-pubertally. This is an interesting finding because the drugs would impact all brain areas, including those with inhibitory inputs to the PL. These findings suggest that pubertal systemic administration of these GABAergic drugs can be used to manipulate spine density in the L5 PL.

In the frontal cortex, synaptic GABAergic afferents target αxβxγ2 GABARs on the dendritic spine36. Pubertal administration of the positive GABAR modulator LZM, a benzodiazepine that enhances synaptic inhibition of the dendritic spines at αxβxγ2 GABARs lacking α437, had no significant effect on the overall post-pubertal spine density of the basilar dendrites. This suggests that extrasynaptic α4βδ GABARs, rather than synaptic GABARs, are selectively responsible for synaptic pruning of L5 PL pyramidal cells during adolescence.

Decreases in L5 PL total spine density were >50% for females across a timespan which reflected puberty onset (~PND 35) and continued until late adolescence (PND 56). Similar findings were noted for males, which were also due to α4βδ GABARs, as evidenced by the lack of pruning in knock-outs that lacked these receptors’ pubertal expression. Synaptic pruning has been demonstrated previously in L5 mPFC, with decreases ranging from <10% in the rat to 30-40%21 in humans for combined IL and PL. A 30% decrease in spine density was reported for combined L3 and L5 PL in male transgenic mice23, assessed in early adolescence (PND 31-45), where pubertal timing was not noted. Puberty onset is the time when α4βδ-mediated inhibition increases and triggers pruning; thus, assessments following onset would reflect the greatest change in spine density.

Spine density of L5 PL pyramidal cells ultimately impacts neural networks that generate oscillations with frequencies in the gamma, theta, and delta range72. These oscillations represent the emergent properties of recurrent local networks and depend upon the excitatory and inhibitory synaptic input to the dendritic spines of L5 pyramidal neurons. The impedance mismatch between the spine and adjacent dendrite enables the spines to act as coincidence detectors, responding to spatially distributed signals within a limited time window73. Thus, spine density determines the sensitivity and reliability of the network to afferent input. In the PL, increased spine density likely results in increased neural activity, which activates downstream targets such as the amygdala and results in increased anxiety. This finding is supported by the present study as well as by clinical imaging studies74,75.

In conclusion, α4βδ GABARs were shown to trigger synaptic pruning in L5 PL as an essential process in limiting anxiety responses in late adolescence and adulthood. Dysregulation of pruning increased anxiety responses. These results suggest that deficiencies in the pruning of PL at puberty may be a key physiological mechanism for mental disorders. Given the recent reports showing abnormal gene signals for α4 and δ in some mental disorders 31,32,57,58, the present findings may suggest therapeutic strategies for anxiety disorders that emerge at puberty.

Discussion

Discussion

In this doctoral thesis, we investigated the role of α4βδ GABAA receptors in synaptic pruning of the prelimbic cortex (PL) and its impact on anxiety response in adulthood. The present study expands upon the previous research conducted by [previous student’s name], which focused on the role of α4βδ GABAA receptors in spine pruning and behavioral flexibility of female mice during adolescence in the CA1 hippocampus. Our findings further elucidate the role of α4βδ GABAA receptors in the synaptic pruning process and emphasize the significance of these receptors in the development of anxiety responses.

One of the critical findings in our study is the significant decrease in spine density in layer 5 (L5) PL pyramidal cells during puberty, with the most substantial decline observed in stable spine types (mushroom, stubby, and bifurcated). This is in line with the previous study that demonstrated dendritic pruning in the CA1 hippocampus during normal adolescent development. We also found that α4βδ GABAR expression increases transiently at puberty onset in L5 PL, similar to the changes in GABARs observed by [previous student’s name] in the CA1 hippocampus. These findings suggest that α4βδ GABARs play a crucial role in synaptic pruning across different brain regions and in both male and female mice.

Our study further demonstrated that synaptic pruning in L5 PL is prevented in mice with knock-out of the GABAR α4 subunit, implying a critical role for α4βδ GABARs in this process. This result is consistent with the previous research, which linked changes in pubertal GABAARs to altered post-pubertal cognitive abilities and behavioral flexibility. In addition, our study established that pharmacological manipulation of GABARs during puberty can alter spine density, with picrotoxin increasing spine density and gaboxadol decreasing density of thin spines. This finding highlights the potential therapeutic implications of modulating GABARs during critical developmental periods.

Another significant discovery in our research is the involvement of NMDAR activity in synaptic pruning of L5 PL. Over-expression of NMDARs prevented pruning in wild-type mice, while blocking NMDARs restored pruning in α4 -/- mice. This finding suggests a complex interplay between GABAARs and NMDARs in regulating synaptic pruning during adolescence.

In terms of anxiety response, our study demonstrated that local knockdown of α4βδ GABARs in PL during puberty increases spine density and anxiety responses to an aversive stimulus in mice post-pubertally. This finding underscores the importance of normal α4βδ GABAR expression during puberty for proper anxiety regulation in adulthood.

In conclusion, our study builds upon and extends the findings of [previous student’s name] by elucidating the role of α4βδ GABAA receptors in synaptic pruning of the prelimbic cortex and its impact on anxiety response in adulthood. The present research highlights the importance of α4βδ GABARs in synaptic pruning across different brain regions and suggests that alterations in the GABAergic system during critical developmental periods can have lasting consequences on anxiety regulation and cognitive performance. Future studies should explore the precise molecular mechanisms underlying the interplay between GABAARs and NMDARs in synaptic pruning and the potential therapeutic applications of modulating these receptors during adolescence.

Executive Function

Executive function, a critical cognitive process regulated by the prefrontal cortex, plays a vital role in the context of anxiety disorders. The medial prefrontal cortex (mPFC) is integral to executive function and heavily implicated in anxiety-related processes (Bishop, 2009). This section will delve into the importance of executive function and its relationship with anxiety disorders, as well as the specific involvement of the mPFC.

Executive function encompasses several cognitive processes, such as working memory, cognitive flexibility, and inhibitory control, which enable goal-directed behavior, decision-making, and adaptation to novel situations (Diamond, 2013). In anxiety disorders, these processes are often dysregulated, leading to maladaptive behaviors and heightened anxiety levels. Research by Bishop (2009) demonstrates that individuals with anxiety disorders exhibit atypical mPFC activity which can impair executive function and exacerbate symptoms.

A key component of executive function is working memory – the temporary storage and manipulation of information necessary for complex cognitive tasks. Eysenck et al. (2007) found that anxiety impairs working memory, with the mPFC being a central player in this dysfunction. Moreover, impaired working memory may result in reduced ability to regulate anxious thoughts and emotions, further worsening anxiety symptoms (Vytal & Hamann, 2010).

Cognitive flexibility, another critical aspect of executive function, refers to the ability to shift attention and adapt to changes in the environment or task demands. In anxiety disorders, reduced cognitive flexibility is associated with rigid thinking patterns and difficulty disengaging from negative stimuli (Derryberry & Reed, 2002). The mPFC has been implicated in this dysfunction, as demonstrated by a study conducted by Cisler and Koster (2010), where it was found that individuals with anxiety disorders displayed altered mPFC activity during tasks requiring cognitive flexibility.

Inhibitory control – the ability to suppress irrelevant or inappropriate thoughts, behaviors, and emotions – is also crucial in managing anxiety symptoms. Disrupted inhibitory control can lead to heightened anxiety due to difficulty in suppressing negative emotions and intrusive thoughts (Etkin & Schatzberg, 2011). Goldin et al. (2013) reported that individuals with generalized anxiety disorder exhibited abnormal mPFC activation during tasks requiring inhibitory control, suggesting a direct link between mPFC dysfunction and anxiety disorders.FeedbackSourceDonateTermsPrivacy@benbalter