Neuronal subtypes and connectivity of the adult mouse paralaminar amygdala.

The paralaminar nucleus of the amygdala (PL) is comprised of neurons which exhibit delayed maturation. PL neurons are born during gestation but mature during adolescent ages, differentiating into excitatory neurons. The PL is prominent in the adult amygdala, contributing to its increased neuron number and relative size compared to childhood. However, the function of the PL is unknown, as the region has only recently begun to be characterized in detail. In this study, we investigated key defining features of the adult PL; the intrinsic morpho-electric properties of its neurons, and its input and output connectivity. We identify two subtypes of excitatory neurons in the PL based on unsupervised clustering of electrophysiological properties. These subtypes are defined by differential action potential firing properties and dendritic architecture, suggesting divergent functional roles. We further uncover major axonal inputs to the adult PL from the main olfactory network and basolateral amygdala. We also find that axonal outputs from the PL project reciprocally to major inputs, and to diverse targets including the amygdala, frontal cortex, hippocampus, hypothalamus, and brainstem. Thus, the adult PL is centrally placed to play a major role in the integration of olfactory sensory information, likely coordinating affective and autonomic behavioral responses to salient odor stimuli. Significance Statement: Mammalian amygdala development includes a growth period from childhood to adulthood, believed to support emotional and social learning. This amygdala growth is partly due to the maturation of neurons during adolescence in the paralaminar amygdala. However, the functional properties of these neurons are unknown. In our recent studies, we characterized the paralaminar amygdala in the mouse. Here, we investigate the properties of the adult PL in the mouse, revealing the existence of two neuronal subtypes that may play distinct functional roles in the adult brain. We further reveal the brain-wide input and output connectivity of the PL, indicating that the PL combines olfactory cues for emotional processing and delivers information to regions associated with reward and autonomic states.

Delayed maturation and migration of excitatory neurons in the juvenile mouse paralaminar amygdala.

The human amygdala paralaminar nucleus (PL) contains many immature excitatory neurons that undergo prolonged maturation from birth to adulthood. We describe a previously unidentified homologous PL region in mice that contains immature excitatory neurons and has previously been considered part of the amygdala intercalated cell clusters or ventral endopiriform cortex. Mouse PL neurons are born embryonically, not from postnatal neurogenesis, despite a subset retaining immature molecular and morphological features in adults. During juvenile-adolescent ages (P21-P35), the majority of PL neurons undergo molecular, structural, and physiological maturation, and a subset of excitatory PL neurons migrate into the adjacent endopiriform cortex. Alongside these changes, PL neurons develop responses to aversive and appetitive olfactory stimuli. The presence of this homologous region in both humans and mice points to the significance of this conserved mechanism of neuronal maturation and migration during adolescence, a key time period for amygdala circuit maturation and related behavioral changes.

Connectivity and molecular profiles of Foxp2- and Dbx1-lineage neurons in the accessory olfactory bulb and medial amygdala.

In terrestrial vertebrates, the olfactory system is divided into main (MOS) and accessory (AOS) components that process both volatile and nonvolatile cues to generate appropriate behavioral responses. While much is known regarding the molecular diversity of neurons that comprise the MOS, less is known about the AOS. Here, focusing on the vomeronasal organ (VNO), the accessory olfactory bulb (AOB), and the medial amygdala (MeA), we reveal that populations of neurons in the AOS can be molecularly subdivided based on their ongoing or prior expression of the transcription factors Foxp2 or Dbx1, which delineate separate populations of GABAergic output neurons in the MeA. We show that a majority of AOB neurons that project directly to the MeA are of the Foxp2 lineage. Using single-neuron patch-clamp electrophysiology, we further reveal that in addition to sex-specific differences across lineage, the frequency of excitatory input to MeA Dbx1- and Foxp2-lineage neurons differs between sexes. Together, this work uncovers a novel molecular diversity of AOS neurons, and lineage and sex differences in patterns of connectivity.

Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors.

Social behaviors are innate and supported by dedicated neural circuits, but the molecular identities of these circuits and how they are established developmentally and shaped by experience remain unclear. Here we show that medial amygdala (MeA) cells originating from two embryonically parcellated developmental lineages have distinct response patterns and functions in social behavior in male mice. MeA cells expressing the transcription factor Foxp2 (MeAFoxp2) are specialized for processing male conspecific cues and are essential for adult inter-male aggression. By contrast, MeA cells derived from the Dbx1 lineage (MeADbx1) respond broadly to social cues, respond strongly during ejaculation and are not essential for male aggression. Furthermore, MeAFoxp2 and MeADbx1 cells show differential anatomical and functional connectivity. Altogether, our results suggest a developmentally hardwired aggression circuit at the MeA level and a lineage-based circuit organization by which a cell's embryonic transcription factor profile determines its social information representation and behavioral relevance during adulthood.

Hardwired to attack: Transcriptionally defined amygdala subpopulations play distinct roles in innate social behaviors.

Social behaviors are innate and supported by dedicated neural circuits, but it remains unclear whether these circuits are developmentally hardwired or established through social experience. Here, we revealed distinct response patterns and functions in social behavior of medial amygdala (MeA) cells originating from two embryonically parcellated developmental lineages. MeA cells in male mice that express the transcription factor Foxp2 (MeAFoxp2) are specialized for processing male conspecific cues even before puberty and are essential for adult inter-male aggression. In contrast, MeA cells derived from the Dbx1-lineage (MeADbx1) respond broadly to social cues and are non-essential for male aggression. Furthermore, MeAFoxp2 and MeADbx1 cells show differential anatomical and functional connectivity. Altogether, our results support a developmentally hardwired aggression circuit at the level of the MeA and we propose a lineage-based circuit organization by which a cell's embryonic transcription factor profile determines its social information representation and behavior relevance during adulthood.

Sex-Specific Social Behavior and Amygdala Proteomic Deficits in Foxp2 +/- Mutant Mice.

In humans, mutations in the transcription factor encoding gene, FOXP2, are associated with language and Autism Spectrum Disorders (ASD), the latter characterized by deficits in social interactions. However, little is known regarding the function of Foxp2 in male or female social behavior. Our previous studies in mice revealed high expression of Foxp2 within the medial subnucleus of the amygdala (MeA), a limbic brain region highly implicated in innate social behaviors such as mating, aggression, and parental care. Here, using a comprehensive panel of behavioral tests in male and female Foxp2 +/- heterozygous mice, we investigated the role Foxp2 plays in MeA-linked innate social behaviors. We reveal significant deficits in olfactory processing, social interaction, mating, aggressive, and parental behaviors. Interestingly, some of these deficits are displayed in a sex-specific manner. To examine the consequences of Foxp2 loss of function specifically in the MeA, we conducted a proteomic analysis of microdissected MeA tissue. This analyses revealed putative sex differences expression of a host of proteins implicated in neuronal communication, connectivity, and dopamine signaling. Consistent with this, we discovered that MeA Foxp2-lineage cells were responsive to dopamine with differences between males and females. Thus, our findings reveal a central and sex-specific role for Foxp2 in social behavior and MeA function.

Sex Differences in Biophysical Signatures across Molecularly Defined Medial Amygdala Neuronal Subpopulations.

The medial amygdala (MeA) is essential for processing innate social and non-social behaviors, such as territorial aggression and mating, which display in a sex-specific manner. While sex differences in cell numbers and neuronal morphology in the MeA are well established, if and how these differences extend to the biophysical level remain unknown. Our previous studies revealed that expression of the transcription factors, Dbx1 and Foxp2, during embryogenesis defines separate progenitor pools destined to generate different subclasses of MEA inhibitory output neurons. We have also previously shown that Dbx1-lineage and Foxp2-lineage neurons display different responses to innate olfactory cues and in a sex-specific manner. To examine whether these neurons also possess sex-specific biophysical signatures, we conducted a multidimensional analysis of the intrinsic electrophysiological profiles of these transcription factor defined neurons in the male and female MeA. We observed striking differences in the action potential (AP) spiking patterns across lineages, and across sex within each lineage, properties known to be modified by different voltage-gated ion channels. To identify the potential mechanism underlying the observed lineage-specific and sex-specific differences in spiking adaptation, we conducted a phase plot analysis to narrow down putative ion channel candidates. Of these candidates, we found a subset expressed in a lineage-biased and/or sex-biased manner. Thus, our results uncover neuronal subpopulation and sex differences in the biophysical signatures of developmentally defined MeA output neurons, providing a potential physiological substrate for how the male and female MeA may process social and non-social cues that trigger innate behavioral responses.

Identification of amygdala-expressed genes associated with autism spectrum disorder.

BACKGROUND: Studies of individuals with autism spectrum disorder (ASD) have revealed a strong multigenic basis with the identification of hundreds of ASD susceptibility genes. ASD is characterized by social deficits and a range of other phenotypes, implicating complex genetics and involvement of a variety of brain regions. However, how mutations and mis-expression of select gene sets are associated with the behavioral components of ASD remains unknown. We reasoned that for genes to be associated with ASD core behaviors they must be: (1) expressed in brain regions relevant to ASD social behaviors and (2) expressed during the ASD susceptible window of brain development. METHODS: Focusing on the amygdala, a brain region whose dysfunction has been highly implicated in the social component of ASD, we mined publicly available gene expression databases to identify ASD-susceptibility genes expressed during human and mouse amygdala development. We found that a large cohort of known ASD susceptibility genes is expressed in the developing human and mouse amygdala. We further performed analysis of single-nucleus RNA-seq (snRNA-seq) data from microdissected amygdala tissue from five ASD and five control human postmortem brains ranging in age from 4 to 20 years to elucidate cell type specificity of amygdala-expressed genes and their dysregulation in ASD. RESULTS: Our analyses revealed that of the high-ranking ASD susceptibility genes, 80 are expressed in both human and mouse amygdala during fetal to early postnatal stages of development. Our human snRNA-seq analyses revealed cohorts of genes with altered expression in the ASD amygdala postnatally, especially within excitatory neurons, with dysregulated expression of seven genes predicted from our datamining pipeline. LIMITATIONS: We were limited by the ages for which we were able to obtain human tissue; therefore, the results from our datamining pipeline approach will require validation, to the extent possible, in human tissue from earlier developmental stages. CONCLUSIONS: Our pipeline narrows down the number of amygdala-expressed genes possibly involved in the social pathophysiology of ASD. Our human single-nucleus gene expression analyses revealed that ASD is characterized by changes in gene expression in specific cell types in the early postnatal amygdala.

Kcnn2 blockade reverses learning deficits in a mouse model of fetal alcohol spectrum disorders.

Learning disabilities are hallmarks of congenital conditions caused by prenatal exposure to harmful agents. These include fetal alcohol spectrum disorders (FASDs) with a wide range of cognitive deficiencies, including impaired motor skill development. Although these effects have been well characterized, the molecular effects that bring about these behavioral consequences remain to be determined. We previously found that the acute molecular responses to alcohol in the embryonic brain are stochastic, varying among neural progenitor cells. However, the pathophysiological consequences stemming from these heterogeneous responses remain unknown. Here we show that acute responses to alcohol in progenitor cells altered gene expression in their descendant neurons. Among the altered genes, an increase of the calcium-activated potassium channel Kcnn2 in the motor cortex correlated with motor learning deficits in a mouse model of FASD. Pharmacologic blockade of Kcnn2 improves these learning deficits, suggesting Kcnn2 blockers as a new intervention for learning disabilities in FASD.

Rigor and reproducibility in rodent behavioral research.

Behavioral neuroscience research incorporates the identical high level of meticulous methodologies and exacting attention to detail as all other scientific disciplines. To achieve maximal rigor and reproducibility of findings, well-trained investigators employ a variety of established best practices. Here we explicate some of the requirements for rigorous experimental design and accurate data analysis in conducting mouse and rat behavioral tests. Novel object recognition is used as an example of a cognitive assay which has been conducted successfully with a range of methods, all based on common principles of appropriate procedures, controls, and statistics. Directors of Rodent Core facilities within Intellectual and Developmental Disabilities Research Centers contribute key aspects of their own novel object recognition protocols, offering insights into essential similarities and less-critical differences. Literature cited in this review article will lead the interested reader to source papers that provide step-by-step protocols which illustrate optimized methods for many standard rodent behavioral assays. Adhering to best practices in behavioral neuroscience will enhance the value of animal models for the multiple goals of understanding biological mechanisms, evaluating consequences of genetic mutations, and discovering efficacious therapeutics.

PAC1R Genotype to Phenotype Correlations in Autism Spectrum Disorder.

Amygdala dysfunction has been implicated in numerous neurodevelopmental disorders, including autism spectrum disorder (ASD). Previous studies in mice and humans, respectively, have linked Pac1r/PAC1R function to social behavior and PTSD-susceptibility. Based on this connection to social and emotional processing and the central role played by the amygdala in ASD, we examined a putative role for PAC1R in social deficits in ASD and determined the pattern of gene expression in the developing mouse and human amygdala. We reveal that Pac1r/PAC1R is expressed in both mouse and human amygdala from mid-neurogenesis through early postnatal stages, critical time points when altered brain trajectories are hypothesized to unfold in ASD. We further find that parents of autistic children carrying a previously identified PTSD-risk genotype (CC) report greater reciprocal social deficits compared to those carrying the non-risk GC genotype. Additionally, by exploring resting-state functional connectivity differences in a subsample of the larger behavioral sample, we find higher functional connectivity between the amygdala and right middle temporal gyrus in individuals with the CC risk genotype. Thus, using multimodal approaches, our data reveal that the amygdala-expressed PAC1R gene may be linked to severity of ASD social phenotype and possible alterations in brain connectivity, therefore potentially acting as a modifier of amygdala-related phenotypes. Autism Res 2019, 12: 200-211 © 2018 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: In this multimodal study across mouse and human, we examined expression patterns of Pac1r/PAC1R, a gene implicated in social behavior, and further explored whether a previously identified human PTSD-linked mutation in PAC1R can predict brain connectivity and social deficits in ASD. We find that PAC1R is highly expressed in the both the mouse and human amygdala. Furthermore, our human data suggest that PAC1R genotype is linked to severity of social deficits and functional amygdala connectivity in ASD.

Amygdala Corticofugal Input Shapes Mitral Cell Responses in the Accessory Olfactory Bulb.

Interconnections between the olfactory bulb and the amygdala are a major pathway for triggering strong behavioral responses to a variety of odorants. However, while this broad mapping has been established, the patterns of amygdala feedback connectivity and the influence on olfactory circuitry remain unknown. Here, using a combination of neuronal tracing approaches, we dissect the connectivity of a cortical amygdala [posteromedial cortical nucleus (PmCo)] feedback circuit innervating the mouse accessory olfactory bulb. Optogenetic activation of PmCo feedback mainly results in feedforward mitral cell (MC) inhibition through direct excitation of GABAergic granule cells. In addition, LED-driven activity of corticofugal afferents increases the gain of MC responses to olfactory nerve stimulation. Thus, through corticofugal pathways, the PmCo likely regulates primary olfactory and social odor processing.

Loss of CLOCK Results in Dysfunction of Brain Circuits Underlying Focal Epilepsy.

Because molecular mechanisms underlying refractory focal epilepsy are poorly defined, we performed transcriptome analysis on human epileptogenic tissue. Compared with controls, expression of Circadian Locomotor Output Cycles Kaput (CLOCK) is decreased in epileptogenic tissue. To define the function of CLOCK, we generated and tested the Emx-Cre; Clockflox/flox and PV-Cre; Clockflox/flox mouse lines with targeted deletions of the Clock gene in excitatory and parvalbumin (PV)-expressing inhibitory neurons, respectively. The Emx-Cre; Clockflox/flox mouse line alone has decreased seizure thresholds, but no laminar or dendritic defects in the cortex. However, excitatory neurons from the Emx-Cre; Clockflox/flox mouse have spontaneous epileptiform discharges. Both neurons from Emx-Cre; Clockflox/flox mouse and human epileptogenic tissue exhibit decreased spontaneous inhibitory postsynaptic currents. Finally, video-EEG of Emx-Cre; Clockflox/flox mice reveals epileptiform discharges during sleep and also seizures arising from sleep. Altogether, these data show that disruption of CLOCK alters cortical circuits and may lead to generation of focal epilepsy.

Embryonic transcription factor expression in mice predicts medial amygdala neuronal identity and sex-specific responses to innate behavioral cues.

The medial subnucleus of the amygdala (MeA) plays a central role in processing sensory cues required for innate behaviors. However, whether there is a link between developmental programs and the emergence of inborn behaviors remains unknown. Our previous studies revealed that the telencephalic preoptic area (POA) embryonic niche is a novel source of MeA destined progenitors. Here, we show that the POA is comprised of distinct progenitor pools complementarily marked by the transcription factors Dbx1 and Foxp2. As determined by molecular and electrophysiological criteria this embryonic parcellation predicts postnatal MeA inhibitory neuronal subtype identity. We further find that Dbx1-derived and Foxp2+ cells in the MeA are differentially activated in response to innate behavioral cues in a sex-specific manner. Thus, developmental transcription factor expression is predictive of MeA neuronal identity and sex-specific neuronal responses, providing a potential developmental logic for how innate behaviors could be processed by different MeA neuronal subtypes.

Molecular and behavioral profiling of Dbx1-derived neurons in the arcuate, lateral and ventromedial hypothalamic nuclei.

BACKGROUND: Neurons in the hypothalamus function to regulate the state of the animal during both learned and innate behaviors, and alterations in hypothalamic development may contribute to pathological conditions such as anxiety, depression or obesity. Despite many studies of hypothalamic development and function, the link between embryonic development and innate behaviors remains unexplored. Here, focusing on the embryonically expressed homeodomain-containing gene Developing Brain Homeobox 1 (Dbx1), we explored the relationship between embryonic lineage, post-natal neuronal identity and lineage-specific responses to innate cues. We found that Dbx1 is widely expressed across multiple developing hypothalamic subdomains. Using standard and inducible fate-mapping to trace the Dbx1-derived neurons, we identified their contribution to specific neuronal subtypes across hypothalamic nuclei and further mapped their activation patterns in response to a series of well-defined innate behaviors. RESULTS: Dbx1-derived neurons occupy multiple postnatal hypothalamic nuclei including the lateral hypothalamus (LH), arcuate nucleus (Arc) and the ventral medial hypothalamus (VMH). Within these nuclei, Dbx1 (+) progenitors generate a large proportion of the Pmch-, Nesfatin-, Cart-, Hcrt-, Agrp- and ERα-expressing neuronal populations, and to a lesser extent the Pomc-, TH- and Aromatase-expressing populations. Inducible fate-mapping reveals distinct temporal windows for development of the Dbx1-derived LH and Arc populations, with Agrp(+) and Cart(+) populations in the Arc arising early (E7.5-E9.5), while Pmch(+) and Hcrt(+) populations in the LH derived from progenitors expressing Dbx1 later (E9.5-E11.5). Moreover, as revealed by c-Fos labeling, Dbx1-derived cells in male and female LH, Arc and VMH are responsive during mating and aggression. In contrast, Dbx1-lineage cells in the Arc and LH have a broader behavioral tuning, which includes responding to fasting and predator odor cues. CONCLUSION: We define a novel fate map of the hypothalamus with respect to Dbx1 expression in hypothalamic progenitor zones. We demonstrate that in a temporally regulated manner, Dbx1-derived neurons contribute to molecularly distinct neuronal populations in the LH, Arc and VMH that have been implicated in a variety of hypothalamic-driven behaviors. Consistent with this, Dbx1-derived neurons in the LH, Arc and VMH are activated during stress and other innate behavioral responses, implicating their involvement in these diverse behaviors.

Rescue of deficient amygdala tonic γ-aminobutyric acidergic currents in the Fmr-/y mouse model of fragile X syndrome by a novel γ-aminobutyric acid type A receptor-positive allosteric modulator.

Alterations in the ratio of excitatory to inhibitory transmission are emerging as a common component of many nervous system disorders, including autism spectrum disorders (ASDs). Tonic γ-aminobutyric acidergic (GABAergic) transmission provided by peri- and extrasynaptic GABA type A (GABAA ) receptors powerfully controls neuronal excitability and plasticity and, therefore, provides a rational therapeutic target for normalizing hyperexcitable networks across a variety of disorders, including ASDs. Our previous studies revealed tonic GABAergic deficits in principal excitatory neurons in the basolateral amygdala (BLA) in the Fmr1(-/y) knockout (KO) mouse model fragile X syndrome. To correct amygdala deficits in tonic GABAergic neurotransmission in Fmr1(-/y) KO mice, we developed a novel positive allosteric modulator of GABAA receptors, SGE-872, based on endogenously active neurosteroids. This study shows that SGE-872 is nearly as potent and twice as efficacious for positively modulating GABAA receptors as its parent molecule, allopregnanolone. Furthermore, at submicromolar concentrations (≤1 µM), SGE-872 is selective for tonic, extrasynaptic α4ß3δ-containing GABAA receptors over typical synaptic α1ß2γ2 receptors. We further find that SGE-872 strikingly rescues the tonic GABAergic transmission deficit in principal excitatory neurons in the Fmr1(-/y) KO BLA, a structure heavily implicated in the neuropathology of ASDs. Therefore, the potent and selective action of SGE-872 on tonic GABAA receptors containing α4 subunits may represent a novel and highly useful therapeutic avenue for ASDs and related disorders involving hyperexcitability of neuronal networks.

Specification of select hypothalamic circuits and innate behaviors by the embryonic patterning gene dbx1.

The hypothalamus integrates information required for the production of a variety of innate behaviors such as feeding, mating, aggression, and predator avoidance. Despite an extensive knowledge of hypothalamic function, how embryonic genetic programs specify circuits that regulate these behaviors remains unknown. Here, we find that in the hypothalamus the developmentally regulated homeodomain-containing transcription factor Dbx1 is required for the generation of specific subclasses of neurons within the lateral hypothalamic area/zona incerta (LH) and the arcuate (Arc) nucleus. Consistent with this specific developmental role, Dbx1 hypothalamic-specific conditional-knockout mice display attenuated responses to predator odor and feeding stressors but do not display deficits in other innate behaviors such as mating or conspecific aggression. Thus, activity of a single developmentally regulated gene, Dbx1, is a shared requirement for the specification of hypothalamic nuclei governing a subset of innate behaviors. VIDEO ABSTRACT.

Neonatal NMDA receptor blockade disrupts spike timing and glutamatergic synapses in fast spiking interneurons in a NMDA receptor hypofunction model of schizophrenia.

The dysfunction of parvalbumin-positive, fast-spiking interneurons (FSI) is considered a primary contributor to the pathophysiology of schizophrenia (SZ), but deficits in FSI physiology have not been explicitly characterized. We show for the first time, that a widely-employed model of schizophrenia minimizes first spike latency and increases GluN2B-mediated current in neocortical FSIs. The reduction in FSI first-spike latency coincides with reduced expression of the Kv1.1 potassium channel subunit which provides a biophysical explanation for the abnormal spiking behavior. Similarly, the increase in NMDA current coincides with enhanced expression of the GluN2B NMDA receptor subunit, specifically in FSIs. In this study mice were treated with the NMDA receptor antagonist, MK-801, during the first week of life. During adolescence, we detected reduced spike latency and increased GluN2B-mediated NMDA current in FSIs, which suggests transient disruption of NMDA signaling during neonatal development exerts lasting changes in the cellular and synaptic physiology of neocortical FSIs. Overall, we propose these physiological disturbances represent a general impairment to the physiological maturation of FSIs which may contribute to schizophrenia-like behaviors produced by this model.

Deficient tonic GABAergic conductance and synaptic balance in the fragile X syndrome amygdala.

Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability. Comorbidities of FXS such as autism are increasingly linked to imbalances in excitation and inhibition (E/I) as well as dysfunction in GABAergic transmission in a number of brain regions including the amygdala. However, the link between E/I imbalance and GABAergic transmission deficits in the FXS amygdala is poorly understood. Here we reveal that normal tonic GABAA receptor-mediated neurotransmission in principal neurons (PNs) of the basolateral amygdala (BLA) is comprised of both δ- and α5-subunit-containing GABAA receptors. Furthermore, tonic GABAergic capacity is reduced in these neurons in the Fmr1 knockout (KO) mouse model of FXS (1.5-fold total, 3-fold δ-subunit, and 2-fold α5-subunit mediated) as indicated by application of gabazine (50 µM), 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP, 1 µM), and α5ia (1.5 µM) in whole cell patch-clamp recordings. Moreover, α5-containing tonic GABAA receptors appear to preferentially modulate nonsomatic compartments of BLA PNs. Examination of evoked feedforward synaptic transmission in these cells surprisingly revealed no differences in overall synaptic conductance or E/I balance between wild-type (WT) and Fmr1 KO mice. Instead, we observed altered feedforward kinetics in Fmr1 KO PNs that supports a subtle yet significant decrease in E/I balance at the peak of excitatory conductance. Blockade of α5-subunit-containing GABAA receptors replicated this condition in WT PNs. Therefore, our data suggest that tonic GABAA receptor-mediated neurotransmission can modulate synaptic E/I balance and timing established by feedforward inhibition and thus may represent a therapeutic target to enhance amygdala function in FXS.

Developmental changes in expression of inhibitory neuronal proteins in the Fragile X Syndrome mouse basolateral amygdala.

In humans, Fragile X Syndrome (FXS) is characterized by enhanced fear, hyperactivity, social anxiety, and, in a subset of individuals, autism. Many of the emotional and social deficits point to defects in the amygdala. We have previously shown defects in inhibitory neuron drive onto excitatory projection neurons in the basolateral amygdala (BLA) of juvenile Fmr1(-/y) knockout (KO) mice. Using pharmacological approaches, we have also previously revealed dynamic functional deficits in α1, α2, and α3 subunit-containing GABAA receptors (GABAARs α1, α2, and α3) during early postnatal development. In this study, we sought to determine whether these defects in GABAAR function are accompanied by changes in protein expression of GABAARs α1, α2, and α3 and the post-synaptic GABAAR-clustering protein gephyrin. Interestingly, we found that while the expression of these proteins did not significantly differ between wildtype (WT) and KO mice at each time point, the timing of developmental expression of GABAAR α1, α2, and gephyrin was altered. Collectively, these data reveal novel defects in inhibitory synapse protein expression during critical periods of early postnatal development that could contribute to observed inhibitory neurotransmission deficits in the KO mouse BLA.