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.

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.

Homeostatic responses fail to correct defective amygdala inhibitory circuit maturation in fragile X syndrome.

Fragile X syndrome (FXS) is a debilitating neurodevelopmental disorder thought to arise from disrupted synaptic communication in several key brain regions, including the amygdala, a central processing center for information with emotional and social relevance. Recent studies reveal defects in both excitatory and inhibitory neurotransmission in mature amygdala circuits in Fmr1(-/y) mutants, the animal model of FXS. However, whether these defects are the result of altered synaptic development or simply faulty mature circuits remains unknown. Using a combination of electrophysiological and genetic approaches, we show the development of both presynaptic and postsynaptic components of inhibitory neurotransmission in the FXS amygdala is dynamically altered during critical stages of neural circuit formation. Surprisingly, we observe that there is a homeostatic correction of defective inhibition, which, despite transiently restoring inhibitory synaptic efficacy to levels at or beyond those of control, ultimately fails to be maintained. Using inhibitory interneuron-specific conditional knock-out and rescue mice, we further reveal that fragile X mental retardation protein function in amygdala inhibitory microcircuits can be segregated into distinct presynaptic and postsynaptic components. Collectively, these studies reveal a previously unrecognized complexity of disrupted neuronal development in FXS and therefore have direct implications for establishing novel temporal and region-specific targeted therapies to ameliorate core amygdala-based behavioral symptoms.

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.

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.

Neuronal Subtypes and Connectivity of the Adult Mouse Paralaminar Amygdala.

The paralaminar nucleus of the amygdala (PL) comprises neurons that exhibit delayed maturation. PL neurons are born during gestation but mature during adolescent ages, differentiating into excitatory neurons. These late-maturing PL neurons contribute to the increase in size and cell number of the amygdala between birth and adulthood. However, the function of the PL upon maturation 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 mouse PL; the intrinsic morpho-electric properties of its neurons, and its input and output circuit 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 these inputs and to diverse targets including the amygdala, frontal cortex, hippocampus, hypothalamus, and brainstem. Thus, the adult mouse PL is centrally placed to play a major role in the integration of olfactory sensory information, to coordinate affective and autonomic behavioral responses to salient odor stimuli.

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.

The iron exporter ferroportin 1 is essential for development of the mouse embryo, forebrain patterning and neural tube closure.

Neural tube defects (NTDs) are some of the most common birth defects observed in humans. The incidence of NTDs can be reduced by peri-conceptional folic acid supplementation alone and reduced even further by supplementation with folic acid plus a multivitamin. Here, we present evidence that iron maybe an important nutrient necessary for normal development of the neural tube. Following implantation of the mouse embryo, ferroportin 1 (Fpn1) is essential for the transport of iron from the mother to the fetus and is expressed in the visceral endoderm, yolk sac and placenta. The flatiron (ffe) mutant mouse line harbors a hypomorphic mutation in Fpn1 and we have created an allelic series of Fpn1 mutations that result in graded developmental defects. A null mutation in the Fpn1 gene is embryonic lethal before gastrulation, hypomorphic Fpn1(ffe/ffe) mutants exhibit NTDs consisting of exencephaly, spina bifida and forebrain truncations, while Fpn1(ffe/KI) mutants exhibit even more severe NTDs. We show that Fpn1 is not required in the embryo proper but rather in the extra-embryonic visceral endoderm. Our data indicate that loss of Fpn1 results in abnormal morphogenesis of the anterior visceral endoderm (AVE). Defects in the development of the forebrain in Fpn1 mutants are compounded by defects in multiple signaling centers required for maintenance of the forebrain, including the anterior definitive endoderm (ADE), anterior mesendoderm (AME) and anterior neural ridge (ANR). Finally, we demonstrate that this loss of forebrain maintenance is due in part to the iron deficiency that results from the absence of fully functional Fpn1.

Programmed cell death is impaired in the developing brain of FMR1 mutants.

Fragile X syndrome (FXS), due to transcriptional silencing of fragile X mental retardation protein (FMRP), is characterized by excess synaptic connections and impaired dendrite maturation. Programmed cell death (PCD) is critical for synaptogenesis and elimination of aberrant neuronal connections in the developing brain; however, the role of FMRP in PCD is unknown. The aim of this work was to assess the intrinsic apoptosis pathway in the developing brain of Fmr1 mutants. To accomplish this, we evaluated two different Fmr1 mutant strains of 10-day-old male mice compared with appropriate controls. We performed immunohistochemistry for activated caspase-3 and TUNEL assays, quantified the number of neurons in neocortex and hippocampus, determined cytochrome c peroxidase activity, measured the amount of cytochrome c release from forebrain mitochondria, and assessed levels of key pro- and antiapoptotic mediators with immunoblot analysis. Both Fmr1 mutant strains demonstrated decreased apoptosis in neocortex, hippocampus, and basolateral amygdala, impaired cytochrome c and procaspase-9 release from mitochondria despite intact Bax translocation, increased expression of the antiapoptotic protein, BCL-xL, and increased number of neurons. Taken together, the data suggest that PCD is impaired due to increased BCL-xL expression and is associated with excess neurons in the developing brain of FMRP-deficient mice. It is possible that deficient PCD prevents neuron elimination and results in abnormal retention of developmentally transient neurons. Thus, defective PCD may contribute to the excess synaptic connections known to exist in Fmr1 mutants and could play a role in the behavioral phenotype of children with FXS.

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.

Pax6 is required at the telencephalic pallial-subpallial boundary for the generation of neuronal diversity in the postnatal limbic system.

During embryogenesis, the pallial-subpallial boundary (PSB) divides the two main progenitor domains in the telencephalon: the pallium, the major source of excitatory neurons, and the subpallium, the major source of inhibitory neurons. The PSB is formed at the molecular interface between the pallial (high Pax6+) and subpallial (high Gsx2+) ventricular zone (VZ) compartments. Initially, the PSB contains cells that express both Pax6 and Gsx2, but during later stages of development this boundary is largely refined into two separate compartments. In this study we examined the developmental mechanisms underlying PSB boundary formation and the postnatal consequences of conditional loss of Pax6 function at the PSB on neuronal fate in the amygdala and olfactory bulb, two targets of PSB-derived migratory populations. Our cell fate and time-lapse imaging analyses reveal that the sorting of Pax6+ and Gsx2+ progenitors during embryogenesis is the result of a combination of changes in gene expression and cell movements. Interestingly, we find that in addition to giving rise to inhibitory neurons in the amygdala and olfactory bulb, Gsx2+ progenitors generate a subpopulation of amygdala excitatory neurons. Consistent with this finding, targeted conditional ablation of Pax6 in Gsx2+ progenitors results in discrete local embryonic patterning defects that are linked to changes in the generation of subsets of postnatal excitatory and inhibitory neurons in the amygdala and inhibitory neurons in the olfactory bulb. Thus, in PSB progenitors, Pax6 plays an important role in the generation of multiple subtypes of neurons that contribute to the amygdala and olfactory bulb.

Defective GABAergic neurotransmission and pharmacological rescue of neuronal hyperexcitability in the amygdala in a mouse model of fragile X syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by variable cognitive impairment and behavioral disturbances such as exaggerated fear, anxiety and gaze avoidance. Consistent with this, findings from human brain imaging studies suggest dysfunction of the amygdala. Underlying alterations in amygdala synaptic function in the Fmr1 knock-out (KO) mouse model of FXS, however, remain largely unexplored. Utilizing a combination of approaches, we uncover profound alterations in inhibitory neurotransmission in the amygdala of Fmr1 KO mice. We demonstrate a dramatic reduction in the frequency and amplitude of phasic IPSCs, tonic inhibitory currents, as well as in the number of inhibitory synapses in Fmr1 KO mice. Furthermore, we observe significant alterations in GABA availability, both intracellularly and at the synaptic cleft. Together, these findings identify abnormalities in basal and action potential-dependent inhibitory neurotransmission. Additionally, we reveal a significant neuronal hyperexcitability in principal neurons of the amygdala in Fmr1 KO mice, which is strikingly rescued by pharmacological augmentation of tonic inhibitory tone using the GABA agonist gaboxadol (THIP). Thus, our study reveals relevant inhibitory synaptic abnormalities in the amygdala in the Fmr1 KO brain and supports the notion that pharmacological approaches targeting the GABAergic system may be a viable therapeutic approach toward correcting amygdala-based symptoms in FXS.

Developmental origin of preBötzinger complex respiratory neurons.

A subset of preBötzinger Complex (preBötC) neurokinin 1 receptor (NK1R) and somatostatin peptide (SST)-expressing neurons are necessary for breathing in adult rats, in vivo. Their developmental origins and relationship to other preBötC glutamatergic neurons are unknown. Here we show, in mice, that the "core" of preBötC SST(+)/NK1R(+)/SST 2a receptor(+) (SST2aR) neurons, are derived from Dbx1-expressing progenitors. We also show that Dbx1-derived neurons heterogeneously coexpress NK1R and SST2aR within and beyond the borders of preBötC. More striking, we find that nearly all non-catecholaminergic glutamatergic neurons of the ventrolateral medulla (VLM) are also Dbx1 derived. PreBötC SST(+) neurons are born between E9.5 and E11.5 in the same proportion as non-SST-expressing neurons. Additionally, preBötC Dbx1 neurons are respiratory modulated and show an early inspiratory phase of firing in rhythmically active slice preparations. Loss of Dbx1 eliminates all glutamatergic neurons from the respiratory VLM including preBötC NK1R(+)/SST(+) neurons. Dbx1 mutant mice do not express any spontaneous respiratory behaviors in vivo. Moreover, they do not generate rhythmic inspiratory activity in isolated en bloc preparations even after acidic or serotonergic stimulation. These data indicate that preBötC core neurons represent a subset of a larger, more heterogeneous population of VLM Dbx1-derived neurons. These data indicate that Dbx1-derived neurons are essential for the expression and, we hypothesize, are responsible for the generation of respiratory behavior both in vitro and in vivo.

Impaired inhibitory control of cortical synchronization in fragile X syndrome.

Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by severe cognitive impairments, sensory hypersensitivity, and comorbidities with autism and epilepsy. Fmr1 knockout (KO) mouse models of FXS exhibit alterations in excitatory and inhibitory neurotransmission, but it is largely unknown how aberrant function of specific neuronal subtypes contributes to these deficits. In this study we show specific inhibitory circuit dysfunction in layer II/III of somatosensory cortex of Fmr1 KO mice. We demonstrate reduced activation of somatostatin-expressing low-threshold-spiking (LTS) interneurons in response to the group I metabotropic glutamate receptor (mGluR) agonist 3,5-dihydroxyphenylglycine (DHPG) in Fmr1 KO mice, resulting in impaired synaptic inhibition. Paired recordings from pyramidal neurons revealed reductions in synchronized synaptic inhibition and coordinated spike synchrony in response to DHPG, indicating a weakened LTS interneuron network in Fmr1 KO mice. Together, these findings reveal a functional defect in a single subtype of cortical interneuron in Fmr1 KO mice. This defect is linked to altered activity of the cortical network in line with the FXS phenotype.

Amygdala regulation of fear and emotionality in fragile X syndrome.

Fear is a universal response to a threat to one's body or social status. Disruption in the detection and response of the brain's fear system is commonly observed in a variety of neurodevelopmental disorders, including fragile X syndrome (FXS), a brain disorder characterized by variable cognitive impairment and behavioral disturbances such as social avoidance and anxiety. The amygdala is highly involved in mediating fear processing, and increasing evidence supports the idea that inhibitory circuits play a key role in regulating the flow of information associated with fear conditioning in the amygdala. Here, we review the known and potential importance of amygdala fear circuits in FXS, and how developmental studies are critical to understand the formation and function of neuronal circuits that modulate amygdala-based behaviors.

The GABA(A) receptor agonist THIP ameliorates specific behavioral deficits in the mouse model of fragile X syndrome.

Hyperactivity, hypersensitivity to auditory stimuli, and exaggerated fear are common behavioral abnormalities observed in individuals with fragile X syndrome (FXS), a neurodevelopmental disorder that is the most common genetic cause of autism. Evidence from studies of the Fmr1 knockout (KO) mouse model of FXS supports the notion that impaired GABAergic transmission in different brain regions such as the amygdala, striatum or cerebral cortex is central to FXS behavioral abnormalities. This suggests that the GABAergic system might be an intriguing target to ameliorate some of the phenotypes in FXS. Our recent work revealed that THIP (gaboxadol), a GABA(A) receptor agonist, can restore principal neuron excitability deficits in the Fmr1 KO amygdala, suggesting that THIP may also restore some of the key behavioral abnormalities in Fmr1 KO mice. Here, we reveal that THIP significantly attenuated hyperactivity in Fmr1 KO mice, and reduced prepulse inhibition in a volume-dependent manner. In contrast, THIP did not reverse the deficits in cued fear or startle response. Thus, this study shows that enhancing GABAergic transmission can correct specific behavioral phenotypes of the Fmr1 KO mouse further supporting that targeting the GABAergic system, and specifically tonic inhibition, might be important for correcting or ameliorating some key behaviors in FXS.

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.

Emx1-lineage progenitors differentially contribute to neural diversity in the striatum and amygdala.

In the developing mammalian basal telencephalon, neural progenitors from the subpallium generate the majority of inhibitory medium spiny neurons (MSNs) in the striatum, while both pallial- and subpallial-derived progenitors contribute to excitatory and inhibitory neuronal diversity in the amygdala. Using a combination of approaches, including genetic fate mapping, cell birth dating, cell migration assays, and electrophysiology, we find that cells derived from the Emx1 lineage contribute to two distinct neuronal populations in the mature basal forebrain: inhibitory MSNs in the striatum and functionally distinct subclasses of excitatory neurons in the amygdala. Our cell birth-dating studies reveal that these two populations are born at different times during early neurogenesis, with the amygdala population born before the MSNs. In the striatum, Emx1-lineage neurons represent a unique subpopulation of MSNs: they are disproportionately localized to the dorsal striatum, are found in dopamine receiving, reelin-positive patches, and are born throughout striatal neurogenesis. In addition, our data suggest that a subpopulation of these Emx1-lineage cells originate in the pallium and subsequently migrate to the developing striatum and amygdala. Our intersectional fate-mapping analysis further reveals that Emx1-lineage cells that coexpress Dlx exclusively generate MSNs but do not contribute to the excitatory neurons in the amygdala. Thus, both the timing of neurogenesis and differential combinatorial gene expression appear to be key determinants of striatal versus amygdala fate decisions of Emx1-lineage cells.