Excess neonatal testosterone causes male-specific social and fear memory deficits in wild-type mice.

Neurodevelopmental disorders (ND) disproportionately affect males compared to females, and Autism Spectrum Disorder (ASD) in particular exhibits a 4:1 male bias. The biological mechanisms of this female protection or male susceptibility have not been identified. There is some evidence to suggest that fetal/neonatal gonadal hormones, which play pivotal roles in many aspects of development, may contribute. Here, we investigate the role of testosterone administration during a critical period of development, and its effects on social approach and fear learning in C57BL/6J wildtype mice. Male, but not female mice treated with testosterone on the day of birth (PN0) exhibited deficits in both social behavior and contextual fear conditioning, whereas mice treated with the same dose of testosterone on postnatal day 18 (PN18) did not display such impairments. Testosterone administration did not induce anxiogenic effects or lead to changes in weight compared to the testosterone-treated group. These impairments are relevant to ND and may help identify novel treatment targets.

The γ-Protocadherins Interact Physically and Functionally with Neuroligin-2 to Negatively Regulate Inhibitory Synapse Density and Are Required for Normal Social Interaction.

Cell adhesion molecules (CAMs) are key players in the formation of neural circuits during development. The γ-protocadherins (γ-Pcdhs), a family of 22 CAMs encoded by the Pcdhg gene cluster, are known to play important roles in dendrite arborization, axon targeting, and synapse development. We showed previously that multiple γ-Pcdhs interact physically with the autism-associated CAM neuroligin-1, and inhibit the latter's ability to promote excitatory synapse maturation. Here, we show that γ-Pcdhs can also interact physically with the related neuroligin-2, and inhibit this CAM's ability to promote inhibitory synapse development. In an artificial synapse assay, γ-Pcdhs co-expressed with neuroligin-2 in non-neuronal cells reduce inhibitory presynaptic maturation in contacting hippocampal axons. Mice lacking the γ-Pcdhs from the forebrain (including the cortex, the hippocampus, and portions of the amygdala) exhibit increased inhibitory synapse density and increased co-localization of neuroligin-2 with inhibitory postsynaptic markers in vivo. These Pcdhg mutants also exhibit defective social affiliation and an anxiety-like phenotype in behavioral assays. Together, these results suggest that γ-Pcdhs negatively regulate neuroligins to limit synapse density in a manner that is important for normal behavior.

Age- and sex-specific fear conditioning deficits in mice lacking Pcdh10, an Autism Associated Gene.

PCDH10 is a gene associated with Autism Spectrum Disorder. It is involved in the growth of thalamocortical projections and dendritic spine elimination. Previously, we characterized Pcdh10 haploinsufficient mice (Pcdh10+/- mice) and found male-specific social deficits and dark phase hypoactivity. Pcdh10+/- males exhibit increased dendritic spine density of immature morphology, decreased NMDAR expression, and decreased gamma synchronization in the basolateral amygdala (BLA). Here, we further characterize Pcdh10+/- mice by testing for fear memory, which relies on BLA function. We used both male and female Pcdh10+/- mice and their wild-type littermates at two ages, juvenile and adult, and in two learning paradigms, cued and contextual fear conditioning. We found that males at both ages and in both assays exhibited fear conditioning deficits, but females were only impaired as adults in the cued condition. These data are further evidence for male-specific alterations in BLA-related behaviors in Pcdh10+/- mice and suggest that these mice may be a useful model for dissecting male specific brain and behavioral phenotypes relevant to social and emotional behaviors.

Comprehensive Behavioral Phenotyping of a 16p11.2 Del Mouse Model for Neurodevelopmental Disorders.

The microdeletion of copy number variant 16p11.2 is one of the most common genetic mutations associated with neurodevelopmental disorders, such as Autism Spectrum Disorders (ASDs). Here, we describe our comprehensive behavioral phenotyping of the 16p11.2 deletion line developed by Alea Mills on a C57BL/6J and 129S1/SvImJ F1 background (Delm ). Male and female Delm mice were tested in developmental milestones as preweanlings (PND2-PND12), and were tested in open field activity, elevated zero maze, rotarod, novel object recognition, fear conditioning, social approach, and other measures during post-weaning (PND21), adolescence (PND42), and adulthood (>PND70). Developmentally, Delm mice show distinct weight reduction that persists into adulthood. Delm males also have reduced grasp reflexes and limb strength during development, but no other reflexive deficits whereas Delm females show limb strength deficits and decreased sensitivity to heat. In a modified version of a rotarod task that measures balance and coordinated motor activity, Delm males, but not females, show improved performance at high speeds. Delm males and females also show age-specific reductions in anxiety-like behavior compared with WTs, but neither sex show deficits in a social preference task. When assessing learning and memory, Delm males and females show age-specific impairments in a novel object or spatial object recognition, but no deficits in contextual fear memory. This work extends the understanding of the behavioral phenotypes seen with 16p11.2 deletion by emphasizing age and sex-specific deficits; important variables to consider when studying mouse models for neurodevelopmental disorders. LAY SUMMARY: Autism spectrum disorder is a common neurodevelopmental disorder that causes repetitive behavior and impairments in social interaction and communication. Here, we assess the effects of one of the most common genetic alterations in ASDs, a deletion of one copy of 29 genes, using a mouse model. These animals show differences in behavior between males and females and across ages compared with control animals, including changes in development, cognition, and motor coordination. Autism Res 2020, 13: 1670-1684. © 2020 International Society for Autism Research and Wiley Periodicals LLC.

Nolz1 expression is required in dopaminergic axon guidance and striatal innervation.

Midbrain dopaminergic (DA) axons make long longitudinal projections towards the striatum. Despite the importance of DA striatal innervation, processes involved in establishment of DA axonal connectivity remain largely unknown. Here we demonstrate a striatal-specific requirement of transcriptional regulator Nolz1 in establishing DA circuitry formation. DA projections are misguided and fail to innervate the striatum in both constitutive and striatal-specific Nolz1 mutant embryos. The lack of striatal Nolz1 expression results in nigral to pallidal lineage conversion of striatal projection neuron subtypes. This lineage switch alters the composition of secreted factors influencing DA axonal tract formation and renders the striatum non-permissive for dopaminergic and other forebrain tracts. Furthermore, transcriptomic analysis of wild-type and Nolz1-/- mutant striatal tissue led to the identification of several secreted factors that underlie the observed guidance defects and proteins that promote DA axonal outgrowth. Together, our data demonstrate the involvement of the striatum in orchestrating dopaminergic circuitry formation.

The Role of Synaptic Cell Adhesion Molecules and Associated Scaffolding Proteins in Social Affiliative Behaviors.

Social affiliative behaviors-engagement in positive (i.e., nonaggressive) social approach and reciprocal social interactions with a conspecific-comprise a construct within the National Institute of Mental Health Research Domain Criteria Social Processes Domain. These behaviors are disrupted in multiple human neurodevelopmental and neuropsychiatric disorders, such as autism, schizophrenia, social phobia, and others. Human genetic studies have strongly implicated synaptic cell adhesion molecules (sCAMs) in several such disorders that involve marked reductions, or other dysregulations, of social affiliative behaviors. Here, we review the literature on the role of sCAMs in social affiliative behaviors. We integrate findings pertaining to synapse structure and morphology, neurotransmission, postsynaptic signaling pathways, and neural circuitry to propose a multilevel model that addresses the impact of a diverse group of sCAMs, including neurexins, neuroligins, protocadherins, immunoglobulin superfamily proteins, and leucine-rich repeat proteins, as well as their associated scaffolding proteins, including SHANKs and others, on social affiliative behaviors. This review finds that the disruption of sCAMs often manifests in changes in social affiliative behaviors, likely through alterations in synaptic maturity, pruning, and specificity, leading to excitation/inhibition imbalance in several key regions, namely the medial prefrontal cortex, basolateral amygdala, hippocampus, anterior cingulate cortex, and ventral tegmental area. Unraveling the complex network of interacting sCAMs in glutamatergic synapses will be an important strategy for elucidating the mechanisms of social affiliative behaviors and the alteration of these behaviors in many neuropsychiatric and neurodevelopmental disorders.

Sociability development in mice with cell-specific deletion of the NMDA receptor NR1 subunit gene.

Social affiliative behavior is an important component of everyday life in many species and is likely to be disrupted in disabling ways in various neurodevelopmental and neuropsychiatric disorders. Therefore, determining the mechanisms involved in these processes is crucial. A link between N-methyl-d-aspartate (NMDA) receptor function and social behaviors has been clearly established. The cell types in which NMDA receptors are critical for social affiliative behavior, however, remain unclear. Here, we use mice carrying a conditional allele of the NMDA R1 subunit to address this question. Mice bearing a floxed NMDAR1 (NR1) allele were crossed with transgenic calcium/calmodulin-dependent kinase IIα (CaMKIIα)-Cre mice or parvalbumin (PV)-Cre mice targeting postnatal excitatory forebrain or PV-expressing interneurons, respectively, and assessed using the three-chambered Social Approach Test. We found that deletion of NR1 in PV-positive interneurons had no effect on social sniffing, but deletion of NR1 in glutamatergic pyramidal cells resulted in a significant increase in social approach behavior, regardless of age or sex. Therefore, forebrain excitatory neurons expressing NR1 play an important role in regulating social affiliative behavior.

Home-cage hypoactivity in mouse genetic models of autism spectrum disorder.

Genome-wide association and whole exome sequencing studies from Autism Spectrum Disorder (ASD) patient populations have implicated numerous risk factor genes whose mutation or deletion results in significantly increased incidence of ASD. Behavioral studies of monogenic mutant mouse models of ASD-associated genes have been useful for identifying aberrant neural circuitry. However, behavioral results often differ from lab to lab, and studies incorporating both males and females are often not performed despite the significant sex-bias of ASD. In this study, we sought to investigate the simple, passive behavior of home-cage activity monitoring across multiple 24-h days in four different monogenic mouse models of ASD: Shank3b-/-, Cntnap2-/-, Pcdh10+/-, and Fmr1 knockout mice. Relative to sex-matched wildtype (WT) littermates, we discovered significant home-cage hypoactivity, particularly in the dark (active) phase of the light/dark cycle, in male mice of all four ASD-associated transgenic models. For Cntnap2-/- and Pcdh10+/- mice, these activity alterations were sex-specific, as female mice did not exhibit home-cage activity differences relative to sex-matched WT controls. These home-cage hypoactivity alterations differ from activity findings previously reported using short-term activity measurements in a novel open field. Despite circadian problems reported in human ASD patients, none of the mouse models studied had alterations in free-running circadian period. Together, these findings highlight a shared phenotype across several monogenic mouse models of ASD, outline the importance of methodology on behavioral interpretation, and in some genetic lines parallel the male-enhanced phenotypic presentation observed in human ASDs.

Sex Differences in Autism Spectrum Disorder: a Review.

PURPOSE OF REVIEW: Neurodevelopmental disorders disproportionately affect males. The mechanisms underlying male vulnerability or female protection are not known and remain understudied. Determining the processes involved is crucial to understanding the etiology and advancing treatment of neurodevelopmental disorders. Here, we review current findings and theories that contribute to male preponderance of neurodevelopmental disorders, with a focus on autism. RECENT FINDINGS: Recent work on the biological basis of the male preponderance of autism and other neurodevelopmental disorders includes discussion of a higher genetic burden in females and sex-specific gene mutations or epigenetic changes that differentially confer risk to males or protection to females. Other mechanisms discussed are sex chromosome and sex hormone involvement. Specifically, fetal testosterone is involved in many aspects of development and may interact with neurotransmitter, neuropeptide, or immune pathways to contribute to male vulnerability. Finally, the possibilities of female underdiagnosis and a multi-hit hypothesis are discussed. This review highlights current theories of male bias in developmental disorders. Topics include environmental, genetic, and epigenetic mechanisms; theories of sex chromosomes, hormones, neuroendocrine, and immune function; underdiagnosis of females; and a multi-hit hypothesis.

Sociability Deficits and Altered Amygdala Circuits in Mice Lacking Pcdh10, an Autism Associated Gene.

BACKGROUND: Behavioral symptoms in individuals with autism spectrum disorder (ASD) have been attributed to abnormal neuronal connectivity, but the molecular bases of these behavioral and brain phenotypes are largely unknown. Human genetic studies have implicated PCDH10, a member of the δ2 subfamily of nonclustered protocadherin genes, in ASD. PCDH10 expression is enriched in the basolateral amygdala, a brain region implicated in the social deficits of ASD. Previous reports indicate that Pcdh10 plays a role in axon outgrowth and glutamatergic synapse elimination, but its roles in social behaviors and amygdala neuronal connectivity are unknown. We hypothesized that haploinsufficiency of Pcdh10 would reduce social approach behavior and alter the structure and function of amygdala circuits. METHODS: Mice lacking one copy of Pcdh10 (Pcdh10+/-) and wild-type littermates were assessed for social approach and other behaviors. The lateral/basolateral amygdala was assessed for dendritic spine number and morphology, and amygdala circuit function was studied using voltage-sensitive dye imaging. Expression of Pcdh10 and N-methyl-D-aspartate receptor (NMDAR) subunits was assessed in postsynaptic density fractions of the amygdala. RESULTS: Male Pcdh10+/- mice have reduced social approach behavior, as well as impaired gamma synchronization, abnormal spine morphology, and reduced levels of NMDAR subunits in the amygdala. Social approach deficits in Pcdh10+/- male mice were rescued with acute treatment with the NMDAR partial agonist d-cycloserine. CONCLUSIONS: Our studies reveal that male Pcdh10+/- mice have synaptic and behavioral deficits, and establish Pcdh10+/- mice as a novel genetic model for investigating neural circuitry and behavioral changes relevant to ASD.

Sleep deprivation causes memory deficits by negatively impacting neuronal connectivity in hippocampal area CA1.

Brief periods of sleep loss have long-lasting consequences such as impaired memory consolidation. Structural changes in synaptic connectivity have been proposed as a substrate of memory storage. Here, we examine the impact of brief periods of sleep deprivation on dendritic structure. In mice, we find that five hours of sleep deprivation decreases dendritic spine numbers selectively in hippocampal area CA1 and increased activity of the filamentous actin severing protein cofilin. Recovery sleep normalizes these structural alterations. Suppression of cofilin function prevents spine loss, deficits in hippocampal synaptic plasticity, and impairments in long-term memory caused by sleep deprivation. The elevated cofilin activity is caused by cAMP-degrading phosphodiesterase-4A5 (PDE4A5), which hampers cAMP-PKA-LIMK signaling. Attenuating PDE4A5 function prevents changes in cAMP-PKA-LIMK-cofilin signaling and cognitive deficits associated with sleep deprivation. Our work demonstrates the necessity of an intact cAMP-PDE4-PKA-LIMK-cofilin activation-signaling pathway for sleep deprivation-induced memory disruption and reduction in hippocampal spine density.

Activation of basolateral amygdala in juvenile C57BL/6J mice during social approach behavior.

There is a strong need to better understand the neurobiology of juvenile sociability (tendency to seek social interaction), a phenotype of central relevance to autism spectrum disorders (ASD). Although numerous genetic mouse models of ASD showing reduced sociability have been reported, and certain brain regions, such as the amygdala, have been implicated in sociability, there has been little emphasis on delineating brain structures and circuits activated during social interactions in the critical juvenile period of the mouse strain that serves as the most common genetic background for these models-the highly sociable C57BL/6J (B6) strain. We measured expression of the immediate early genes Fos and Egr-1 to map activation of brain regions following the Social Approach Test (SAT) in juvenile male B6 mice. We hypothesized that juvenile B6 mice would show activation of the amygdala during social interactions. The basolateral amygdala (BLA) was activated by social exposure in highly sociable, 4-week-old B6 mice. In light of these data, and the many lines of evidence indicating alteration of amygdala circuits in human ASD, future studies are warranted to assess structural and functional alterations in the BLA, particularly at BLA synapses, in various mouse models of ASD.

Transiently increasing cAMP levels selectively in hippocampal excitatory neurons during sleep deprivation prevents memory deficits caused by sleep loss.

The hippocampus is particularly sensitive to sleep loss. Although previous work has indicated that sleep deprivation impairs hippocampal cAMP signaling, it remains to be determined whether the cognitive deficits associated with sleep deprivation are caused by attenuated cAMP signaling in the hippocampus. Further, it is unclear which cell types are responsible for the memory impairments associated with sleep deprivation. Transgenic approaches lack the spatial resolution to manipulate specific signaling pathways selectively in the hippocampus, while pharmacological strategies are limited in terms of cell-type specificity. Therefore, we used a pharmacogenetic approach based on a virus-mediated expression of a Gαs-coupled Drosophila octopamine receptor selectively in mouse hippocampal excitatory neurons in vivo. With this approach, a systemic injection with the receptor ligand octopamine leads to increased cAMP levels in this specific set of hippocampal neurons. We assessed whether transiently increasing cAMP levels during sleep deprivation prevents memory consolidation deficits associated with sleep loss in an object-location task. Five hours of total sleep deprivation directly following training impaired the formation of object-location memories. Transiently increasing cAMP levels in hippocampal neurons during the course of sleep deprivation prevented these memory consolidation deficits. These findings demonstrate that attenuated cAMP signaling in hippocampal excitatory neurons is a critical component underlying the memory deficits in hippocampus-dependent learning tasks associated with sleep deprivation.

Estradiol regulates markers of synaptic plasticity in the hypothalamic ventromedial nucleus and amygdala of female rats.

Ovarian hormones act in multiple brain regions to modulate specific behaviors and emotional states. For example, ovarian hormones promote female sexual receptivity in the hypothalamic ventromedial nucleus (VMH) and modulate anxiety in the amygdala. Hormone-induced changes within the VMH include structural modifications, such as changes in dendritic spines, dendrite length and the number of synapses. In some situations, dendrite remodeling requires actin polymerization, which depends on phospho-deactivation of the enzyme cofilin, or the ionotropic AMPA-type glutamate receptors, especially the GluA1 and GluA2 subunits. The present experiments used immunohistochemistry to test the hypothesis that ovarian hormone-induced neural plasticity in the VMH and amygdala involves the regulation of phospho-cofilin, GluA1 and GluA2. These proteins were assessed acutely after estradiol administration (0.5, 1.0 and 4.0h), as well as three days after hormone treatment. Both brain regions displayed rapid (4.0h or less) and transient estradiol-induced increases in the level of phospho-cofilin. At the behaviorally relevant time point of three days, differential changes in AMPA receptor subunits were observed. Using Golgi impregnation, the effect of estradiol on amygdala dendrites was examined. Three days after estradiol treatment, an increase in the length of dendrites in the central nucleus of the amygdala was observed. Thus, estradiol initiates structural changes in dendrites in both the VMH and amygdala associated with an early phospho-deactivation of cofilin, followed by dynamic, brain region-specific changes in AMPA receptor composition.

Dendritic arbor of neurons in the hypothalamic ventromedial nucleus in female prairie voles (Microtus ochrogaster).

Female mating behavior in rats is associated with hormone-induced changes in the dendritic arbor of neurons in the ventromedial nucleus of the hypothalamus (VMH), particularly the ventrolateral portion. Regulation of mating behavior in female prairie voles differs substantially from that in rats; therefore, we examined the dendritic morphology of VMH neurons in this species. Sexually naïve adult female prairie voles were housed with a male to activate the females' reproductive endocrine system. Following 48 h of cohabitation, females were tested for evidence of reproductive activation by assessing the level of male sexual interest, after which their brains were processed using Golgi impregnation, which allowed ventrolateral VMH neurons to be visualized and analyzed. Dendritic arborization in the female prairie vole VMH neurons was strikingly similar to that of female rats. The key difference was that in the prairie voles the long primary dendrites extended considerably further than those observed in rats. Although most female voles paired with males exhibited sexual activation, some females did not. These two groups displayed specific differences in their VMH dendrites. In particular, the long primary dendrites were longer in the reproductively active females compared with those in the non-activated females. Overall, dendrite lengths were positively correlated with plasma estradiol levels in females exposed to males, but not in unpaired females. Although causal relationships between the neuroendocrine events, dendrite length, and the outward, behavioral manifestation of reproductive activation cannot be determined from this study, these results suggest an association between ventrolateral VMH dendrite morphology and female mating behavior in prairie voles, akin to what has been observed in female rats.

Oxytocin and dendrite remodeling in the hypothalamus.

For most people, their quality of life depends on their successful interdependence with others, which requires sophisticated social cognition, communication, and emotional bonds. Across the lifespan, new bonds must be forged and maintained, and conspecific menaces must be managed. The dynamic nature of the human social landscape suggests ongoing specific alterations in neural circuitry across several brain systems to subserve social behavior. To discover the biological mechanisms that contribute to normal social activities, animal models of social behavior have been developed. One valuable model system has been female rat sexual behavior, which is governed by cyclic variation of ovarian hormones. This behavior is modulated by the neuropeptide oxytocin (OT) through its actions in the hypothalamic ventromedial nucleus (VMH). The fluctuation of this behavior is associated with dendrite remodeling, like several other examples of behavioral plasticity. This review compares hormone-induced plasticity in the VMH with other examples of dendrite plasticity across the mammalian nervous system, namely the neurobehavioral paradigms of environmental enrichment, chronic stress, and incentive sensitization, which affect the neocortex, hippocampal formation, and ventral striatum, respectively. This comparison suggests that the effects of ovarian hormones on VMH neurons in rats, given the simple dendritic arbor and short time course for dendrite remodeling, provide a dual opportunity for mechanistic and functional studies that will shed light on i) the neural actions of OT that regulate social behavior and, ii) behaviorally relevant dendrite regulation in a variety of brain structures. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

fruitless Gene products truncated of their male-like qualities promote neural and behavioral maleness in Drosophila if these proteins are produced in the right places at the right times.

To bring GAL4 production under the control of the sex promoter (P1) contained within Drosophila's fruitless gene, a gal4 cassette was previously inserted downstream of P1. This insert should eliminate male-specific FRU(M) proteins, which normally contain 101 amino acids (aa's) at their N termini. Thus males homozygous for the P1-gal4 insert should be courtless, as was briefly stated to be so in the initial report of this transgenic type. But XY flies whose only fru form is P1-gal4 have now been found to court vigorously. P1-gal4 females displayed no appreciable male-like actions except courtship rejection behaviors; yet, they developed a male-specific abdominal muscle. No immunoreactivity against the male-specific aa's was detectable in P1-gal4 flies. But male-like neural signals were observed in XY or XX P1-gal4 pupae and adults after applying an antibody that detects all FRU isoforms; transgenic females displayed reduced expression of such proteins. RT-PCR's rationalized these findings: P1 transcripts include anomalous splice forms from which gal4 was removed, allowing FRU's lacking M aa's to be produced in male-like patterns in both sexes. Within males, such defective proteins promote neural differentiation and function that is sufficient to support spirited P1-gal4 courtship. But dispensability of the male-specific FRU N-terminus is tempered by the finding that intra-fru sequences encoding these 101 aa's are highly conserved among interspecific relatives of D. melanogaster.

Functional analysis of fruitless gene expression by transgenic manipulations of Drosophila courtship.

A gal4-containing enhancer-trap called C309 was previously shown to cause subnormal courtship of Drosophila males toward females and courtship among males when driving a conditional disrupter of synaptic transmission (shi(TS)). We extended these manipulations to analyze all features of male-specific behavior, including courtship song, which was almost eliminated by driving shi(TS) at high temperature. In the context of singing defects and homosexual courtship affected by mutations in the fru gene, a tra-regulated component of the sex-determination hierarchy, we found a C309/tra(F) combination also to induce high levels of courtship between pairs of males and "chaining" behavior in groups; however, these doubly transgenic males sang normally. Because production of male-specific FRU(M) protein is regulated by TRA, we hypothesized that a fru-derived transgene encoding the male (M) form of an Inhibitory RNA (fru(MIR)) would mimic the effects of tra(F); but C309/fru(MIR) males exhibited no courtship chaining, although they courted other males in single-pair tests. Double-labeling of neurons in which GFP was driven by C309 revealed that 10 of the 20 CNS clusters containing FRU(M) in wild-type males included coexpressing neurons. Histological analysis of the developing CNS could not rationalize the absence of tra(F) or fru(MIR) effects on courtship song, because we found C309 to be coexpressed with FRU(M) within the same 10 neuronal clusters in pupae. Thus, we hypothesize that elimination of singing behavior by the C309/shi(TS) combination involves neurons acting downstream of FRU(M) cells.