Cocaine Exposure Increases Excitatory Synaptic Transmission and Intrinsic Excitability in the Basolateral Amygdala in Male and Female Rats and across the Estrous Cycle.

INTRODUCTION: Sex and ovarian hormones influence cocaine seeking and relapse vulnerability, but less is known regarding the cellular and synaptic mechanisms contributing to these behavioral sex differences. One factor thought to influence cue-induced seeking behavior following withdrawal is cocaine-induced changes in the spontaneous activity of pyramidal neurons in the basolateral amygdala (BLA). However, the mechanisms underlying these changes, including potential sex or estrous cycle effects, are unknown. METHODS: Ex vivo whole-cell patch clamp electrophysiology was conducted to investigate the effects of cocaine exposure, sex, and estrous cycle fluctuations on two properties that can influence spontaneous activity of BLA pyramidal neurons: (1) frequency and amplitude of spontaneous excitatory postsynaptic currents (sEPSCs) and (2) intrinsic excitability. Recordings of BLA pyramidal neurons were conducted in adult male and female rats and across the estrous cycle following 2-4 weeks of withdrawal from extended-access cocaine self-administration (6 h/day for 10 days) or drug-naïve conditions. RESULTS: In both sexes, cocaine exposure increased the frequency, but not amplitude, of sEPSCs and neuronal intrinsic excitability. Across the estrous cycle, sEPSC frequency and intrinsic excitability were significantly elevated only in cocaine-exposed females in the estrus stage of the cycle, a stage when cocaine-seeking behavior is known to be enhanced. CONCLUSIONS: Here, we identify potential mechanisms underlying cocaine-induced alterations in the spontaneous activity of BLA pyramidal neurons in both sexes along with changes in these properties across the estrous cycle.

Pet-1 Switches Transcriptional Targets Postnatally to Regulate Maturation of Serotonin Neuron Excitability.

Newborn neurons enter an extended maturation stage, during which they acquire excitability characteristics crucial for development of presynaptic and postsynaptic connectivity. In contrast to earlier specification programs, little is known about the regulatory mechanisms that control neuronal maturation. The Pet-1 ETS (E26 transformation-specific) factor is continuously expressed in serotonin (5-HT) neurons and initially acts in postmitotic precursors to control acquisition of 5-HT transmitter identity. Using a combination of RNA sequencing, electrophysiology, and conditional targeting approaches, we determined gene expression patterns in maturing flow-sorted 5-HT neurons and the temporal requirements for Pet-1 in shaping these patterns for functional maturation of mouse 5-HT neurons. We report a profound disruption of postmitotic expression trajectories in Pet-1(-/-) neurons, which prevented postnatal maturation of 5-HT neuron passive and active intrinsic membrane properties, G-protein signaling, and synaptic responses to glutamatergic, lysophosphatidic, and adrenergic agonists. Unexpectedly, conditional targeting revealed a postnatal stage-specific switch in Pet-1 targets from 5-HT synthesis genes to transmitter receptor genes required for afferent modulation of 5-HT neuron excitability. Five-HT1a autoreceptor expression depended transiently on Pet-1, thus revealing an early postnatal sensitive period for control of 5-HT excitability genes. Chromatin immunoprecipitation followed by sequencing revealed that Pet-1 regulates 5-HT neuron maturation through direct gene activation and repression. Moreover, Pet-1 directly regulates the 5-HT neuron maturation factor Engrailed 1, which suggests Pet-1 orchestrates maturation through secondary postmitotic regulatory factors. The early postnatal switch in Pet-1 targets uncovers a distinct neonatal stage-specific function for Pet-1, during which it promotes maturation of 5-HT neuron excitability. SIGNIFICANCE STATEMENT: The regulatory mechanisms that control functional maturation of neurons are poorly understood. We show that in addition to inducing brain serotonin (5-HT) synthesis and reuptake, the Pet-1 ETS (E26 transformation-specific) factor subsequently globally coordinates postmitotic expression trajectories of genes necessary for maturation of 5-HT neuron excitability. Further, Pet-1 switches its transcriptional targets as 5-HT neurons mature from 5-HT synthesis genes to G-protein-coupled receptors, which are necessary for afferent synaptic modulation of 5-HT neuron excitability. Our findings uncover gene-specific switching of downstream targets as a previously unrecognized regulatory strategy through which continuously expressed transcription factors control acquisition of neuronal identity at different stages of development.

Dorsal raphe serotonin neurons in mice: immature hyperexcitability transitions to adult state during first three postnatal weeks suggesting sensitive period for environmental perturbation.

Trauma during early life is a major risk factor for the development of anxiety disorders and suggests that the developing brain may be particularly sensitive to perturbation. Increased vulnerability most likely involves altering neural circuits involved in emotional regulation. The role of serotonin in emotional regulation is well established, but little is known about the postnatal development of the raphe where serotonin is made. Using whole-cell patch-clamp recording and immunohistochemistry, we tested whether serotonin circuitry in the dorsal and median raphe was functionally mature during the first 3 postnatal weeks in mice. Serotonin neurons at postnatal day 4 (P4) were hyperexcitable. The increased excitability was due to depolarized resting membrane potential, increased resistance, increased firing rate, lack of 5-HT1A autoreceptor response, and lack of GABA synaptic activity. Over the next 2 weeks, membrane resistance decreased and resting membrane potential hyperpolarized due in part to potassium current activation. The 5-HT1A autoreceptor-mediated inhibition did not develop until P21. The frequency of spontaneous inhibitory and excitatory events increased as neurons extended and refined their dendritic arbor. Serotonin colocalized with vGlut3 at P4 as in adulthood, suggesting enhanced release of glutamate alongside enhanced serotonin release. Because serotonin affects circuit development in other brain regions, altering the developmental trajectory of serotonin neuron excitability and release could have many downstream consequences. We conclude that serotonin neuron structure and function change substantially during the first 3 weeks of life during which external stressors could potentially alter circuit formation.

Sex-Specific Role for Dopamine Receptor D2 in Dorsal Raphe Serotonergic Neuron Modulation of Defensive Acoustic Startle and Dominance Behavior.

Brain networks underlying states of social and sensory alertness are normally adaptive, influenced by serotonin and dopamine (DA), and abnormal in neuropsychiatric disorders, often with sex-specific manifestations. Underlying circuits, cells, and molecules are just beginning to be delineated. Implicated is a subtype of serotonergic neuron denoted Drd2-Pet1, distinguished by expression of the type-2 DA receptor (Drd2) gene, inhibited cell-autonomously by DRD2 agonism in slice, and, when constitutively silenced in male mice, affects levels of defensive and exploratory behaviors (Niederkofler et al., 2016). Unknown has been whether DRD2 signaling in these Pet1 neurons contributes to their capacity for shaping defensive behaviors. To address this, we generated mice in which Drd2 gene sequences were deleted selectively in Pet1 neurons. We found that Drd2Pet1-CKO males, but not females, demonstrated increased winning against sex-matched controls in a social dominance assay. Drd2Pet1-CKO females, but not males, exhibited blunting of the acoustic startle response, a protective, defensive reflex. Indistinguishable from controls were auditory brainstem responses (ABRs), locomotion, cognition, and anxiety-like and depression-like behaviors. Analyzing wild-type Drd2-Pet1 neurons, we found sex-specific differences in the proportional distribution of axonal collaterals, in action potential (AP) duration, and in transcript levels of Gad2, important for GABA synthesis. Drd2Pet1-CKO cells displayed sex-specific differences in the percentage of cells harboring Gad2 transcripts. Our results suggest that DRD2 function in Drd2-Pet1 neurons is required for normal defensive/protective behaviors in a sex-specific manner, which may be influenced by the identified sex-specific molecular and cellular features. Related behaviors in humans too show sex differences, suggesting translational relevance.

Identification of Serotonergic Neuronal Modules that Affect Aggressive Behavior.

Escalated aggression can have devastating societal consequences, yet underlying neurobiological mechanisms are poorly understood. Here, we show significantly increased inter-male mouse aggression when neurotransmission is constitutively blocked from either of two subsets of serotonergic, Pet1+ neurons: one identified by dopamine receptor D1(Drd1a)::cre-driven activity perinatally, and the other by Drd2::cre from pre-adolescence onward. Blocking neurotransmission from other Pet1+ neuron subsets of similar size and/or overlapping anatomical domains had no effect on aggression compared with controls, suggesting subtype-specific serotonergic neuron influences on aggression. Using established and novel intersectional genetic tools, we further characterized these subtypes across multiple parameters, showing both overlapping and distinct features in axonal projection targets, gene expression, electrophysiological properties, and effects on non-aggressive behaviors. Notably, Drd2::cre marked 5-HT neurons exhibited D2-dependent inhibitory responses to dopamine in slices, suggesting direct and specific interplay between inhibitory dopaminergic signaling and a serotonergic subpopulation. Thus, we identify specific serotonergic modules that shape aggression.

Unexpected effects of perinatal gonadal hormone manipulations on sexual differentiation of the extrahypothalamic arginine-vasopressin system in prairie voles.

The sexually dimorphic extrahypothalamic arginine-vasopressin (AVP) projections from the bed nucleus of the stria terminalis to the lateral septum (LS) and lateral habenula (LHb) are denser in males than females and, in rats, require males' perinatal exposure to gonadal hormones but the absence of such exposure in females. We examined perinatal hormone effects on development of this sex difference in prairie voles (Microtus ochrogaster), which show atypical effects of hormones on sexual differentiation of some reproductive behaviors. Neonatal castration reduced the number of AVP mRNA-expressing cells in the bed nucleus of the stria terminalis and AVP immunoreactivity (ir) in the LS and LHb. Surprisingly, daily injections of 1000 microg of testosterone propionate (TP) during the first postnatal week did not maintain high levels of AVP-ir in neonatally castrated males. Furthermore, perinatal treatments with TP (75, 500, or 1000 microg), testosterone (100 microg), or dihydrotestosterone (200 microg) did not masculinize AVP-ir in the female LS or LHb. In fact, 1000 microg TP reduced it in some cases. However, 1000 microg TP lengthened anogenital distance, indicating that TP was biologically active. Neonatal estrogen receptor antagonism with tamoxifen reduced AVP-ir in the male LS, whereas treating neonatal females with the synthetic estrogen diethylstilbestrol increased septal AVP-ir. Tamoxifen and diethylstilbestrol had no effects in the LHb. Similar to rats, therefore, postnatal estrogen influences some components of the extrahypothalamic AVP system in prairie voles, but this developing system appears to be insensitive to exogenous androgens, including aromatizable androgens. Such insensitivity is atypical for a sexually dimorphic neural system in a rodent and may reflect the unusual effects of hormones on sexual differentiation of some behaviors in prairie voles.

Multi-Scale Molecular Deconstruction of the Serotonin Neuron System.

Serotonergic (5HT) neurons modulate diverse behaviors and physiology and are implicated in distinct clinical disorders. Corresponding diversity in 5HT neuronal phenotypes is becoming apparent and is likely rooted in molecular differences, yet a comprehensive approach characterizing molecular variation across the 5HT system is lacking, as is concomitant linkage to cellular phenotypes. Here we combine intersectional fate mapping, neuron sorting, and genome-wide RNA-seq to deconstruct the mouse 5HT system at multiple levels of granularity-from anatomy, to genetic sublineages, to single neurons. Our unbiased analyses reveal principles underlying system organization, 5HT neuron subtypes, constellations of differentially expressed genes distinguishing subtypes, and predictions of subtype-specific functions. Using electrophysiology, subtype-specific neuron silencing, and conditional gene knockout, we show that these molecularly defined 5HT neuron subtypes are functionally distinct. Collectively, this resource classifies molecular diversity across the 5HT system and discovers sertonergic subtypes, markers, organizing principles, and subtype-specific functions with potential disease relevance.

Site of origin of and sex differences in the vasopressin innervation of the mouse (Mus musculus) brain.

Defining how arginine vasopressin (AVP) acts centrally to regulate homeostasis and behavior is problematic, as AVP is made in multiple nuclei in the hypothalamus (i.e., paraventricular [PVN], supraoptic [SON], and suprachiasmatic [SCN]) and extended amygdala (i.e., bed nucleus of the stria terminalis [BNST] and medial amygdala [MeA]), and these groups of neurons have extensive projections throughout the brain. To understand the function of AVP, it is essential to know the site of origin of various projections. In mice, we used gonadectomy to eliminate gonadal steroid hormone-dependent expression of AVP in the BNST and MeA and electrolytic lesions to eliminate the SCN, effectively eliminating those AVP-immunoreactive projections; we also quantified AVP-immunoreactive fiber density in gonadectomized and sham-operated male and female mice to examine sex differences in AVP innervation. Our results suggest that the BNST/MeA AVP system innervates regions containing major modulatory neurotransmitters (e.g., serotonin and dopamine) and thus may be involved in regulating behavioral state. Furthermore, this system may be biased toward the regulation of male behavior, given the numerous regions in which males have a denser AVP-immunoreactive innervation than females. AVP from the SCN is found in regions important for the regulation of hormone output and behavior. Innervation from the PVN and SON is found in brain regions that likely work in concert with the well-known peripheral AVP actions of controlling homeostasis and stress response; female-biased sex differences in this system may be related to the heightened stress response observed in females.

Vasopressin innervation of the mouse (Mus musculus) brain and spinal cord.

The neuropeptide vasopressin (AVP) has been implicated in the regulation of numerous physiological and behavioral processes. Although mice have become an important model for studying this regulation, there is no comprehensive description of AVP distribution in the mouse brain and spinal cord. With C57BL/6 mice, we used immunohistochemistry to corroborate the location of AVP-containing cells and to define the location of AVP-containing fibers throughout the mouse central nervous system. We describe AVP-immunoreactive (-ir) fibers in midbrain, hindbrain, and spinal cord areas, which have not previously been reported in mice, including innervation of the ventral tegmental area, dorsal and median raphe, lateral and medial parabrachial, solitary, ventrolateral periaqueductal gray, and interfascicular nuclei. We also provide a detailed description of AVP-ir innervation in heterogenous regions such as the amygdala, bed nucleus of the stria terminalis, and ventral forebrain. In general, our results suggest that, compared with other species, the mouse has a particularly robust and widespread distribution of AVP-ir fibers, which, as in other species, originates from a number of different cell groups in the telencephalon and diencephalon. Our data also highlight the robust nature of AVP innervation in specific regulatory nuclei, such as the ventral tegmental area and dorsal raphe nucleus among others, that are implicated in the regulation of many behaviors.

Parental responsiveness is feminized after neonatal castration in virgin male prairie voles, but is not masculinized by perinatal testosterone in virgin females.

We previously found a large sex difference in the parental responsiveness of adult virgin prairie voles (Microtus ochrogaster) such that most males are spontaneously parental, whereas most females are not. Because this sex difference is independent of the gonadal hormones normally circulating in adult virgin voles, the present study examined whether perinatal hormones influence the development of this sex difference. Males were treated prenatally (via their pregnant dam) with both the androgen receptor blocker flutamide (5 mg/day/dam) and the aromatase inhibitor ATD (1 mg/day/dam), or oil, for the last 2 weeks of gestation. Half of the subjects from each group were castrated on the day of birth and the other half received a sham surgery. As adults, intact males were castrated and all males received a silastic capsule filled with testosterone. Prenatal treatment with flutamide and ATD had no effect on males' behavior toward pups, but neonatal castration significantly reduced the percentage of males acting parentally. In a second experiment, females were exposed to testosterone propionate (TP; 50 microg/day/dam) or oil via their dam during the last 2 weeks of gestation. For the first neonatal week, half of the females from each group were injected with TP (1 mg/day) and the other half oil. As adults, females were ovariectomized and half from each group received a testosterone-filled capsule and the other half received an empty capsule. None of the perinatal TP treatments increased females' parental responsiveness, although females from all groups that received testosterone capsules as adults were highly parental. Therefore, although postnatal testicular hormones are necessary for high parental responsiveness in males, the behavior of females is not influenced by perinatal exposure to testosterone.