Daytime Light Deficiency Leads to Sex- and Brain Region-Specific Neuroinflammatory Responses in a Diurnal Rodent.

Seasonal changes in peripheral inflammation are well documented in both humans and animal models, but seasonal changes in neuroinflammation, especially the impact of seasonal lighting environment on neuroinflammation remain unclear. To address this question, the present study examined the effects of environmental lighting conditions on neuroinflammation in a diurnal rodent model, Nile grass rats (Arvicanthis niloticus). Male and female grass rats were housed in either bright (brLD) or dim (dimLD) light during the day to simulate a summer or winter light condition, respectively. After 4 weeks, microglia markers Iba-1 and CD11b, as well as pro-inflammatory cytokines TNF-α and IL-6, were examined in the anterior cingulate cortex (ACC), basolateral amygdala (BLA), and dorsal hippocampus (dHipp). The results revealed that winter-like dim light during the day leads to indicators of increased neuroinflammation in a brain site- and sex-specific manner. Specifically, relatively few changes in the neuroinflammatory markers were observed in the ACC, while numerous changes were found in the BLA and dHipp. In the BLA, winter-like dimLD resulted in hyper-ramified microglia morphology and increased expression of the pro-inflammatory cytokine IL-6, but only in males. In the dHipp, dimLD led to a higher number and hyper-ramified morphology of microglia as well as increased expression of CD11b and TNF-α, but only in females. Neuroinflammatory state is thus influenced by environmental light, differently in males and females, and could play a role in sex differences in the prevalence and symptoms of psychiatric or neurological disorders that are influenced by season or other environmental light conditions. Diurnal Nile grass rats were housed under bright or dim light during the day for 4 weeks, simulating seasonal fluctuations in daytime lighting environment. Dim light housing resulted in hyper-ramified morphology of microglia (scale bar, 15 µm) and altered expression of pro-inflammatory cytokines (TNF-α) in a sex- and brain region-specific manner.

Oxytocin receptors in the midbrain dorsal raphe are essential for postpartum maternal social and affective behaviors.

Oxytocin receptors (OTRs) in the midbrain dorsal raphe (DR; the source of most forebrain serotonin) have recently been identified as a potential pharmacological target for treating numerous psychiatric disorders. However, almost all research on this topic has been conducted on males and the role of DR OTRs in female social and affective behaviors is mostly unknown. This may be particularly relevant during early motherhood, which is a time of high endogenous oxytocin signaling, but also a time of elevated risk for psychiatric dysfunction. To investigate whether OTRs in the DR are necessary for postpartum female social and affective behaviors, we constructed and then injected into the DR an adeno-associated virus permanently expressing an shRNA targeting OTR mRNA. We then observed a suite of social and affective behaviors postpartum. OTR knockdown in the maternal DR led to pup loss after parturition, decreased nursing, increased aggression, and increased behavioral despair. These effects of OTR knockdown in the DR may be due to disrupted neuroplasticity in the primary somatosensory cortex (S1), which mediates maternal sensitivity to the tactile cues from young, as we found significantly more plasticity-restricting perineuronal nets (PNNs) in the S1 rostral barrel field and fewer PNNs in the caudal barrel field of OTR-knockdown mothers. These results demonstrate that OTRs in the midbrain DR are essential for postpartum maternal social and affective behaviors, are involved in postpartum cortical plasticity, and suggest that pharmacotherapies targeting OTRs in the DR could be effective treatments for some peripartum affective disorders.

Fos expression in the medial preoptic area and nucleus accumbens of female Japanese quail (Coturnix japonica) after maternal induction and interaction with chicks.

The neural system underlying maternal caregiving has often been studied using laboratory rodents and a few other mammalian species. This research shows that the medial preoptic area (mPOA) integrates sensory cues from the young that, along with hormonal and other environmental signals, control maternal acceptance of neonates. The mPOA then activates the mesolimbic system to drive maternal motivation and caregiving activities. How components of this neural system respond to maternal experience and exposure to young in non-mammals has rarely been examined. To gain more insight into this question, virgin female Japanese quail (Coturnix japonica) were induced to be maternal through four days of continuous exposure to chicks (Maternal), or were not exposed to chicks (Non-Maternal). Chicks were removed overnight from the Maternal group and half the females from each group were then exposed to chicks for 90 minutes (Exposed), or not exposed to chicks (Non-Exposed), before euthanasia. The number of Fos-immunoreactive (Fos-ir) cells was examined as a marker of neuronal activation. As expected, repeated exposure to chicks induced caregiving behavior in the Maternal females, which persisted after the overnight separation, suggesting the formation of a maternal memory. In contrast, Non-Maternal females were aggressive and rejected the chicks when exposed to them. Exposed females, whether or not they were given prior experience with chicks (i.e., regardless if they accepted or rejected chicks during the exposure before euthanasia), had more Fos-ir cells in the mPOA compared to Non-Exposed females. In the nucleus accumbens (NAC), the number of Fos-ir cells was high in all Maternal females whether or not they were Exposed to chicks again before euthanasia. In the lateral bed nucleus of the stria terminalis, a site involved in general stress responding, groups did not differ in the number of Fos-ir cells. These data indicate a conserved role for the mPOA and NAC in maternal caregiving across vertebrates, with the mPOA acutely responding to the salience rather than valence of offspring cues, and the NAC showing longer-term changes in activity after a positive maternal experience even without a recent exposure to young.

Low Daytime Light Intensity Disrupts Male Copulatory Behavior, and Upregulates Medial Preoptic Area Steroid Hormone and Dopamine Receptor Expression, in a Diurnal Rodent Model of Seasonal Affective Disorder.

Seasonal affective disorder (SAD) involves a number of psychological and behavioral impairments that emerge during the low daytime light intensity associated with winter, but which remit during the high daytime light intensity associated with summer. One symptom frequently reported by SAD patients is reduced sexual interest and activity, but the endocrine and neural bases of this particular impairment during low daylight intensity is unknown. Using a diurnal laboratory rodent, the Nile grass rat (Arvicanthis niloticus), we determined how chronic housing under a 12:12 h day/night cycle involving dim low-intensity daylight (50 lux) or bright high-intensity daylight (1,000 lux) affects males' copulatory behavior, reproductive organ weight, and circulating testosterone. We also examined the expression of mRNAs for the aromatase enzyme, estrogen receptor 1 (ESR1), and androgen receptor (AR) in the medial preoptic area (mPOA; brain site involved in the sensory and hormonal control of copulation), and mRNAs for the dopamine (DA) D1 and D2 receptors in both the mPOA and nucleus accumbens (NAC; brain site involved in stimulus salience and motivation to respond to reward). Compared to male grass rats housed in high-intensity daylight, males in low-intensity daylight displayed fewer mounts and intromissions when interacting with females, but the groups did not differ in their testes or seminal vesicle weights, or in their circulating levels of testosterone. Males in low-intensity daylight unexpectedly had higher ESR1, AR and D1 receptor mRNA in the mPOA, but did not differ from high-intensity daylight males in D1 or D2 mRNA expression in the NAC. Reminiscent of humans with SAD, dim winter-like daylight intensity impairs aspects of sexual behavior in a male diurnal rodent. This effect is not due to reduced circulating testosterone and is associated with upregulation of mPOA steroid and DA receptors that may help maintain some sexual motivation and behavior under winter-like lighting conditions.

Decreased daytime illumination leads to anxiety-like behaviors and HPA axis dysregulation in the diurnal grass rat (Arvicanthis niloticus).

The impact of ambient light on mood and anxiety is best exemplified in seasonal affective disorder, in which patients experience depression and anxiety in winter when there is less light in the environment. However, the brain mechanisms underlying light-dependent changes in affective state remain unclear. Our previous work revealed increased depression-like behaviors in the diurnal Nile grass rat (Arvicanthis niloticus) housed in a dim light-dark (dim-LD) cycle as compared to the controls housed in a bright light-dark (bright-LD) condition. As depression is often comorbid with anxiety and is associated with dysregulation of the body's stress response system, the present study examined the anxiety-like behaviors as well as indicators of the hypothalamic-pituitary-adrenal (HPA) axis functioning in the grass rats. Animals housed in dim-LD showed increased anxiety-like behaviors compared to bright-LD controls, as revealed by fewer entries and less time spent at the center in the open field test and more marbles buried during the marble-burying test. Following the marble-burying test, dim-LD animals showed higher plasma corticosterone (CORT) levels and hippocampal Fos expression. Although the daily CORT rhythm was comparable between bright-LD and dim-LD groups, the day/night variation of corticotropin-releasing hormone mRNA expression in the paraventricular nucleus was diminished in dim-LD animals. In addition, glucocorticoid receptor and mineralocorticoid receptor mRNA expression were higher in the hippocampus of dim-LD animals. The results suggest that in diurnal species, reduced daytime illumination can lead to increased anxiety-like behaviors and altered HPA axis functioning, providing insights into the link between decreased environmental illumination and negative emotion.

Progesterone receptor expression in the brain of the socially monogamous and paternal male prairie vole.

Differences in the social organization and behavior of male mammals are attributable to species differences in neurochemistry, including differential expression of steroid hormone receptors. However, the distribution of progestin receptors (PR) in a socially monogamous and spontaneously parental male rodent has never been examined. Here we determined if PR exists and is regulated by testicular hormones in forebrain sites traditionally influencing socioreproductive behaviors in male prairie voles (Microtus ochrogaster). We hypothesized that PR expression in male prairie voles would differ from that described in other male rodents because PR activity inhibits parental behaviors and social memory in laboratory mice and rats. Adult male prairie voles received a sham surgery, were gonadectomized, or were gonadectomized and implanted with a testosterone-filled capsule. PR immunoreactivity (PRir) was measured four weeks later in areas of the hypothalamus and extended amygdala. A group of gonadally intact female prairie voles was included to reveal possible sex differences. We found considerable PRir in all sites examined. Castration reduced PRir in males' medial preoptic nucleus, anteroventral periventricular nucleus, ventromedial hypothalamus, and posterodorsal medial amygdala, and it was maintained in these sites by testosterone. This is the first study to examine PR expression in brain sites involved in socioreproductive behaviors in a socially monogamous and spontaneously paternal male rodent. Our results mostly reveal cross-species conservation in the distribution and hormone sensitivity of PR expression. Because PR interferes with aspects of sociality in other male rodents, PR may eventually be found to have different neurobiological actions in male prairie voles.

Social novelty increases tyrosine hydroxylase immunoreactivity in the extended olfactory amygdala of female prairie voles.

The monogamous social behaviors of prairie voles (Microtus ochrogaster) require olfactory inputs, which are processed by the posterodorsal medial amygdala (MeApd) and principal bed nucleus of the stria terminalis (pBST). The male prairie vole MeApd and pBST contain hundreds of cells densely immunoreactive for tyrosine hydroxylase (TH-ir). Female prairie voles have relatively few of these cells, but we previously found that the number of these TH-ir cells is greatly increased in females by exogenous estradiol. We here hypothesized that the number of TH-ir cells in their MeApd and pBST would also increase during the natural hormone surges associated with females' induced estrus. We found that the number of TH-ir cells in both sites did significantly increase after females cohabitated for two days with an unfamiliar male. However, this increase did not require the presence of ovaries and even tended to occur in the pBST of females cohabitating for two days with unfamiliar females. We then determined if the greater number of TH-ir cells after heterosexual pairing was transient by examining two groups of long-term pairbonded females (primiparous and multiparous), and found these females also had significantly more TH-ir cells in the pBST and/or MeApd compared to unmated controls. Thus, social novelty arising from cohabitation with unfamiliar conspecifics produces a reoccurring increase in the number of TH-ir cells in the female prairie vole extended olfactory amygdala. Ovarian hormones are not necessarily required. This increase in catecholaminergic cells may facilitate acquisition and retention of olfactory memories necessary for social recognition in this species.

Sex differences and effects of neonatal aromatase inhibition on masculine and feminine copulatory potentials in prairie voles.

Copulatory behaviors in most rodents are highly sexually dimorphic, even when circulating hormones are equated between the sexes. Prairie voles (Microtus ochrogaster) are monomorphic in their display of some social behaviors, including partner preferences and parenting, but differences between the sexes in their masculine and feminine copulatory behavior potentials have not been studied in detail. Furthermore, the role of neonatal aromatization of testosterone to estradiol on the development of prairie vole sexual behavior potentials or their brain is unknown. To address these issues, prairie vole pups were injected daily for the first week after birth with 0.5 mg of the aromatase inhibitor 1,4,6-androstatriene-3,17-dione (ATD) or oil. Masculine and feminine copulatory behaviors in response to testosterone or estradiol were later examined in both sexes. Males and females showed high mounting and thrusting in response to testosterone, but only males reliably showed ejaculatory behavior. Conversely, males never showed feminine copulatory behaviors in response to estradiol. Sex differences in these behaviors were not affected by neonatal ATD, but ATD-treated females received fewer mounts and thrusts than controls, possibly indicating reduced attractiveness to males. In other groups of subjects, neonatal ATD demasculinized males' tyrosine hydroxylase expression in the anteroventral periventricular preoptic area, and estrogen receptor alpha expression in the medial preoptic area. Thus, although sexual behavior in both sexes of prairie voles is highly masculinized, aromatase during neonatal life is necessary only for females' femininity. Furthermore, copulatory behavior potentials and at least some aspects of brain development in male prairie voles are dissociable by their requirement for neonatal aromatase.

Contact with infants modulates anxiety-generated c-fos activity in the brains of postpartum rats.

The postpartum period is associated with many behavioral changes, including a reduction in anxiety, which is thought to be necessary for mothers' ability to appropriately care for infants. In laboratory rats, this reduction in anxiety requires recent contact with pups, but areas of the brain where infant contact influences neural activity to reduce anxiety are mostly unknown. We examined c-fos expression in lactating rats whose pups were removed for 4h to increase mothers' anxiety, or not removed to maintain low anxiety in mothers, followed by exposure to the anxiogenic stimuli of either brief handling or handling followed by exposure to an elevated plus maze. Control animals had their litters removed or not, but no further stimulation. A large number of neural sites traditionally implicated in regulating anxiety in male rats were examined, and similar to what is found in male rats, most showed increased Fos expression after handling and/or elevated plus-maze exposure. Litter presence before testing affected Fos expression due to handling or elevated plus-maze exposure only in the ventral bed nucleus of the stria terminalis, dorsal and ventral preoptic area, ventromedial hypothalamus, lateral habenula, and supramammillary nucleus. Contrary to expectations, prior litter presence was associated with more Fos expression in most of these sites after handling and/or elevated plus-maze stimulation, and only after such stimulation. These sites may be of particular importance for how sensory inputs from infants modulate anxiety and other mood states during the postpartum period.

Tyrosine hydroxylase-synthesizing cells in the hypothalamus of prairie voles (Microtus ochrogaster): sex differences in the anteroventral periventricular preoptic area and effects of adult gonadectomy or neonatal gonadal hormones.

The vertebrate hypothalamus and surrounding region contain a large population of cells expressing tyrosine hydroxylase (TH), the rate limiting enzyme for synthesis of dopamine and other catecholamines. Some of these populations are sexually dimorphic in rats. We here examined sex differences in TH-immunoreactive populations in the forebrain of gonadally intact and gonadectomized prairie voles (Microtus ochrogaster), a species that sometimes shows unusual sexual differentiation of brain and behavior. A sex difference was found in the anteroventral periventricular preoptic area (AVPV; likely analogous to the rat rostral A14) only in gonadectomized subjects, which was due to a 50% reduction in the number of TH-immunoreactive cells after castration in males. There was no significant sex difference or effects of gonadectomy on the number of TH-immunoreactive cells in the anteroventral preoptic area (AVP), periventricular anterior hypothalamus (caudal A14), arcuate nucleus (A12), zona incerta (A13), or posterodorsal hypothalamus (A11). In a second experiment, testosterone propionate (TP; 500 microg), diethylstilbestrol (DES; 1 microg), or estradiol benzoate (EB; 30 microg) injected daily during the first week after birth each significantly reduced later TH expression in the AVPV of females by approximately 40-65% compared to oil-treated controls. Unlike rats, therefore, a sex difference in TH expression in the prairie vole AVPV is found only after removal of circulating gonadal hormones in males. Furthermore, unlike our previous findings on the generation of sex differences in extra-hypothalamic arginine-vasopressin expression in prairie voles, TH expression in the AVPV of female prairie voles can be highly masculinized by neonatal exposure to either aromatizable androgens or estrogens.

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.

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.