Neuroanatomical characterization of Gαi2-expressing neurons in the hypothalamic paraventricular nucleus of male and female Sprague-Dawley rats.

Hypertension is a global health burden. The hypothalamic paraventricular nucleus (PVN) is an essential component of the neuronal network that regulates sodium homeostasis and blood pressure (BP). Previously, we have shown PVN-specific G protein-coupled receptor-coupled Gαi2 subunit proteins are essential to counter the development of salt-sensitive hypertension by mediating the sympathoinhibitory and natriuretic responses to increased dietary sodium intake to maintain sodium homeostasis and normotension. However, the cellular localization and identity of PVN Gαi2-expressing neurons are currently unknown. In this study using in situ hybridization, we determined the neuroanatomical characterization of Gαi2-expressing PVN neurons in 3-mo-old male and female Sprague-Dawley rats. We observed that Gαi2-expressing neurons containing Gnai2 mRNA are highly localized in the parvocellular region of the hypothalamic PVN. At level 2 of the hypothalamic PVN, Gnai2 mRNA colocalized with ∼ 85% of GABA-expressing neurons and ∼28% of glutamatergic neurons. Additionally, within level 2 Gnai2 mRNA colocalized with ∼75% of corticotrophin-releasing hormone PVN neurons. Gnai2 neurons had lower colocalization with tyrosine hydroxylase (∼33%)-, oxytocin (∼6%)-, and arginine vasopressin (∼10%)-expressing parvocellular neurons in level 2 PVN. Colocalization was similar among male and female rats. The high colocalization of Gnai2 mRNA with GABAergic neurons, in conjunction with our previous findings that PVN Gαi2 proteins mediate sympathoinhibition, suggests that Gαi2 proteins potentially modulate GABAergic signaling to impact sympathetic outflow and BP.

Increased anxiety and decreased sociability induced by paternal deprivation involve the PVN-PrL OTergic pathway.

Early adverse experiences often have devastating consequences. However, whether preweaning paternal deprivation (PD) affects emotional and social behaviors and their underlying neural mechanisms remain unexplored. Using monogamous mandarin voles, we found that PD increased anxiety-like behavior and attenuated social preference in adulthood. PD also decreased the number of oxytocin (OT)-positive neurons projecting from the paraventricular nucleus (PVN) and reduced the levels of the medial prefrontal cortex OT receptor protein in females and of the OT receptor and V1a receptor proteins in males. Intra-prelimbic cortical OT injections reversed the PD-induced changes in anxiety-like behavior and social preferences. Optogenetic activation of the prelimbic cortex OT terminals from PVN OT neurons reversed the PD-induced changes in emotion and social preference behaviors, whereas optogenetic inhibition was anxiogenic and impaired social preference in naive voles. These findings demonstrate that PD increases anxiety-like behavior and attenuates social preferences through the involvement of PVN OT neuron projections to the prelimbic cortex.

Mapping the co-localization of the circadian proteins PER2 and BMAL1 with enkephalin and substance P throughout the rodent forebrain.

Despite rhythmic expression of clock genes being found throughout the central nervous system, very little is known about their function outside of the suprachiasmatic nucleus. Determining the pattern of clock gene expression across neuronal subpopulations is a key step in understanding their regulation and how they may influence the functions of various brain structures. Using immunofluorescence and confocal microscopy, we quantified the co-expression of the clock proteins BMAL1 and PER2 with two neuropeptides, Substance P (SubP) and Enkephalin (Enk), expressed in distinct neuronal populations throughout the forebrain. Regions examined included the limbic forebrain (dorsal striatum, nucleus accumbens, amygdala, stria terminalis), thalamus medial habenula of the thalamus, paraventricular nucleus and arcuate nucleus of the hypothalamus and the olfactory bulb. In most regions examined, BMAL1 was homogeneously expressed in nearly all neurons (~90%), and PER2 was expressed in a slightly lower proportion of cells. There was no specific correlation to SubP- or Enk- expressing subpopulations. The olfactory bulb was unique in that PER2 and BMAL1 were expressed in a much smaller percentage of cells, and Enk was rarely found in the same cells that expressed the clock proteins (SubP was undetectable). These results indicate that clock genes are not unique to specific cell types, and further studies will be required to determine the factors that contribute to the regulation of clock gene expression throughout the brain.

Commentary on: Saper CB, Loewy AD, Swanson LW, Cowan WM. (1976) Direct hypothalamo-autonomic connections. Brain Research 117:305-312.

The 1970s saw the introduction of new technologies for tracing axons both anterogradely and retrogradely. These methods allowed us to visualize fine, unmyelinated pathways for the first time, such as the hypothalamic pathways that control the autonomic nervous system. As a result, we were able to identify the paraventricular nucleus and lateral hypothalamus as the key sites that provide direct inputs to the autonomic preganglionic neurons in the medulla and spinal cord. These findings revolutionized our understanding of hypothalamic control of the autonomic nervous system.

Control of food intake and energy expenditure by Nos1 neurons of the paraventricular hypothalamus.

The paraventricular nucleus of the hypothalamus (PVH) contains a heterogeneous cluster of Sim1-expressing cell types that comprise a major autonomic output nucleus and play critical roles in the control of food intake and energy homeostasis. The roles of specific PVH neuronal subtypes in energy balance have yet to be defined, however. The PVH contains nitric oxide synthase-1 (Nos1)-expressing (Nos1(PVH)) neurons of unknown function; these represent a subset of the larger population of Sim1-expressing PVH (Sim1(PVH)) neurons. To determine the role of Nos1(PVH) neurons in energy balance, we used Cre-dependent viral vectors to both map their efferent projections and test their functional output in mice. Here we show that Nos1(PVH) neurons project to hindbrain and spinal cord regions important for food intake and energy expenditure control. Moreover, pharmacogenetic activation of Nos1(PVH) neurons suppresses feeding to a similar extent as Sim1(PVH) neurons, and increases energy expenditure and activity. Furthermore, we found that oxytocin-expressing PVH neurons (OXT(PVH)) are a subset of Nos1(PVH) neurons. OXT(PVH) cells project to preganglionic, sympathetic neurons in the thoracic spinal cord and increase energy expenditure upon activation, though not to the same extent as Nos1(PVH) neurons; their activation fails to alter feeding, however. Thus, Nos1(PVH) neurons promote negative energy balance through changes in feeding and energy expenditure, whereas OXT(PVH) neurons regulate energy expenditure alone, suggesting a crucial role for non-OXT Nos1(PVH) neurons in feeding regulation.

Reciprocal connections between CART-immunoreactive, hypothalamic paraventricular neurons and serotonergic dorsal raphe cells in the rat: Light microscopic study.

Based on the overlapping physiological roles of cocaine- and amphetamine-regulated transcript (CART) peptides and serotonin, the present study examined the anatomical connection between the hypothalamic paraventricular nucleus (PVN) and the dorsal raphe (DR). The first series of experiments were performed to investigate descending projections from the CART-immunoreactive (CART-ir) PVN to serotonergic DR cells. CART-ir varicosities made contact with serotonergic DR neurons. An anterograde tracing study revealed that varicosities originating from the PVN formed close appositions to serotonergic neuronal profiles along the entire rostro-caudal extent of the DR. A retrograde study demonstrated that CART neurons projecting to the DR were mainly localized in the caudal parvicellular PVN, comprising approximately 3.0%±0.4% (n=8) of total CART cells. A second series of experiments was performed to investigate ascending projections from the DR to CART-ir PVN cells. Serotonin transporter-ir boutons made contact with CART-ir PVN neurons. Anterograde tracing revealed that varicosities originating from the DR formed close appositions to CART-ir PVN cells. Retrograde examination demonstrated that serotonergic neurons projecting to the parvicellular PVN were located along the entire rostro-caudal extent of the DR. The present observation provided an anatomical basis for accumulating evidence in the literature that suggests a functional interaction between the CART and serotonin systems during the regulation of energy balance, emotional behavior, and arousal.

Bilateral descending hypothalamic projections to the spinal trigeminal nucleus caudalis in rats.

Several lines of evidence suggest that the hypothalamus is involved in trigeminal pain processing. However, the organization of descending hypothalamic projections to the spinal trigeminal nucleus caudalis (Sp5C) remains poorly understood. Microinjections of the retrograde tracer, fluorogold (FG), into the Sp5C, in rats, reveal that five hypothalamic nuclei project to the Sp5C: the paraventricular nucleus, the lateral hypothalamic area, the perifornical hypothalamic area, the A11 nucleus and the retrochiasmatic area. Descending hypothalamic projections to the Sp5C are bilateral, except those from the paraventricular nucleus which exhibit a clear ipsilateral predominance. Moreover, the density of retrogradely FG-labeled neurons in the hypothalamus varies according to the dorso-ventral localization of the Sp5C injection site. There are much more labeled neurons after injections into the ventrolateral part of the Sp5C (where ophthalmic afferents project) than after injections into its dorsomedial or intermediate parts (where mandibular and maxillary afferents, respectively, project). These results demonstrate that the organization of descending hypothalamic projections to the spinal dorsal horn and Sp5C are different. Whereas the former are ipsilateral, the latter are bilateral. Moreover, hypothalamic projections to the Sp5C display somatotopy, suggesting that these projections are preferentially involved in the processing of meningeal and cutaneous inputs from the ophthalmic branch of the trigeminal nerve in rats. Therefore, our results suggest that the control of trigeminal and spinal dorsal horn processing of nociceptive information by hypothalamic neurons is different and raise the question of the role of bilateral, rather than unilateral, hypothalamic control.

Inner capillary diameter of hypothalamic paraventricular nucleus of female rat increases during lactation.

BACKGROUND: The role of the endothelial cell (EC) in blood flow regulation within the central nervous system has been little studied. Here, we explored EC participation in morphological changes of the anterior hypothalamic paraventricular nucleus (PVN) microvasculature of female rats at two reproductive stages with different metabolic demand (virginity and lactation). We measured the inner capillary diameter (ICD) of 800 capillaries from either the magnocellular or parvocellular regions. The space occupied by neural (somas, dendrites and axons) and glial, but excluding vascular elements of the neurovascular compartment was also measured in 100-µm2 sample fields of both PVN subdivisions. RESULTS: The PVN of both groups of animals showed ICDs that ranged from 3 to 10 microns. The virgin group presented mostly capillaries with small ICD, whereas the lactating females exhibited a significant increment in the percentage of capillaries with larger ICD. The space occupied by the neural and glial elements of the neurovascular compartment did not show changes with lactation. CONCLUSIONS: Our findings suggest that during lactation the microvasculature of the PVN of female rats undergoes dynamic, transitory changes in blood flow as represented by an increment in the ICD through a self-cytoplasmic volume modification reflected by EC changes. A model of this process is proposed.

Endogenous hydrogen peroxide in the hypothalamic paraventricular nucleus regulates sympathetic nerve activity responses to L-glutamate.

The hypothalamic paraventricular nucleus (PVN) is important for maintenance of sympathetic nerve activity (SNA) and cardiovascular function. PVN-mediated increases of SNA often involve the excitatory amino acid L-glutamate (L-glu), whose actions can be positively and negatively modulated by a variety of factors, including reactive oxygen species. Here, we determined modulatory effects of the highly diffusible reactive oxygen species hydrogen peroxide (H(2)O(2)) on responses to PVN L-glu. Renal SNA (RSNA), arterial blood pressure, and heart rate were recorded in anesthetized rats. L-Glu (0.2 nmol in 100 nl) microinjected unilaterally into PVN increased RSNA (P < 0.05), without affecting mean arterial blood pressure or heart rate. Effects of endogenously generated H(2)O(2) were determined by comparing responses to PVN L-glu before and after PVN injection of the catalase inhibitor 3-amino-1,2,4-triazole (ATZ; 100 nmol/200 nl, n = 5). ATZ alone was without effect on recorded variables, but attenuated the increase of RSNA elicited by PVN L-glu (P < 0.05). PVN injection of exogenous H(2)O(2) (5 nmol in 100 nl, n = 4) and vehicle (artificial cerebrospinal fluid) were without affect, but H(2)O(2), like ATZ, attenuated the increase of RSNA to PVN L-glu (P < 0.05). Tonic effects of endogenous H(2)O(2) were determined by PVN injection of polyethylene glycol-catalase (1.0 IU in 200 nl, n = 5). Whereas polyethylene glycol-catalase alone was without effect, increases of RSNA to subsequent PVN injection of L-glu were increased (P < 0.05). From these data, we conclude that PVN H(2)O(2) tonically, but submaximally, suppresses RSNA responses to L-glu, supporting the idea that a change of H(2)O(2) availability within PVN could influence SNA regulation under physiological and/or disease conditions.

The projection and synaptic organisation of NTS afferent connections with presympathetic neurons, GABA and nNOS neurons in the paraventricular nucleus of the hypothalamus.

Elevated sympathetic nerve activity, strongly associated with cardiovascular disease, is partly generated from the presympathetic neurons of the paraventricular nucleus of the hypothalamus (PVN). The PVN-presympathetic neurons regulating cardiac and vasomotor sympathetic activity receive information about cardiovascular status from receptors in the heart and circulation. These receptors signal changes via afferent neurons terminating in the nucleus tractus solitarius (NTS), some of which may result in excitation or inhibition of PVN-presympathetic neurons. Understanding the anatomy and neurochemistry of NTS afferent connections within the PVN could provide important clues to the impairment in homeostasis cardiovascular control associated with disease. Transynaptic labelling has shown the presence of neuronal nitric oxide synthase (nNOS)-containing neurons and GABA interneurons that terminate on presympathetic PVN neurons any of which may be the target for NTS afferents. So far NTS connections to these diverse neuronal pools have not been demonstrated and were investigated in this study. Anterograde (biotin dextran amine - BDA) labelling of the ascending projection from the NTS and retrograde (fluorogold - FG or cholera toxin B subunit - CTB) labelling of PVN presympathetic neurons combined with immunohistochemistry for GABA and nNOS was used to identify the terminal neuronal targets of the ascending projection from the NTS. It was shown that NTS afferent terminals are apposed to either PVN-GABA interneurons or to nitric oxide producing neurons or even directly to presympathetic neurons. Furthermore, there was evidence that some NTS axons were positive for vesicular glutamate transporter 2 (vGLUT2). The data provide an anatomical basis for the different functions of cardiovascular receptors that mediate their actions via the NTS-PVN pathways.

Centrally administered bombesin activates COX-containing spinally projecting neurons of the PVN in anesthetized rats.

The paraventricular nucleus (PVN) of the hypothalamus has a heterogenous structure containing different types of output neurons that project to the median eminence, posterior pituitary, brain stem autonomic centers and sympathetic preganglionic neurons in the spinal cord. Presympathetic neurons in the PVN send mono- and poly-synaptic projections to the spinal cord. In the present study using urethane-anesthetized rats, we examined the effects of centrally administered bombesin (a homologue of the mammalian gastrin-releasing peptide) on the mono-synaptic spinally projecting PVN neurons pre-labeled with a retrograde tracer Fluoro-Gold (FG) injected into T8 level of the spinal cord, with regard to the immunoreactivity for cyclooxygenase (COX) isozymes (COX-1/COX-2) and Fos (a marker of neuronal activation). FG-labeled spinally projecting neurons were abundantly observed in the dorsal cap, ventral part and posterior part of the PVN. The immunoreactivity of each COX-1 and COX-2 was detected in FG-labeled spinally projecting PVN neurons in the vehicle (10 µl of saline/animal, i.c.v.)-treated group, while bombesin (1 nmol/animal, i.c.v.) had no effect on the number of these immunoreactive neurons for each COX isozyme with labeling of FG. On the other hand, the peptide significantly increased the number of double-immunoreactive neurons for Fos and COX-1/COX-2 with FG-labeling in the PVN (except triple-labeled neurons for FG, COX-2 and Fos in the dorsal cap of the PVN), as compared to those of vehicle-treated group. These results suggest that centrally administered bombesin activates spinally projecting PVN neurons containing COX-1 and COX-2 in rats.

The vasculature within the paraventricular nucleus of the hypothalamus in mice varies as a function of development, subnuclear location, and GABA signaling.

The paraventricular nucleus of the hypothalamus (PVN) is a cell group that plays important roles in regulating sympathetic vasomotor tone, food intake, neuroendocrine and autonomic stress responses, and cardiovascular function. The developing PVN is surrounded by neuronal elements containing, and presumably secreting, gamma-aminobutyric acid (GABA). The vasculature of the adult PVN is notably denser than in other brain regions or in the PVN during perinatal development. To characterize the postnatal angiogenic process in mice, blood vessels were analyzed at P8, 20, and 50 in rostral, mid, and caudal divisions of the PVN in males and females. Vascular changes relative to disruption of the R1 subunit of the GABA(B) receptor were evaluated at P8 and P20. For defined regions of interest within the PVN there were age dependent increases in blood vessel lengths and branching from P8 to 20 to 50 with the most notable increases in the middle region. Loss of GABA(B) receptors did not influence vascular characteristics at P8 in any region, but by P20 there was significantly (20%) less blood vessel length and branching in the mid-PVN region vs. wild type. These findings suggest that the loss of GABA(B) signaling may lead to a late developing defect in angiogenesis. The loss of vascularity with defective GABA(B) signaling suggests that neurovascular relationships in the PVN may be an important locus for understanding disorders of the hypothalamic-pituitary-adrenal axis with potential impact for psychiatric mood disorders along with other comorbid disorders that may be regulated by cells in the PVN.

Genistein stimulates the hypothalamo-pituitary-adrenal axis in adult rats: morphological and hormonal study.

Genistein, the soy isoflavone structurally similar to estradiol, is widely consumed for putative beneficial health effects. However, there is a lack of data about the genisteins' effects in adult males, especially its effects on the hipothalamo-pituitary-adrenal (HPA) axis. Therefore, the present study was carried out to investigate the effects of genistein on the HPA axis in orchidectomized adult rats, and to create a parallel with those of estradiol. Changes in the hypothalamic corticotrophin-releasing hormone (CRH) neurons and pituitary corticotrophs (ACTH cells) were evaluated stereologically, while corticosterone and ACTH levels were determined biochemically. Orchidectomy (Orx) provoked the enlargement (p<0.05) of: hypothalamic paraventricular nucleus volume (60%), percentage of CRH neurons (23%), percentage of activated CRH neurons (45%); pituitary weight (15%) and ACTH level (57%). In comparison with Orx, estradiol treatment provoked the enlargement (p<0.05) of: percentage of CRH neurons (28%), percentage of activated CRH neurons (2.7-fold), pituitary weight (131%) and volume (82%), ACTH level (69%), the serum (103%) and adrenal tissue (4.8 fold) level of corticosterone. Clearly, Orx has induced the increase in HPA axis activity, which even augments after estradiol treatment. Also, compared to Orx, genistein treatment provoked the enhancement (p<0.05) of: percentage of activated CRH neurons (2.3-fold), pituitary weight (28%) and volume (21%), total number of ACTH cells (22%) ACTH level (45%), the serum (2.6-fold) and adrenal tissue (2.8 fold) level of corticosterone. It can be concluded that an identical tendency, concerning the HPA axis parameters, follows estradiol and genistein administration to the orchidectomized adult rats.

MC4R dimerization in the paraventricular nucleus and GHSR/MC3R heterodimerization in the arcuate nucleus: is there relevance for body weight regulation?

The worldwide obesity epidemic is increasing, yet at this time, no long-acting and specific pharmaceutical therapies are available. Peripheral hormonal signals communicate metabolic status to the hypothalamus by activating their corresponding receptors in the arcuate nucleus (ARC). In this brain region, a variety of G protein-coupled receptors (GPCRs) are expressed that are potentially involved in weight regulation, but so far, the detailed function of most hypothalamic GPCRs is only partially understood. An important and underappreciated feature of GPCRs is the capacity for regulation via di- and heterodimerization. Increasing evidence implicates that heterodimerization of GPCRs results in profound functional consequences. Recently, we could demonstrate that interaction of the melanocortin 3 receptor (MC3R) and the growth hormone secretagogue receptor (GHSR)-1a results in a modulation of function in both receptors. Although the physiological role of GPCR-GPCR interaction in the hypothalamus is yet to be elucidated, this concept promises new avenues for investigation and understanding of hypothalamic functions dependent on GPCR signaling. Since GPCRs are important targets for drugs to combat many diseases, identification of heterodimers may be a prerequisite for highly specific drugs. Therefore, a detailed understanding of the mechanisms and their involvement in weight regulation is necessary. Fundamental to this understanding is the interplay of GPCR-GPCR in the hypothalamic nuclei in energy metabolism. In this review, we summarize the current knowledge on melanocortin receptors and GHSR-1a in hypothalamic weight regulation, especially as they pertain to possible drug targets. Furthermore, we include available evidence for the participation and significance of GPCR dimerization.

Hypocretin/orexin signaling in the hypothalamic paraventricular nucleus is essential for the expression of nicotine withdrawal.

BACKGROUND: Hypocretin (orexin) signaling is involved in drug addiction. In this study, we investigated the role of these hypothalamic neuropeptides in nicotine withdrawal by using behavioral and neuroanatomical approaches. METHODS: Nicotine withdrawal syndrome was precipitated by mecamylamine (2 mg/kg, subcutaneous) in C57BL/6J nicotine-dependent mice (25 mg/kg/day for 14 days) pretreated with the hypocretin receptor 1 (Hcrtr-1) antagonist SB334867 (5 and 10 mg/kg, intraperitoneal), the hypocretin receptor 2 antagonist TCSOX229 (5 and 10 mg/kg, intraperitoneal), and in preprohypocretin knockout mice. c-Fos expression was analyzed in several brain areas related to nicotine dependence by immunofluorescence techniques. Retrograde tracing with rhodamine-labeled fluorescent latex microspheres was used to determine whether the hypocretin neurons project directly to the paraventricular nucleus of the hypothalamus (PVN), and SB334867 was locally administered intra-PVN (10 nmol/side) to test the specific involvement of Hcrtr-1 in this brain area during nicotine withdrawal. RESULTS: Somatic signs of nicotine withdrawal were attenuated in mice pretreated with SB334867 and in preprohypocretin knockout mice. No changes were found in TCSOX229 pretreated animals. Nicotine withdrawal increased the percentage of hypocretin cells expressing c-Fos in the perifornical, dorsomedial, and lateral hypothalamus. In addition, the increased c-Fos expression in the PVN during withdrawal was dependent on hypocretin transmission through Hcrtr-1 activation. Hypocretin neurons directly innervate the PVN and the local infusion of SB334867 into the PVN decreased the expression of nicotine withdrawal. CONCLUSIONS: These data demonstrate that hypocretin signaling acting on Hcrtr-1 in the PVN plays a crucial role in the expression of nicotine withdrawal.

Cyto- and chemoarchitecture of the hypothalamic paraventricular nucleus in the C57BL/6J male mouse: a study of immunostaining and multiple fluorescent tract tracing.

The paraventricular nucleus of the hypothalamus (PVH) plays a critical role in the regulation of autonomic, neuroendocrine, and behavioral activities. This understanding has come from extensive characterization of the PVH in rats, and for this mammalian species we now have a robust model of basic PVH neuroanatomy and function. However, in mice, whose use as a model research animal has burgeoned with the increasing sophistication of tools for genetic manipulation, a comparable level of PVH characterization has not been achieved. To address this, we employed a variety of fluorescent tract tracing and immunostaining techniques in several different combinations to determine the neuronal connections and cyto- and chemoarchitecture of the PVH in the commonly used C57BL/6J male mouse. Our findings reveal a distinct organization in the mouse PVH that is substantially different from the PVH of male rats. The differences are particularly evident with respect to the spatial relations of two principal neuroendocrine divisions (magnocellular and parvicellular) and three descending preautonomic populations in the PVH. We discuss these data in relation to what is known about PVH function and provide the work as a resource for further studies of the neuronal architecture and function of the mouse PVH.

Brain arginine vasotocin immunoreactivity differs between urban and desert curve-billed thrashers, Toxostoma curvirostre: relationships with territoriality and stress physiology.

The neuropeptide arginine vasotocin (AVT: the avian homolog of vasopressin) has numerous functional roles including mediating social behaviors, coregulating the adrenocortical stress response and maintaining water balance. These functions of AVT make it susceptible to environmental influence, yet little is understood concerning the variation in the AVT system across habitats. In this study, AVT immunoreactivity was compared between male curve-billed thrashers, Toxostoma curvirostre, from native Sonoran Desert locations and those within the city of Phoenix, Ariz. Previous research found that urban thrashers are more responsive to territorial intrusion, secrete more corticosterone (CORT) during capture stress, and they may also have greater access to water than desert counterparts. Variation in AVT immunoreactivity was also related to levels of plasma CORT and osmolality, and with behavioral responses to a simulated territorial intrusion. Birds from these two habitats showed different AVT immunoreactive patterns in two brain regions: the paraventricular nucleus of the hypothalamus and the medial bed nucleus of the stria terminalis (BSTM), a part of the limbic system. Immunoreactive AVT within the paraventricular nucleus was associated with plasma CORT levels in urban, but not desert, birds, but no such association with osmolality was observed in birds from either habitat. The total number of BSTM AVT-immunoreactive cells was related to a decreased responsiveness to territorial intrusion. These data suggest that divergence in the AVT system between urban and desert thrashers may help explain observed differences in both the adrenocortical stress response and territorial behavior between populations. Whether differences in water availability between habitats contribute to population differences in the brain AVT system is unknown.

Social status and sex effects on neural morphology in Damaraland mole-rats, Fukomys damarensis.

We previously reported that in a eusocial rodent, the naked mole-rat (Heterocephalus glaber), traditional neural sex differences were absent; instead, neural dimorphisms were associated with breeding status. Here we examined the same neural regions previously studied in naked mole-rats in a second eusocial species, the Damaraland mole-rat (Fukomys damarensis). Damaraland mole-rats live in social groups with breeding restricted to a small number of animals. However, colony sizes are much smaller in Damaraland mole-rats than in naked mole-rats and there is consequently less reproductive skew. In this sense, Damaraland mole-rats may be considered intermediate in social organization between naked mole-rats and more traditional laboratory rodents. We report that, as in naked mole-rats, breeding Damaraland mole-rats have larger volumes of the principal nucleus of the bed nucleus of the stria terminalis and paraventricular nucleus of the hypothalamus than do subordinates, with no effect of sex on these measures. Thus, these structures may play special roles in breeders of eusocial species. However, in contrast to what was seen in naked mole-rats, we also found sex differences in Damaraland mole-rats: volume of the medial amygdala and motoneuron number in Onuf's nucleus were both greater in males than in females, with no significant effect of breeding status. Thus, both sex and breeding status influence neural morphology in Damaraland mole-rats. These findings are in accord with the observed sex differences in body weight and genitalia in Damaraland but not naked mole-rats. We hypothesize that the increased sexual dimorphism in Damaraland mole-rats relative to naked mole-rats is related to reduced reproductive skew.

Forebrain origins of glutamatergic innervation to the rat paraventricular nucleus of the hypothalamus: differential inputs to the anterior versus posterior subregions.

The hypothalamic paraventricular nucleus (PVN) regulates numerous homeostatic systems and functions largely under the influence of forebrain inputs. Glutamate is a major neurotransmitter in forebrain, and glutamate neurosignaling in the PVN is known to mediate many of its functions. Previous work showed that vesicular glutamate transporters (VGluTs; specific markers for glutamatergic neurons) are expressed in forebrain sites that project to the PVN; however, the extent of this presumed glutamatergic innervation to the PVN is not clear. In the present study retrograde FluoroGold (FG) labeling of PVN-projecting neurons was combined with in situ hybridization for VGluT1 and VGluT2 mRNAs to identify forebrain regions that provide glutamatergic innervation to the PVN and its immediate surround in rats, with special consideration for the sources to the anterior versus posterior PVN. VGluT1 mRNA colocalization with retrogradely labeled FG neurons was sparse. VGluT2 mRNA colocalization with FG neurons was most abundant in the ventromedial hypothalamus after anterior PVN FG injections, and in the lateral, posterior, dorsomedial, and ventromedial hypothalamic nuclei after posterior PVN injections. Anterograde tract tracing combined with VGluT2 immunolabeling showed that 1) ventromedial nucleus-derived glutamatergic inputs occur in both the anterior and posterior PVN; 2) posterior nucleus-derived glutamatergic inputs occur predominantly in the posterior PVN; and 3) medial preoptic nucleus-derived inputs to the PVN are not glutamatergic, thereby corroborating the innervation pattern seen with retrograde tracing. The results suggest that PVN subregions are influenced by varying amounts and sources of forebrain glutamatergic regulation, consistent with functional differentiation of glutamate projections.

Projections of the suprachiasmatic nucleus and ventral subparaventricular zone in the Nile grass rat (Arvicanthis niloticus).

The phases of many circadian rhythms differ between diurnal and nocturnal species. However, rhythms within the hypothalamic suprachiasmatic nucleus (SCN), which contains the central circadian pacemaker, are very similar, suggesting that the mechanisms underlying phase preference lie downstream of the SCN. Rhythms in Fos expression in the ventral subparaventricular zone (vSPVZ), a major target of the SCN, differ substantially between diurnal Nile grass rats and nocturnal lab rats, raising the possibility that the vSPVZ modulates the effects of SCN signals at its targets. To understand better how and where the SCN and vSPVZ communicate circadian signals within the grass rat brain, we mapped their projections using the anterograde tracer biotinylated dextran amine (BDA). Adult female grass rats received unilateral BDA injections directed at the SCN or vSPVZ and their brains were perfusion-fixed several days later. Immunohistochemistry revealed that the distribution patterns of SCN and vSPVZ efferents were very similar. Labeled fibers originating in each region were heavily concentrated in the medial preoptic area, paraventricular thalamic nucleus, the subparaventricular zone, and the hypothalamic paraventricular and dorsomedial nuclei. BDA-labeled fibers from the SCN and vSPVZ formed appositions with orexin neurons and gonadotropin-releasing hormone neurons, two cell populations whose rhythms in Fos expression track temporally reversed patterns of locomotor and reproductive behavior, respectively, in diurnal and nocturnal rodents. These data demonstrate that projections of the SCN and vSPVZ are highly conserved in diurnal and nocturnal rodents, and the vSPVZ projections may enable it to modulate the responsiveness of target cells to signals from the SCN.