Fos-positive Nuclei Research Articles

Electroacupuncture Attenuates Anxiety-Like Behaviors in a Rat Model of Post-traumatic Stress Disorder: The Role of the Ventromedial Prefrontal Cortex.

Electroacupuncture (EA) is a promising clinical approach to treating posttraumatic stress disorder (PTSD), yet the mechanisms whereby EA can alleviate anxiety and other PTSD symptoms have yet to be clarified. In the present report, rats underwent EA for 14 consecutive days following modified single prolonged stress (MSPS) exposure. These animals were then evaluated in open field and elevated plus maze tests (OFT and EPM), while Fos immunohistochemical staining was performed to assess ventromedial prefrontal cortex (vmPFC) functional activation. In addition, an extracellular recording and stimulation system was used to analyze vmPFC inputs into the ventral tegmental area (VTA) in these rats. Temporary vmPFC inactivation was further performed to assess whether this was sufficient to reverse the anxiolytic effects of EA. Overall, rats that underwent EA treatment spent more time in the central region (OFT) and the open arm (EPM) relative to MSPS model animals (P < 0.05). These MSPS model animals also exhibited significantly fewer activated Fos-positive nuclei in the vmPFC following behavioral testing, while EA was associated with a significant relative increase in c-Fos expression in this region. The transient inactivation of the vmPFC was sufficient to reverse the effects of EA treatment on anxiety-like behaviors in MSPS model rats. MSPS and SEA rats exhibiting no differences in bursting activity between baseline and vmPFC stimulation, whereas bursting activity rose relative to baseline upon ventral mPFC stimulation in EA treated and control rats. Together, these findings indicate that the vmPFC and its inputs into the VTA are functionally linked to the anxiolytic activity of EA, implicating this pathway in the EA-mediated treatment of PTSD.

Integration of Hypernatremia and Angiotensin II by the Organum Vasculosum of the Lamina Terminalis Regulates Thirst.

The organum vasculosum of the lamina terminalis (OVLT) contains NaCl-sensitive neurons to regulate thirst, neuroendocrine function, and autonomic outflow. The OVLT also expresses the angiotensin II (AngII) type1 receptor, and AngII increases Fos expression in OVLT neurons. The present study tested whether individual OVLT neurons sensed both NaCl and AngII to regulate thirst and body fluid homeostasis. A multifaceted approach, including in vitro whole-cell patch recordings, in vivo single-unit recordings, and optogenetic manipulation of OVLT neurons, was used in adult, male Sprague Dawley rats. First, acute intravenous infusion of hypertonic NaCl or AngII produced anatomically distinct patterns of Fos-positive nuclei in the OVLT largely restricted to the dorsal cap versus vascular core, respectively. However, in vitro patch-clamp recordings indicate 66% (23 of 35) of OVLT neurons were excited by bath application of both hypertonic NaCl and AngII. Similarly, in vivo single-unit recordings revealed that 52% (23 of 44) of OVLT neurons displayed an increased discharge to intracarotid injection of both hypertonic NaCl and AngII. In marked contrast to Fos immunoreactivity, neuroanatomical mapping of Neurobiotin-filled cells from both in vitro and in vivo recordings revealed that NaCl- and AngII-responsive neurons were distributed throughout the OVLT. Next, optogenetic excitation of OVLT neurons stimulated thirst but not salt appetite. Conversely, optogenetic inhibition of OVLT neurons attenuated thirst stimulated by hypernatremia or elevated AngII but not hypovolemia. Collectively, these findings provide the first identification of individual OVLT neurons that respond to both elevated NaCl and AngII concentrations to regulate thirst and body fluid homeostasis.SIGNIFICANCE STATEMENT Body fluid homeostasis requires the integration of neurohumoral signals to coordinate behavior, neuroendocrine function, and autonomic function. Extracellular NaCl concentrations and the peptide hormone angiotensin II (AngII) are two major neurohumoral signals that regulate body fluid homeostasis. Herein, we present the first compelling evidence that individual neurons located in the organum vasculosum of the lamina terminalis detect both NaCl and AngII. Furthermore, optogenetic interrogations demonstrate that these neurons play a pivotal role in the regulation of thirst stimulated by NaCl and AngII. These novel observations lay the foundation for future investigations for how such inputs as well as others converge onto unique organum vasculosum of the lamina terminalis neurons to coordinate body fluid homeostasis and contribute to disorders of fluid balance.

Temporal effect of electroacupuncture on anxiety-like behaviors and c-Fos expression in the anterior cingulate cortex in a rat model of post-traumatic stress disorder

Post-traumatic stress disorder (PTSD) is a psychiatric disease which leads to a series of anxiety-like behaviors. In this study, we investigated the temporal effects of electroacupuncture (EA) at acupoint ST36 on anxiety-like behaviors and the expression of c-Fos in the anterior cingulate cortex (ACC) in a rat model of PTSD. PTSD was induced by a single prolonged stress procedure comprising three stages: restraint for 2 h, forced swim for 20 min, and pentobarbital sodium anesthesia. EA at acupoint ST36 was performed from 7:00-9:00 once a day for 7 consecutive days. Open field test (OFT) and elevated plus maze (EPM) test were used to assess the success of the model and evaluate anxiety-like behaviors. Immunohistochemistry was used to detect Fos-positive nuclei in the ACC. We observed that EA performed from 7:00-9:00 was associated with significantly more time spent in the center area during the OFT and in the open arm during the EPM, as well as lower corticosterone response compared with that of regular EA (P < 0.05). PTSD rats expressed significantly less c-Fos in the ACC. Timed EA significantly increased c-Fos expression in the ACC. The effect of timed EA acting on PTSD rats was linked to altered neuronal activation in the ACC. Compared to regular EA, timed EA exhibited superior therapeutic effects by attenuating anxiety-like behaviors in PTSD rats. These results emphasize the association between temporal parameters of EA manipulation and acupuncture effects. Timed acupuncture therapy may be a novel therapeutic application in the treatment of PTSD.

Effects of lateral olfactory tract stimulation on Fos immunoreactivity in vasopressin neurones of the rat piriform cortex.

In the main olfactory system, odours are registered at the main olfactory epithelium and are then processed at the main olfactory bulb (MOB) and, subsequently, by the anterior olfactory nucleus (AON), the piriform cortex (PC) and the cortical amygdala. Previously, we reported populations of vasopressin neurones in different areas of the rat olfactory system, including the MOB, accessory olfactory bulb (AOB) and the AON and showed that these are involved in the coding of social odour information. Utilising immunohistochemistry and a transgenic rat in which an enhanced green fluorescent protein reporter gene is expressed in vasopressin neurones (eGFP-vasopressin), we now show a population of vasopressin neurones in the PC. The vasopressin neurones are predominantly located in the layer II of the PC and the majority co-express the excitatory transmitter glutamate. Furthermore, there is no sex difference in the number of neurones expressing vasopressin. Electrical stimulation of the lateral olfactory tract leads to a significant increase in the number of Fos-positive nuclei in the PC, MOB, AOB, dorsal AON and supraoptic nucleus (SON). However, there was only a significant increase in Fos expression in vasopressin cells of the PC and SON. Thus, functionally distinct populations of vasopressin cells are implicated in olfactory processing at multiple stages of the olfactory pathway.

Central Administration of Insulin and Leptin Together Enhance Renal Sympathetic Nerve Activity and Fos Production in the Arcuate Nucleus.

There is considerable interest in the central actions of insulin and leptin. Both induce sympatho-excitation. This study (i) investigated whether centrally administered leptin and insulin together elicits greater increases in renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) than when given alone, and (ii) quantified the number of activated neurons in brain regions influencing SNA, to identify potential central sites of interaction. In anesthetised (urethane 1.4–1.6 g/kg iv) male Sprague-Dawley rats, RSNA, MAP, and HR were recorded following intracerebroventricular (ICV) saline (control; n = 5), leptin (7 μg; n = 5), insulin (500 mU; n = 4) and the combination of leptin and insulin; (n = 4). Following leptin or insulin alone, RSNA was significantly increased (74 and 62% respectively). MAP responses were not significantly different between the groups. Insulin alone significantly increased HR. Leptin alone also increased HR but it was significantly less than following insulin alone (P < 0.005). When leptin and insulin were combined, the RSNA increase (124%) was significantly greater than the response to either alone. There were no differences between the groups in MAP responses, however, the increase in HR induced by insulin was attenuated by leptin. Of the brain regions examined, only in the arcuate nucleus did leptin and insulin together increase the number of Fos-positive cell nuclei significantly more than leptin or insulin alone. In the lamina terminalis and rostroventrolateral medulla, leptin and insulin together increased Fos, but the effect was not greater than leptin alone. The results suggest that when central leptin and insulin levels are elevated, the sympatho-excitatory response in RSNA will be greater. The arcuate nucleus may be a common site of cardiovascular integration.

Rats showing low and high sensitization of frequency-modulated 50-kHz vocalization response to amphetamine differ in amphetamine-induced brain Fos expression

Individuals predisposed to addiction constitute a minority of drug users, in both humans and animal models of the disorder, but there are no established characteristics that would allow identifying them beforehand. Our studies demonstrate that sensitization of rat 50-kHz ultrasonic vocalization (USV) response to amphetamine shows marked inter-individual diversity but substantial intra-individual stability. Low sensitization of the response shows relevance to the acquisition of self-administration of this drug and hence might be of predictive value regarding the risk of addiction. We compared amphetamine-induced Fos expression in 16 brain regions considered important for the development of addiction between rats preselected for low and high sensitization of the response and next given nine daily amphetamine doses followed by a 2-week withdrawal and final amphetamine challenge. Ventral tegmental area and nucleus accumbens shell Fos-positive nuclei counts correlated positively with 50-kHz USV response to the challenge in high-sensitized rats. Compared to those in amphetamine-untreated controls, Fos-positive nuclei counts were significantly and markedly (2–6 times) higher in 12 regions in high-sensitized rats, whereas in low-sensitized rats they were significantly higher in the cingulate cortex and dorsomedial striatum only. The difference in the counts between the latter two subsets reached statistical significance in dorsomedial and dorsolateral striatum and three out of four cortical regions studied. The fact that the diversification was most distinct in dorsal striatum that plays a critical role in the transition from controlled to compulsive drug intake suggests that the USV-based categorization may be related to divergent vulnerability of rats to AMPH addiction.

DREADD-induced activation of subfornical organ neurons stimulates thirst and salt appetite.

The subfornical organ (SFO) plays a pivotal role in body fluid homeostasis through its ability to integrate neurohumoral signals and subsequently alter behavior, neuroendocrine function, and autonomic outflow. The purpose of the present study was to evaluate whether selective activation of SFO neurons using virally mediated expression of Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) stimulated thirst and salt appetite. Male C57BL/6 mice (12-15 wk) received an injection of rAAV2-CaMKII-HA-hM3D(Gq)-IRES-mCitrine targeted at the SFO. Two weeks later, acute injection of clozapine N-oxide (CNO) produced dose-dependent increases in water intake of mice with DREADD expression in the SFO. CNO also stimulated the ingestion of 0.3 M NaCl. Acute injection of CNO significantly increased the number of Fos-positive nuclei in the SFO of mice with robust DREADD expression. Furthermore, in vivo single-unit recordings demonstrate that CNO significantly increases the discharge frequency of both ANG II- and NaCl-responsive neurons. In vitro current-clamp recordings confirm that bath application of CNO produces a significant membrane depolarization and increase in action potential frequency. In a final set of experiments, chronic administration of CNO approximately doubled 24-h water intake without an effect on salt appetite. These findings demonstrate that DREADD-induced activation of SFO neurons stimulates thirst and that DREADDs are a useful tool to acutely or chronically manipulate neuronal circuits influencing body fluid homeostasis.

Osmoregulatory thirst in mice lacking the transient receptor potential vanilloid type 1 (TRPV1) and/or type 4 (TRPV4) receptor.

Recent studies suggest the ability of the central nervous system to detect changes in osmolality is mediated by products of the genes encoding the transient receptor potential vanilloid-1 (TRPV1) or vanilloid-4 (TRPV4) channel. The purpose of the present study was to determine whether deletion of TRPV1 and/or TRPV4 channels altered thirst responses to cellular dehydration in mice. Injection of 0.5 or 1.0 M NaCl produced dose-dependent increases in cumulative water intakes of wild-type (WT), TRPV1-/-, TRPV4-/-, and TRPV1-/-V4-/- mice. However, there were no differences in cumulative water intakes between WT versus any other strain despite similar increases in plasma electrolytes and osmolality. Similar results were observed after injection of hypertonic mannitol. This was a consistent finding regardless of the injection route (intraperitoneal vs. subcutaneous) or timed access to water (delayed vs. immediate). There were also no differences in cumulative intakes across strains after injection of 0.15 M NaCl or during a time-controlled period (no injection). Chronic hypernatremia produced by sole access to 2% NaCl for 48 h also produced similar increases in water intake across strains. In a final set of experiments, subcutaneous injection of 0.5 M NaCl produced similar increases in the number of Fos-positive nuclei within the organum vasculosum of the lamina terminalis and median preoptic nucleus across strains but significantly smaller number in the subfornical organ of WT versus TRPV1-/-V4-/- mice. Collectively, these findings suggest that TRPV1 and/or TRPV4 channels are not the primary mechanism by which the central nervous system responds to cellular dehydration during hypernatremia or hyperosmolality to increase thirst.

Effects of electroacupuncture on ethanol-induced impairments of spatial learning and memory and Fos expression in the hippocampus in rats

It is well established that alcohol impairs spatial learning and memory. Here, we investigated the effects of electroacupuncture (EA) at ST36 or nonacupoint on ethanol-induced learning and memory impairment and the expression of Fos in the hippocampus. Ethanol (5g/kg) was administered intragastrically once a day for 5 consecutive days; 2Hz EA was administered immediately after ethanol exposure. After a 2-day ethanol abstinence, for 6 consecutive days, the rats were submitted to Morris water maze training. Probe trials were performed on 1 day after the final training session. We also applied immunohistochemistry to detect Fos-positive nuclei in the hippocampus. We found that 5-day ethanol exposure markedly decreased spatial learning and memory abilities in the Morris water maze task as indicated by escape latency and time in the target quadrant. EA treatment shortened the time of reaching platform and increased times traveled in the target quadrant (P<0.05). Animals administered with ethanol emitted significantly fewer Fos expression in the hippocampal CA1 area. EA increased Fos expression in the hippocampal CA1 area. Significant correlations were obtained between Fos protein expression in CA1 and time in the target quadrant. Altogether, these results suggest that EA protects against ethanol-induced impairments of spatial learning and memory, which may be involved in the hippocampal CA1 area. EA treatment may provide a novel nonpharmacological strategy for ethanol-induced learning and memory impairment.

Acute hypernatremia promotes anxiolysis and attenuates stress-induced activation of the hypothalamic–pituitary–adrenal axis in male mice

Previous investigation by our laboratory found that acute hypernatremia potentiates an oxytocinergic tone that inhibits parvocellular neurosecretory neurons in the paraventricular nucleus of the hypothalamus (PVN), attenuates restraint-induced surges in corticosterone (CORT), and reduces anxiety-like behavior in male rats. To investigate the neural mechanisms mediating these effects and extend our findings to a more versatile species, we repeated our studies using laboratory mice. In response to 2.0M NaCl injections, mice had increased plasma sodium concentrations which were associated with a blunted rise in CORT subsequent to restraint challenge relative to 0.15M NaCl injected controls. Immunofluorescent identification of the immediate early gene product Fos found that 2.0M NaCl treatment increased the number of activated neurons producing oxytocin in the PVN. To evaluate the effect of acute hypernatremia on PVN neurons producing corticotropin-releasing hormone (CRH), we used the Cre-lox system to generate mice that produced the red fluorescent protein, tdTomato, in cells that had Cre-recombinase activity driven by CRH gene expression. Analysis of brain tissue from these CRH-reporter mice revealed that 2.0M NaCl treatment caused a dramatic reduction in Fos-positive nuclei specifically in CRH-producing PVN neurons. This altered pattern of activity was predictive of alleviated anxiety-like behavior as mice administered 2.0M NaCl spent more time exploring the open arms of an elevated-plus maze than 0.15M NaCl treated controls. Taken together, these results further implicate an oxytocin-dependent inhibition of CRH neurons in the PVN and demonstrate the impact that slight elevations in plasma sodium have on hypothalamic–pituitary–adrenocortical axis output and anxiety-like behavior.

Evidence for anti-inflammatory and putative analgesic effects of a monoclonal antibody to calcitonin gene-related peptide

BackgroundCalcitonin gene-related peptide (CGRP) is a powerful pro-inflammatory mediator thought to play a significant role in the development of inflammation and pain. We investigated the role of CGRP in trigeminal inflammatory pain by determining the ability of a monoclonal antibody to CGRP to modify central Fos expression in response to stimulation of the inflamed ferret tooth pulp. We also assessed the effect of the antibody on pulpal inflammation. MethodsTen adult ferrets were prepared under anaesthesia to allow stimulation of the upper and lower left canine pulps, recording from the digastric muscle and intravenous injections at subsequent experiments. In all animals, pulpal inflammation was induced by introducing human caries into a deep buccal cavity. Four days later animals were treated intravenously with either CGRP antibody (n=5) or vehicle (n=5). After a further 2days animals were re-anaesthetised and the tooth pulps stimulated at 10times jaw-opening reflex threshold. Brainstems and tooth pulps were processed immunohistochemically for Fos and the common leucocyte marker CD45, respectively. ResultsFos was expressed in ipsilateral trigeminal subnuclei caudalis (Vc) and oralis (Vo). Significantly fewer Fos-positive nuclei were present within Vc of CGRP antibody-treated animals (p=0.003 vs vehicle-treated). Mean percentage area of staining for CD45 was significantly less in antibody-treated animals (p=0.04 vs vehicle-treated). ConclusionsThis is the first direct evidence that sequestration of CGRP has anti-inflammatory and putative analgesic effects. Previous studies using this Fos model have demonstrated that it is able to predict clinical analgesic efficacy. Thus these data indicate that this antibody may have analgesic effects in dental pain and other types of inflammatory-mediated transmission, and suggest that this is in part due to peripheral anti-inflammatory effects.

Acute behavioural responses to nicotine and nicotine withdrawal syndrome are modified in GABAB1 knockout mice

Nicotine is the main active component of tobacco, and has both acute and chronic pharmacological effects that can contribute to its abuse potential in humans. The aim of the present study was to evaluate a possible role of GABAB receptors in acute and chronic responses to nicotine administration, by comparing GABAB1 knockout mice and their wild-type littermates. In wild-type mice, acute nicotine administration (0.5, 1, 3 and 6 mg/kg, sc) dose-dependently decreased locomotor activity, and induced antinociceptive responses in the tail-immersion and hot-plate tests. In GABAB1 knockout mice, the hypolocomotive effect was observed only with the highest dose of nicotine, and the antinociceptive responses in both tests were significantly reduced in GABAB1 knockout mice compared to their wild-type littermate. Additionally, nicotine elicited anxiolytic- (0.05 mg/kg) and anxiogenic-like (0.8 mg/kg) responses in the elevated plus-maze test in wild-type mice, while selectively the anxiolytic-like effect was abolished in GABAB1 knockout mice. We further investigated nicotine withdrawal in mice chronically treated with nicotine (25 mg/kg/day, sc). Mecamylamine (1 mg/kg, sc) precipitated several somatic signs of nicotine withdrawal in wild-type mice. However, signs of nicotine withdrawal were missing in GABAB1 knockout mice. Finally, there was a decreased immunoreactivity of Fos-positive nuclei in the bed nucleus of the stria terminalis, basolateral amygdaloid nucleus and hippocampal dentate gyrus in abstinent wild-type but not in GABAB1 knockout mice. These results reveal an interaction between the GABAB system and the neurochemical systems through which nicotine exerts its acute and long-term effects.

Electroacupuncture Suppresses Discrete Cue-Evoked Heroin-Seeking and Fos Protein Expression in the Nucleus Accumbens Core in Rats

Relapse to drug seeking was studied using a rodent model of reinstatement induced by exposure to drug-related cues. Here, we used intravenous drug self-administration procedures in rats to further investigate the beneficial effects of electroacupuncture (EA) on heroin-seeking behavior in a reinstatement model of relapse. We trained Sprague-Dawley rats to nose-poke for i.v. heroin either daily for 4 h or 25 infusions for 14 consecutive days. Then the rats were abstinent from heroin for two weeks. 2 Hz EA stimulation was conducted once daily for 14 days during heroin abstinence. We tested these animals for contextual and discrete cue-induced reinstatement of active responses. We also applied immunohistochemistry to detect Fos-positive nuclei in the nucleus accumbens (NACc) core and shell after reinstatement test. We found that active responses elicited by both contextual cues and discrete cues were high in the rats trained with heroin than in saline controls. EA treatment significantly reduced active responses elicited by discrete cues. EA stimulation attenuated Fos expression in the core but not the shell of the NACc. Altogether, these results highlight the therapeutic benefit of EA in preventing relapse to drug addiction.

Differential feeding behavior and neuronal responses to CCK in obesity-prone and -resistant rats

Deficits in satiation signals are strongly suspected of accompanying obesity and contributing to its pathogenesis in both humans and rats. One such satiation signal is cholecystokinin (CCK), whose effects on food intake are diminished in animals adapted to a high fat diet. In this study, we tested the hypothesis that diet-induced obese prone (OP) rats exhibit altered feeding and vagal responses to systemic (IP) administration of CCK-8 compared to diet-induced obese resistant (OR) rats. We found that CCK (4.0 μg/kg) suppressed food intake significantly more in OP than OR rats. To determine whether enhanced suppression of feeding is accompanied by altered vagal sensory responsiveness, we examined dorsal hindbrain expression of Fos-like immunoreactivity (Fos-Li) following IP CCK injection in OP and OR rats. After 4.0 μg/kg CCK, there were significantly more Fos-positive nuclei in the NTS of OP compared to OR rats. Treatment with 8.0 μg/kg CCK resulted in no significant difference in food intake or in Fos-Li between OP and OR rats. Also, we found that OP rats were hyperphagic on a regular chow diet and gained more weight compared to OR rats. Finally OP rats had decreased relative fat pad mass compared to OR rats. Collectively, these results show that OP rats exhibit a different behavioral and vagal neuronal responses to CCK than OR rats.

Involvement of hypothalamic peptides in the anorectic action of the CB1 receptor antagonist rimonabant (SR 141716)

Numerous studies have demonstrated that administration of rimonabant (SR 141716), a CB(1) receptor antagonist, causes a decrease in energy intake. However, the mechanisms by which rimonabant exerts its anorectic actions are unclear. The main focus of the study reported here was to establish the chemical identity of neurons that may subserve the anorectic effects of rimonabant. As such three approaches were utilised: (i) the identification of rimonabant-activated neurons using Fos as a marker of neuronal activity; (ii) the identification of the chemical phenotype of rimonabant-activated neurons by combining immunocytochemical identification of Fos and feeding-related peptides; and (iii) the evaluation of the effect of rimonabant on messenger RNA (mRNA) and protein for a number of feeding-related peptides. Rimonabant-induced Fos-positive nuclei were localized within a range of discrete hypothalamic regions with a predominance in the parvocellular part of the paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus, arcuate nucleus and lateral hypothalamic area. Furthermore, Fos labelling within these hypothalamic regions was colocalized with anorexigenic and orexigenic peptides including melanin-concentrating hormone (MCH), orexin, cocaine- and amphetamine-regulated transcript (CART) and alpha-melanocyte-stimulating hormone (alpha-MSH). Rimonabant specifically induced a decrease in NPY and an increase in CART and alpha-MSH mRNA and protein, consistent with its effect in reducing food intake and increasing energy expenditure. As such these data provide insights into the mechanisms of action that may underpin rimonabant's effects on energy balance and body weight.

Deletion of Both Transient Receptor Potential Vanilloid‐1 (TRPV1) and TRPV4 Genes Disrupts Osmoregulatory Thirst and Central Fos Activation

Recent evidence suggests that TRPV1 and TRPV4 channels are the putative link between plasma hypernatremia and brain osmosensation. However, deletion of either gene does not profoundly disrupt osmoregulatory responses, thereby raising the possibility that the other gene compensates to permit normal osmoregulation. Therefore, we hypothesized that mice lacking both TRPV1 and TRPV4 genes (TRPV1xV4‐/‐, n=9) vs wild‐type (n=10) mice would have attenuated thirst responses to plasma hypernatremia. Injection (0.5mL, sc) of 0.5 M NaCl produced smaller increases in water intake of TRPV1xV4‐/‐ vs wild‐type mice (0.37±0.02 vs 0.49±0.03mL, P&lt;0.05). TRPV1xV4‐/‐ vs wild‐type mice also ingested less water after chronic salt loading by sole access to 2% NaCl for 48 hrs (0.06±0.03 vs 0.24±0.04mL, P&lt;0.01). Yet, hypovolemia produced by 30% polyethylene glycol (0.5mL, sc) produced similar increases in water intake between TRPV1xV4‐/‐ vs wild‐type mice (0.67±0.04 vs 0.72±0.09mL). In addition, TRPV1xV4‐/‐ vs wild‐type mice displayed smaller numbers of Fos‐positive nuclei in brain osmosensory regions after injection of 0.5 M NaCl: organum vasculosum of the lamina terminalis (15±3 vs 33±6, P&lt;0.05) and subfornical organ (4±2 vs 27±4, P&lt;0.05). These findings suggest that TRPV1 and TRPV4 channels act in concert to participate in osmoregulation. Supported by NIH HL090826, AHA 0630202N &amp; 0815372D

Suckling-induced oxytocin increase in the spinal cord of the rat

Oxytocin (OT) is essential for parturition and milk ejection, and OT-containing fibers are present in several regions of the brain and in the spinal cord. During lactation, activation of spinal cord neurons by suckling stimulation involves deep laminae III–X including sympathetic preganglionic neurons of the intermedio-medial cell column. In the present study, experiments were designed to determine if the suckling provided by the litter increased OT levels in the spinal cord of dams, as determined by competitive immunoassay. In addition, we investigated if OT fibers reach neurons of the spinal cord that are known to respond to suckling. The OT content was higher in the hypothalamus than in the spinal cord in animals from all experimental groups. After 6 h of pup separation, OT levels decreased and suckling for 5 min induced a significant increase of OT levels in the spinal cord. Double immunostaining for Fos and OT showed OT-positive fibers adjacent to neurons that had Fos-positive nuclei, located mostly in laminae III, IV, and X. The present data support the notion that OT is released within the spinal cord in response to suckling, suggesting a role for this peptide in modulating the afferent and/or efferent responses generated by suckling.

Estrogen enhances light‐induced activation of dorsal raphe serotonergic neurons

The serotonergic system has been implicated in the modulation of physiological processes including circadian rhythms, learning, memory, mood and food intake. In females, cessation of ovarian function produces deleterious changes in all of these processes and estrogen treatment often ameliorates these conditions. Estrogen may produce these effects by acting on the midbrain raphe, an estrogen-sensitive region that receives direct projections from sensory systems. Here we examined the ability of estradiol to modulate neuronal responses of neurons within raphe nuclei to photic stimulation. Ovariectomized rats treated with estradiol or cholesterol were killed 1 h after the normal onset of light (Zeitgeber time 0) or after a 2-h phase advance (Zeitgeber time 22). In a second study, estradiol-treated ovariectomized rats under constant dark conditions were exposed to light 2 h before the subjective onset of circadian time [(CT)22] and killed 1 h later (CT23). The brains from all animals were processed for Fos and/or serotonin (5-HT) immunocytochemistry. Comparisons showed that the phase shift increased Fos immunoreactivity in all dorsal raphe nucleus (DRN) regions. Although estradiol did not alter the overall number of Fos-positive nuclei, it significantly increased the number of Fos/5-HT double-labelled cells in the medial and lateral DRN. In contrast, neither a phase shift nor estradiol altered the number of Fos-immunoreactive cells or the proportion of Fos-positive 5-HT cells in the median raphe nucleus. Results reveal that the DRN 5-HT system responds to changes in the light : dark cycle and that these responses are modulated by estrogen.

Mineralocorticoid Treatment Upregulates the Hypothalamic Vasopressinergic System of Spontaneously Hypertensive Rats

Mineralocorticoid effects in the brain include the control of cardiovascular functions, induction of salt appetite, interaction with the vasoactive neuropeptides arginine vasopressin (AVP) and angiotensin II and development or aggravation of hypertension. In this regard, mineralocorticoids may play a pathogenic role in rats with a genetic form of hypertension (spontaneously hypertensive rats, SHR). Our objective was to compare the response of the hypothalamic vasopressinergic system to mineralocorticoid administration in SHR and control Wistar-Kyoto (WKY) rats. Sixteen-week-old male SHR showing a systolic blood pressure of 190 ± 5 mm Hg and normotensive WKY rats (130 ± 5 mm Hg) were treated subcutaneously with oil vehicle or a single 10-mg dose of deoxycorticosterone acetate (DOCA). After 2 h, rats were sacrificed and brains prepared for immunocytochemistry of Fos and vasopressin V1a receptor (V1aR) and for non-isotopic in situ hybridization of AVP mRNA. In the basal state, SHR demonstrated a higher number of AVP mRNA- and V1aR-immunopositive cells in the magnocellular division of the paraventricular hypothalamic nucleus (PVN) than WKY rats. After DOCA injection, SHR responded with a significant increase in both parameters with respect to vehicle-injected SHR. In WKY rats, DOCA was without effect on AVP mRNA although it increased the number of V1aR-positive cells. Changes in the number of Fos-positive nuclei were measured in the PVN, median preoptic nucleus (MnPO) and organum vasculosum of the lamina terminalis (OVLT), a circumventricular region showing anatomical connections with the PVN. In vehicle-injected rats, the PVN of SHR showed a higher number of Fos-positive nuclei than in WKY rats, whereas after DOCA treatment, a significant increment occurred in the OVLT but not in the PVN or MnPO of the SHR group only. These data suggest that the enhanced response of the vasopressinergic system to mineralocorticoids may contribute to the abnormal blood pressure of SHR.

Spinal neurons activated with the urethrogenital reflex in the male rat

The urethrogenital (UG) reflex is a spinal ejaculatory-like reflex. The location of spinal neurons activated by the UG reflex was examined in the male rat using the immediate early gene, c- fos. In addition, co localization of neurons containing galanin and choline acetyl transferase (ChAT) and serotonin fibers with fos-immunoreactive (fos-I) nuclei was examined. Activation of the UG reflex resulted in a significant increase in fos positive nuclei in segments T13–S1, compared to controls in which the UG reflex was not activated. Spinal circuits involved in the UG reflex include neurons relaying afferent information from the pudendal sensory nerve, in the dorsal horn and medial cord of L5–S1. Interneurons specifically activated with the UG reflex were identified in the medial, intermediate and lateral gray. A small proportion of parasympathetic and sympathetic preganglionic neurons in the intermediolateral cell column (IML) of L5–S1 and IML and medial gray of T13–L2, respectively, was activated with the UG reflex. A significant increase in the number of galanin containing neurons expressing c- fos in the medial gray of L3–L4 was also observed with the UG reflex. Serotonin fibers and varicosities were found throughout the spinal cord and were especially dense in the ventral horn, IML and medial gray. Fos activated neurons were found in close apposition to serotonin fibers in the IML and medial gray. These studies demonstrate the multisegmental intraspinal circuitry responsible for ejaculatory-like responses and demonstrate the potential involvement of galanin, acetylcholine and serotonin in mediation of the UG reflex.