Fos-immunoreactive Cells Research Articles

Oxytocin shortens spreading depolarization-induced periorbital allodynia

BackgroundMigraine is among the most prevalent and burdensome neurological disorders in the United States based on disability-adjusted life years. Cortical spreading depolarization (SD) is the most likely electrophysiological cause of migraine aura and may be linked to trigeminal nociception. We previously demonstrated, using a minimally invasive optogenetic approach of SD induction (opto-SD), that opto-SD triggers acute periorbital mechanical allodynia that is reversed by 5HT1B/1D receptor agonists, supporting SD-induced activation of migraine-relevant trigeminal pain pathways in mice. Recent data highlight hypothalamic neural circuits in migraine, and SD may activate hypothalamic neurons. Furthermore, neuroanatomical, electrophysiological, and behavioral data suggest a homeostatic analgesic function of hypothalamic neuropeptide hormone, oxytocin. We, therefore, examined the role of hypothalamic paraventricular nucleus (PVN) and oxytocinergic (OXT) signaling in opto-SD-induced trigeminal pain behavior.MethodsWe induced a single opto-SD in adult male and female Thy1-ChR2-YFP transgenic mice and quantified fos immunolabeling in the PVN and supraoptic nucleus (SON) compared with sham controls. Oxytocin expression was also measured in fos-positive neurons in the PVN. Periorbital mechanical allodynia was tested after treatment with selective OXT receptor antagonist L-368,899 (5 to 25 mg/kg i.p.) or vehicle at 1, 2, and 4 h after opto-SD or sham stimulation using von Frey monofilaments.ResultsOpto-SD significantly increased the number of fos immunoreactive cells in the PVN and SON as compared to sham stimulation (p < 0.001, p = 0.018, respectively). A subpopulation of fos-positive neurons also stained positive for oxytocin. Opto-SD evoked periorbital mechanical allodynia 1 h after SD (p = 0.001 vs. sham), which recovered quickly within 2 h (p = 0.638). OXT receptor antagonist L-368,899 dose-dependently prolonged SD-induced periorbital allodynia (p < 0.001). L-368,899 did not affect mechanical thresholds in the absence of opto-SD.ConclusionsThese data support an SD-induced activation of PVN neurons and a role for endogenous OXT in alleviating acute SD-induced trigeminal allodynia by shortening its duration.

The ghrelin agonist, HM01 activates central vagal and enteric cholinergic neurons and reverses gastric inflammatory and ileus responses in rats.

Electrical vagal stimulation alleviates abdominal surgery (AS)-induced intestinal inflammation. Ghrelin receptors (GHS-Rs) are expressed in the brain and peripheral tissues. We investigated the influence of HM01, an orally active ghrelin agonist crossing the blood-brain barrier, on AS-induced gastric inflammation and emptying (GE) in rats. HM01 (6mg/kg) or saline pretreatment was administered per orally (po) or intraperitoneally (ip). We assessed GE, gastric cytokine mRNA, and Fos positive cells in the dorsal motor nucleus of the vagus (DMN) and gastric corpus myenteric plexus (MP) in sham (anesthesia alone) and AS groups. The transcripts of GHS-R1 variants were determined in the medulla oblongata and gastric corpus of naïve rats. In vehicle pretreated rats, HM01 (ip) significantly increased the number of Fos immunoreactive cells in the MP and DMN in 55% and 52% of cholinergic neurons respectively. Hexamethonium did not modify HM01-induced Fos expression in the DMN while reducing it in the MP by 2-fold with values still significantly higher than that in control groups. AS upregulated gastric IL-1β and TNFα expression and inhibited GE by 66.6%. HM01 (po) abolished AS-induced gastric ileus and increased cytokine expression and elevated IL-10 by 4.0-fold versus vehicle/sham. GHS-R1a mRNA level was 5.4-fold higher than the truncated GHS-R1b isoform in the brain medulla and 40-fold higher in the gastric submucosa/muscle layers than in the mucosa. Peripheral HM0 activates central vagal and myenteric cholinergic pathways that may influence both central and peripheral targets to prevent AS-induced gastric inflammatory and ileus.

Site-Specific Regulation of Stress Responses Along the Rostrocaudal Axis of the Insular Cortex in Rats.

The insular cortex (IC) has been described as a part of the central network implicated in the integration and processing of limbic information, being related to the behavioral and physiological responses to stressful events. Besides, a site-specific control of physiological functions has been reported along the rostrocaudal axis of the IC. However, a functional topography of the IC in the regulation of stress responses has never been reported. Therefore, this study aimed to investigate the impact of acute restraint stress in neuronal activation at different sites along the rostrocaudal axis of the IC. Furthermore, we evaluated the involvement of IC rostrocaudal subregions in the cardiovascular responses to acute restraint stress. We observed that an acute session of restraint stress increased the number of Fos-immunoreactive cells in the rostral posterior region of the IC, while fewer activated cells were identified in the anterior and caudal posterior regions. Bilateral injection of the non-selective synaptic inhibitor CoCl2 into the anterior region of the IC did not affect the blood pressure and heart rate increases and the sympathetically mediated cutaneous vasoconstriction to acute restraint stress. However, synaptic ablation of the rostral posterior IC decreased the restraint-evoked arterial pressure increase, whereas tachycardia was reduced in animals in which the caudal posterior IC was inhibited. Taken together, these pieces of evidence indicate a site-specific regulation of cardiovascular stress response along the rostrocaudal axis of the IC.

Site‐specific regulation of emotional stress responses along the rostrocaudal axis of the insular cortex in rats

The insular cortex (IC) has been described as part of the central network implicated in the behavioral and physiological responses to emotional stress. Besides, it has been reported a site‐specific control of physiological functions along the rostrocaudal axis of the IC. Nevertheless, evaluation of a functional topography of the IC in the regulation of the responses to stressful stimuli has never been documented. Therefore, this study aimed to investigate the impact of acute restraint stress in neuronal activation at different sites along the rostrocaudal axis of the IC in rats. Furthermore, we evaluated the involvement of IC rostrocaudal subregions in the cardiovascular responses to acute restraint stress. For this, male Wistar rats (60‐days‐old) were used. The restraint stress was performed by placing the animals in a cylindrical plastic tube for 60 minutes. For the cardiovascular study, different set of animals had cannula‐guide bilaterally implanted into the anterior, rostral posterior of caudal posterior subregions of the IC; and the non‐selective synaptic inhibitor CoCl2(1mM/100nL) was microinjected 10 min before the restraint onset. We observed that acute restraint stress increased the number of Fos‐immunoreactive cells in the rostral posterior region of the IC (t=2.95, df=14, P=0.0106), while fewer activated cells were identified in the anterior (t=2.44, df=14, P= 0.0276) and caudal posterior (t=2.27, df=14, P=0.0388) regions. Treatment of the anterior region of the IC with CoCl2 did not affect the blood pressure (F(1,15)=0.90, P&gt;0.05) and heart rate (F(1,15)=0.3, P&gt;0.05) increases and the sympathetically‐mediated cutaneous vasoconstriction (F(1,15)=2.23, P&gt;0.05) evoked by restraint stress. However, synaptic ablation of the rostral posterior IC decreased the restraint‐evoked arterial pressure increase (F(1,21)=5.3, P=0.0313), but without affecting the tachycardia (F(1,21)=4.1, P=0.0561) and the reduction in tail skin temperature (F(1,21)=0.05, P&gt;0.05). Besides, bilateral microinjection of CoCl2 into the caudal posterior IC decreased the tachycardia to restraint stress (F(1,17)=11.3, P=0.0036), but without affecting the blood pressure (F(1,17)= 0.2, P&gt;0.05) and tail skin temperature (F(1,17)= 1.4, P&gt;0.05) responses. Taken together, these findings indicate a site‐specific regulation of stress response along the rostrocaudal axis of the IC.

Lateral hypothalamus involvement in control of stress response by bed nucleus of the stria terminalis endocannabinoid neurotransmission in male rats

The endocannabinoid neurotransmission acting via local CB1 receptor in the bed nucleus of the stria terminalis (BNST) has been implicated in behavioral and physiological responses to emotional stress. However, the neural network related to this control is poorly understood. In this sense, the lateral hypothalamus (LH) is involved in stress responses, and BNST GABAergic neurons densely innervate this hypothalamic nucleus. However, a role of BNST projections to the LH in physiological responses to stress is unknown. Therefore, using male rats, we investigated the role of LH GABAergic neurotransmission in the regulation of cardiovascular responses to stress by CB1 receptors within the BNST. We observed that microinjection of the selective CB1 receptor antagonist AM251 into the BNST decreased the number of Fos-immunoreactive cells within the LH of rats submitted to acute restraint stress. Treatment of the BNST with AM251 also enhanced restraint-evoked tachycardia. Nevertheless, arterial pressure increase and sympathetically-mediated cutaneous vasoconstriction to restraint was not affected by CB1 receptor antagonism within the BNST. The effect of AM251 in the BNST on restraint-evoked tachycardia was abolished in animals pretreated with the selective GABAA receptor antagonist SR95531 in the LH. These results indicate that regulation of cardiovascular responses to stress by CB1 receptors in the BNST is mediated by GABAergic neurotransmission in the LH. Present data also provide evidence of the BNST endocannabinoid neurotransmission as a mechanism involved in LH neuronal activation during stressful events.

Rapamycin reduces orofacial nociceptive responses and microglial p38 mitogen-activated protein kinase phosphorylation in trigeminal nucleus caudalis in mouse orofacial formalin model.

The mammalian target of rapamycin (mTOR) plays a role in various cellular phenomena, including autophagy, cell proliferation, and differentiation. Although recent studies have reported its involvement in nociceptive responses in several pain models, whether mTOR is involved in orofacial pain processing is currently unexplored. This study determined whether rapamycin, an mTOR inhibitor, reduces nociceptive responses and the number of Fos-immunoreactive (Fos-ir) cells in the trigeminal nucleus caudalis (TNC) in a mouse orofacial formalin model. We also examined whether the glial cell expression and phosphorylated p38 (p-p38) mitogen-activated protein kinases (MAPKs) in the TNC are affected by rapamycin. Mice were intraperitoneally given rapamycin (0.1, 0.3, or 1.0 mg/kg); then, 30 min after, 5% formalin (10 µl) was subcutaneously injected into the right upper lip. The rubbing responses with the ipsilateral forepaw or hindpaw were counted for 45 min. High-dose rapamycin (1.0 mg/kg) produced significant antinociceptive effects in both the first and second phases of formalin test. The number of Fos-ir cells in the ipsilateral TNC was also reduced by high-dose rapamycin compared with vehicle-treated animals. Furthermore, the number of p-p38-ir cells the in ipsilateral TNC was significantly decreased in animals treated with high-dose rapamycin; p-p38 expression was co-localized in microglia, but not neurons and astrocytes. Therefore, the mTOR inhibitor, rapamycin, reduces orofacial nociception and Fos expression in the TNC, and its antinociceptive action on orofacial pain may be associated with the inhibition of p-p38 MAPK in the microglia.

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.

Existence of wild brown rats (Rattus norvegicus) that are indifferent to novel objects.

Exposure to novel objects typically evokes avoidance behavior in wild animals, which is called neophobia. We previously found that wild brown rats (Rattus norvegicus) that were trapped in a park in downtown Tokyo, Japan, exhibited neophobia. We also found that this behavior was accompanied by the activation of the basolateral complex of the amygdala (BLA). Previous studies have suggested that genetic factors are the primary determinants of neophobia. Since rats in cities form populations with distinct genetic characteristics, it is reasonable to assume that wild rats caught at different locations in urban centers will exhibit different levels of neophobia. Here we assessed the intensity of neophobia in wild rats trapped at a wholesale market in Tokyo. Although we performed exactly the same experiment in which neophobia was observed in wild rats trapped at the park, the presence of novel objects did not affect the behaviors of wild rats trapped at the market. Conversely, laboratory rats showed approach and exploratory behaviors as seen in the previous study, suggesting that the experiment was performed appropriately. Compared to the laboratory rats, the lack of behavioral changes in the wild rats was accompanied by fewer Fos immunoreactive cells in the BLA. In addition, the numbers of Fos immunoreactive cells in the bed nucleus of the stria terminalis and ventromedial hypothalamus were similar between the two types of rats. The results demonstrated the existence of wild rats that were indifferent to novel objects.

Acute Mono-Arthritis Activates the Neurohypophysial System and Hypothalamo-Pituitary Adrenal Axis in Rats.

Various types of acute/chronic nociceptive stimuli cause neuroendocrine responses such as activation of the hypothalamo-neurohypophysial [oxytocin (OXT) and arginine vasopressin (AVP)] system and hypothalamo-pituitary adrenal (HPA) axis. Chronic multiple-arthritis activates the OXT/AVP system, but the effects of acute mono-arthritis on the OXT/AVP system in the same animals has not been simultaneously evaluated. Further, AVP, not corticotropin-releasing hormone (CRH), predominantly activates the HPA axis in chronic multiple-arthritis, but the participation of AVP in HPA axis activation in acute mono-arthritis remains unknown. Therefore, we aimed to simultaneously evaluate the effects of acute mono-arthritis on the activity of the OXT/AVP system and the HPA axis. In the present study, we used an acute mono-arthritic model induced by intra-articular injection of carrageenan in a single knee joint of adult male Wistar rats. Acute mono-arthritis was confirmed by a significant increase in knee diameter in the carrageenan-injected knee and a significant decrease in the mechanical nociceptive threshold in the ipsilateral hind paw. Immunohistochemical analysis revealed that the number of Fos-immunoreactive (ir) cells in the ipsilateral lamina I–II of the dorsal horn was significantly increased, and the percentage of OXT-ir and AVP-ir neurons expressing Fos-ir in both sides of the supraoptic (SON) and paraventricular nuclei (PVN) was increased in acute mono-arthritic rats. in situ hybridization histochemistry revealed that levels of OXT mRNA and AVP hnRNA in the SON and PVN, CRH mRNA in the PVN, and proopiomelanocortin mRNA in the anterior pituitary were also significantly increased in acute mono-arthritic rats. Further, plasma OXT, AVP, and corticosterone levels were significantly increased in acute mono-arthritic rats. These results suggest that acute mono-arthritis activates ipsilateral nociceptive afferent pathways at the spinal level and causes simultaneous and integrative activation of the OXT/AVP system. In addition, the HPA axis is activated by both AVP and CRH in acute mono-arthritis with a distinct pattern compared to that in chronic multiple-arthritis.

Association of high-fat diet with neuroinflammation, anxiety-like defensive behavioral responses, and altered thermoregulatory responses in male rats

Overweight and obesity are a worldwide pandemic affecting billions of people. These conditions have been associated with a chronic low-grade inflammatory state that is recognized as a risk factor for a range of somatic diseases as well as neurodevelopmental disorders, anxiety disorders, trauma- and stressor-related disorders, and affective disorders. We previously reported that the ingestion of a high-fat diet (HFD; 45% fat kcal/g) for nine weeks was capable of inducing obesity in rats in association with increased reactivity to stress and increased anxiety-related defensive behavior. In this study, we conducted a nine-week diet protocol to induce obesity in rats, followed by investigation of anxiety-related defensive behavioral responses using the elevated T-maze (ETM), numbers of FOS-immunoreactive cells after exposure of rats to the avoidance or escape task of the ETM, and neuroinflammatory cytokine expression in hypothalamic and amygdaloid nuclei. In addition, we investigated stress-induced cutaneous thermoregulatory responses during exposure to an open-field (OF). Here we demonstrated that nine weeks of HFD intake induced obesity, in association with increased abdominal fat pad weight, increased anxiety-related defensive behavioral responses, and increased proinflammatory cytokines in hypothalamic and amygdaloid nuclei. In addition, HFD exposure altered avoidance- or escape task-induced FOS-immunoreactivity within brain structures involved in control of neuroendocrine, autonomic, and behavioral responses to aversive stimuli, including the basolateral amygdala (BLA) and dorsomedial (DMH), paraventricular (PVN) and ventromedial (VMH) hypothalamic nuclei. Furthermore, rats exposed to HFD, relative to control diet-fed rats, responded with increased tail skin temperature at baseline and throughout exposure to an open-field apparatus. These data are consistent with the hypothesis that HFD induces neuroinflammation, alters excitability of brain nuclei controlling neuroendocrine, autonomic, and behavioral responses to stressful stimuli, and enhances stress reactivity and anxiety-like defensive behavioral responses.

Patterns of preoptic-hypothalamic neuronal activation and LH secretion in female sheep following the introduction and withdrawal of novel males.

The neuroendocrine response of female sheep to a novel male involves neural activation in the hypothalamus. However, if males are removed, the gonadotrophic signal declines, so the neural activity is likely to change. We examined Fos-immunoreactive (IR) cells in hypothalamic tissues from seasonally anovulatory female sheep exposed to males for 2 or 6h, or for 2h followed by 4h isolation from males. Control females were killed in the absence of male exposure. Male introduction increased LH secretion in all females; male removal was associated with a reduction only in mean and basal LH concentrations. Females exposed to males for 2h had more Fos-IR cells in the arcuate nucleus (ARC), ventromedial nucleus of the hypothalamus (VMH) and organum vasculosum of the lamina terminalis (OVLT) than control females. Fos-IR cells in the preoptic area (POA) were only greater than in control females after 6h exposure to a male. Removal of males decreased the number of Fos-IR cells in the ARC, VMH and OVLT, but not in the POA. Thus, hypothalamic neural activation and LH secretion in female sheep are stimulated by males and decline after male removal. However, activation in the POA persists after removal and may explain the incomplete decline in the LH response.

The weaning period promotes alterations in the orexin neuronal population of rats in a suckling-dependent manner.

The orexin-immunoreactive neurons are part of an important arousal-promoting hypothalamic population. Several groups have investigated these neurons during the lactation period, when numerous physiological alterations occur in the dam's body to cope with the newly acquired metabolic needs of the litter. Although those studies have probed this population during the early and intermediate stages of lactation, few works have examined its response to weaning, including the cessation of the tactile suckling stimulus as the litter stops nursing. Using double immunohistochemistry for orexin and FOS combined with three-dimensional reconstruction techniques, we investigated orexin-synthesizing neurons and their activation at different times during weaning, in addition to the role played by the suckling stimulus. We report here that weaning promoted a decline in the anterior population of orexin-immunoreactive neurons and decreased the number of double orexin-FOS neurons labeled in the central dorsomedial hypothalamus, in addition to reducing the overall number of FOS-immunoreactive cells in the whole tuberal hypothalamus. Disruption of the suckling stimulus from the pups impaired the decrease in the number of anteriorly located orexin-immunoreactive neurons, attenuated the activation of orexin-synthesizing cells in the dorsomedial hypothalamus and reduced the number of FOS-immunoreactive neurons across the tuberal hypothalamus. When taken together, our data suggest that the weaning period is necessary to restore neurochemical pathways altered during the lactation period and that the suckling stimulus plays a significant role in this process.

Distribution of Fos-immunoreactive cells in the ventral part of rat medulla following voluntary treadmill exercise.

The ventral part of the medulla, which contains important cardiovascular regions, is reportedly activated during exercise. Nevertheless, it was uncertain which region(s) in the ventral medulla are specifically activated by exercise. The present study aimed to demonstrate a general pattern of exercise-specific distribution of excited neuronal cells in the rat ventral medulla. Via immunohistochemical experiments, we mapped tyrosine hydroxylase- and Fos-immunoreactive cells (TH-IR and Fos-IR cells, respectively) on rat medullary coronal sections following a bout of voluntary treadmill exercise, a comparative control period, or after pharmacologically induced-hypotension under anesthesia. In the ventral medulla at the rostrocaudal level adjacent, but not rostral or caudal, to the caudal edge of the facial nucleus, voluntary treadmill exercise induced significant (P<0.05) increases in Fos expression, similar to hypotension. The rostral ventrolateral medulla (RVLM), as compared with the rostral ventromedial medulla (RVMM), displayed a greater number of Fos-IR cells due to either exercise or hypotension. In the RVLM, either exercise or hypotension induced significant expression of Fos in both TH-IR and TH non-immunoreactive cells. In the caudal ventrolateral medulla (CVLM), hypotension, but not exercise, increased the ratio of Fos-IR cells in the TH-IR population. These findings demonstrate that RVLM adrenergic and non-adrenergic neurons are specifically excited by voluntary exercise in rats, while RVMM or CVLM neurons are not. We suggest that RVLM C1/non-C1 neurons are a major part of central circuitries underlying sympathetic adjustments to exercise.

Profiling coping strategies in male and female rats: Potential neurobehavioral markers of increased resilience to depressive symptoms

Coping strategies have been associated with differential stress responsivity, perhaps providing a valuable neurobiological marker for susceptibility to the emergence of depressogenic symptoms or vulnerability to other anxiety-related disorders. Rats profiled with a flexible coping phenotype, for example, exhibit increased neurobiological markers of emotional regulation compared to active and passive copers (Bardi et al., 2012; Lambert et al., 2014). In the current study, responses of male and female rats to prediction errors in a spatial foraging task (dry land maze; DLM) were examined after animals were exposed to chronic unpredictable stress (CUS). Brains were processed following the DLM training/assessment for fos-activation patterns and several measures of neuroplasticity in relevant areas. Behavioral responses observed during both the CUS and DLM phases of testing suggested that males and females employ different means of gathering information such as increased ambulatory exploration in males and rear responses in females. Fecal samples collected during baseline and following CUS swim exposure revealed higher corticosterone (CORT) in active copers, whereas flexible copers had higher dehydroepiandrosterone (DHEA) and DHEA/CORT ratios, both indications of enhanced emotional regulation. Focusing on the neural analysis, flexible copers exhibited fewer fos-immunoreactive cells in the basolateral amygdala and a trend toward lower activation in the insula while encountering the prediction error associated with the DLM probe trial. Coping profiles also differentially influenced markers of neuroplasticity; specifically, flexible copers exhibited higher levels nestin-immunoreactivity (ir). Further, less hippocampal glucocorticoid receptor-ir was observed in the flexible copers than the active and passive copers. In sum, flexible coping rats exhibited evidence of emotional resilience as indicated by several neurobiological measures; however, despite increased rates of depression and related symptoms reported in human females, sex effects weren't as pervasive as coping strategy profiles in the analysis of neurobiological markers employed in the current study.

Hypovolemic hemorrhage induces Fos expression in the rat hypothalamus: Evidence for involvement of the lateral hypothalamus in the decompensatory phase of hemorrhage

This study tested the hypothesis that the hypothalamus participates in the decompensatory phase of hemorrhage by measuring Fos immunoreactivity and by inhibiting neuronal activity in selected hypothalamic nuclei with lidocaine or cobalt chloride. Previously, we reported that inactivation of the arcuate nucleus inhibited, but did not fully prevent, the fall in arterial pressure evoked by hypotensive hemorrhage. Here, we report that hemorrhage (2.2ml/100g body weight over 20min) induced Fos expression in a high percentage of cells in the paraventricular, supraoptic and arcuate nuclei of the hypothalamus as shown previously. Lower densities of Fos immunoreactive cells were also found in the medial preoptic area (mPOA), anterior hypothalamus, lateral hypothalamus (LH), dorsomedial hypothalamus, ventromedial hypothalamus (VMH) and posterior hypothalamus. Bilateral injection of lidocaine (2%; 0.1μl or 0.3μl) or cobalt chloride (5mM; 0.3μl) into the tuberal portion of the LH immediately before hemorrhage was initiated reduced the magnitude of hemorrhagic hypotension and bradycardia significantly. Lidocaine injection into the VMH also attenuated the fall in arterial pressure and heart rate evoked by hemorrhage although inactivation of the mPOA or rostral LH was ineffective. These findings indicate that hemorrhage activates neurons throughout much of the hypothalamus and that a relatively broad area of the hypothalamus, extending from the arcuate nucleus laterally through the caudal VMH and tuberal LH, plays an important role in the decompensatory phase of hemorrhage.

Differential mesocorticolimbic responses to palatable food in binge eating prone and binge eating resistant female rats

Binge eating is a key symptom of many eating disorders (e.g. binge eating disorder, bulimia nervosa, anorexia nervosa binge/purge type), yet the neurobiological underpinnings of binge eating are poorly understood. The mesocorticolimbic reward circuit, including the nucleus accumbens and the medial prefrontal cortex, is likely involved because this circuit mediates the hedonic value and incentive salience of palatable foods (PF). Here we tested the hypothesis that higher propensity for binge eating is associated with a heightened response (i.e., Fos induction) of the nucleus accumbens and medial prefrontal cortex to PF, using an animal model that identifies binge eating prone (BEP) and binge eating resistant (BER) rats. Forty adult female Sprague–Dawley rats were given intermittent access to PF (high fat pellets) 3×/week for 3weeks. Based on a pattern of either consistently high or consistently low PF consumption across these feeding tests, 8 rats met criteria for categorization as BEP, and 11 rats met criteria for categorization as BER. One week after the final feeding test, BEP and BER rats were either exposed to PF in their home cages or were given no PF in their home cages for 1h prior to perfusion, leading to three experimental groups for the Fos analysis: BEPs given PF, BERs given PF, and a No PF control group. The total number of Fos-immunoreactive (Fos-ir) cells in the nucleus accumbens core and shell, and the cingulate, prelimbic, and infralimbic regions of the medial prefrontal cortex was estimated by stereological analysis. PF induced higher Fos expression in the nucleus accumbens shell and core and in the prelimbic and infralimbic cortex of BEP rats compared to No PF controls. Throughout the nucleus accumbens and medial prefrontal cortex, PF induced higher Fos expression in BEP than in BER rats, even after adjusting for differences in PF intake. Differences in the neural activation pattern between BEP and BER rats were more robust in prefrontal cortex than in nucleus accumbens. These data confirm that PF activates brain regions responsible for encoding the incentive salience and hedonic properties of PF, and suggest that binge eating proneness is associated with enhanced responses to PF in brain regions that exert executive control over food reward.

The ventral portion of the anterior pretectal nucleus controls descending mechanisms that initiate neuropathic pain in rats.

Stimulating the dorsal anterior pretectal nucleus (dAPtN) in rats is more effective than stimulating the ventral APtN (vAPtN) at reducing tail-flick latency, whereas stimulation of the vAPtN is more effective at reducing postoperative pain behaviour. This study examines whether a cell lesion caused by injecting N-methyl-D-aspartate into the dAPtN or vAPtN changes the withdrawal threshold of a rat hind paw during different phases of the tactile hypersensitivity induced by a chronic constriction injury (CCI) of the contralateral sciatic nerve. The number of Fos immunoreactive cells in the APtN was also evaluated. The rats whose vAPtN was lesioned 2 days before CCI had more intense tactile hypersensitivity 2 days after CCI than that of the control group, but the groups were not different 7 days after the CCI. The rats whose vAPtN was lesioned 5 days after CCI had withdrawal thresholds that did not differ significantly 7 days after the CCI. The tactile hypersensitivity of the rats whose dAPtN was lesioned 2 days before or 5 days after CCI was not different from that of the control on the second and seventh days after the CCI. The number of Fos immunoreactive cells in the vAPtN and dAPtN increased 2 days after CCI, but did not differ from that in the control 7 days after CCI. We conclude that vAPtN and dAPtN cells are activated by nerve injury; the vAPtN exerts inhibitory control of the initial phase of neuropathic pain whereas the dAPtN does not appear to exert an inhibitory effect in neuropathic processing.

Activation of the cerebellum by olfactory stimulation in sexually naive male rats

IntroductionThe cerebellum has been linked to multiple functions, such as motor control, cognition, memory, and emotional processing. As for its involvement in the sensory systems, the role of the cerebellum in the sense of smell remains unclear. We suggest that sexually naive male rats will present increased neuronal activity in the cerebellar vermis after being stimulated with almond odour or oestrous odour from receptive females. MethodsWe compared activity in the cerebellar vermis using Fos immunoreactivity after olfactory stimulation. Stimulation took place for 60min in a cube-shaped acrylic chamber with a double bottom. Stimuli were clean woodchip bedding, bedding with almond extract, and bedding taken from a cage of receptive females. Male rats were subsequently anaesthetised with intraperitoneal sodium pentobarbital. Cerebellar tissue was fixed with paraformaldehyde for later immunohistochemical analysis. ResultsThe number of Fos immunoreactive cells in all lobes of the cerebellar vermis was similar between groups stimulated with almond extract and with oestrous odour, and higher than in the clean woodchip group. ConclusionsStimulation of the main olfactory system (almond) and the accessory system (oestrous odour) increases Fos protein production in the granular layer of the cortex of the cerebellar vermis in naive male rats.

Activation of Brainstem and Hypothalamic Areas Related to Increased Tail Skin Temperature in Ovariectomized Rats

Hot flushes are usually treated with estrogen replacement in postmenopausal women but the neural pathways involved are poorly understood. We investigated the activation of brain areas associated with the increase in tail skin temperature in an animal model of menopause. Female Wistar rats were ovariectomized (OVX) and implanted with subcutaneous capsules containing 17ß‐estradiol (E2) (OVX+E2) or corn oil (OVX). Tail skin temperature (TST) was recorded during two hours at ambient temperature at 3, 7 and 14 days after surgery. On day 15th, rats were perfused and the brains were processed for immunohistochemistry. Neuronal activity was evaluated by counting the number of Fos‐immunoreactive (ir) cells. On day 14 after ovariectomy, the TST in OVX rats was higher than in OVX+E2 rats. All the brain areas examined exhibited significantly more Fos‐ir cells in OVX than OVX E2 rats. In the brainstem, OVX rats displayed more Fos/tyrosine hydroxylase doubled‐labeled neurons in the A1, A2 and locus coeruleus compared with OVX+E2 rats. OVX rats exhibited more Fos‐ir neurons than OVX+E2 rats in the rostral preoptic area (POA), anteroventral periventricular nucleus (AVPV), and median preoptic nucleus (MnPO) in the preoptic area and in the paraventricular nucleus (PVN) and arcuate nucleus (ARC) of the hypothalamus. Moreover, there was a high correlation between the TST and Fos expression in the A1, A2, AVPV and POA. Thus, ovariectomy gradually increases TST and induces neuronal activation in the brainstem, preoptic area and hypothalamus. Fos expression in neurons of the A1, A2, AVPV, and POA is directly correlated with TST, suggesting these brain areas are associated with the thermoregulatory effects of estrogen. &#x0D; Financial support: CNPQ, FAPEMIG, CAPES

Neurokinin 3 Receptor-Expressing Neurons in the Median Preoptic Nucleus Modulate Heat-Dissipation Effectors in the Female Rat.

KNDy neurons facilitate tail skin vasodilation and modulate the effects of estradiol on thermoregulation. We hypothesize that KNDy neurons influence cutaneous vasodilation via projections to neurons in the median preoptic nucleus (MnPO) that express the neurokinin 3 receptor (NK3R). In support of this hypothesis, focal microinjections of senktide, an NK3R agonist, into the MnPO lowers core temperature (TCORE) in the female rat. To further study the role of MnPO NK3R neurons in thermoregulation, these neurons were specifically ablated using a conjugate of a selective NK3R agonist and saporin (NK3-SAP). NK3-SAP or blank-SAP (control) was injected into the MnPO/medial septum. Tail skin temperature (TSKIN) and TCORE were measured in ovariectomized rats exposed to 3 ambient temperatures (TAMBIENT) before and after estradiol-17β (E2) treatment. Before killing, we injected senktide (sc), monitored TCORE for 70 minutes, and harvested brains for Fos immunohistochemistry. Ablation of MnPO NK3R neurons lowered TSKIN at neutral and subneutral TAMBIENT regardless of E2 treatment. However, ablation did not prevent the effects of E2 on TCORE and TSKIN. In control rats, senktide injections induced hypothermia with numerous Fos-immunoreactive cells in the MnPO. In contrast, in NK3-SAP rats, senktide did not alter TCORE and minimal Fos-immunoreactive neurons were identified in the MnPO. These data show that NK3R neurons in the MnPO are required for the hypothermic effects of senktide but not for the E2 modulation of thermoregulation. The lower TSKIN in NK3-SAP-injected rats suggests that MnPO NK3R neurons, like KNDy neurons, facilitate cutaneous vasodilation, an important heat-dissipation effector.