Parvocellular Paraventricular Nucleus Research Articles

Adrenalectomy Potentiates Noradrenergic Suppression of GABAergic Transmission in Parvocellular Neurosecretory Neurons of Hypothalamic Paraventricular Nucleus

Glucocorticoids are known to regulate both the noradrenergic and GABAergic inputs to the paraventricular nucleus (PVN). However, little is known about the effects of glucocorticoids on the interaction of these two input systems. Here we examined the effects of bilateral adrenalectomy (ADX) on the noradrenergic modulation of GABAergic transmission in the type II PVN neurons labeled with a retrograde dye injected into the pituitary stalk. Noradrenaline either reduced or augmented the frequency of spontaneous inhibitory postsynaptic current (sIPSC) without changing the amplitude and decay time constant. These effects were blocked by alpha2A- and alpha(1A/1L)-adrenoceptor antagonists, respectively. ADX increased the proportion of the neurons showing the noradrenergic reduction and the extent of reduction in the IPSC frequency. The ADX-induced changes were reversed by supplementation of ADX rats with corticosterone (10-mg pellet). ADX also potentiated the noradrenergic reduction in the frequency of miniature IPSC and paired-pulse facilitation of evoked IPSC. BRL 44408 (3 microM), a alpha2A-adrenoceptor antagonist, blocked the noradrenergic reduction in ADX rats. Corticotropin-releasing hormone and/or vasopressin transcripts were detected in neurons displaying noradrenergic augmentation or reduction of IPSC frequency. ADX enhanced the proportion of neurons expressing corticotropin-releasing hormone. Collectively, the results suggest that depletion of corticosterone by ADX markedly potentiates the noradrenergic suppression of GABAergic transmission mediated by the alpha2A-adrenoceptors on the GABAergic terminals in the parvocellular neurosecretory PVN neurons. These results may provide a novel synaptic mechanism for the glucocorticoid-induced plasticity in the noradrenergic modulation of neuroendocrine function of the PVN.

Augmented Hypothalamic Corticotrophin‐Releasing Hormone mRNA and Corticosterone Responses to Stress in Adult Rats Exposed to Perinatal Hypoxia

Stressful events before or just after parturition alter the subsequent phenotypical response to stress in a general process termed programming. Hypoxia during the period before and during parturition, and in the postnatal period, is one of the most common causes of perinatal distress, morbidity, and mortality. We have found that perinatal hypoxia (prenatal day 19 to postnatal day 14) augmented the corticosterone response to stress and increased basal corticotrophin-releasing hormone (CRH) mRNA levels in the parvocellular portion of the paraventricular nucleus (PVN) in 6-month-old rats. There was no effect on the levels of hypothalamic parvocellular PVN vasopressin mRNA, anterior pituitary pro-opiomelanocortin or CRH receptor-1 mRNA, or hippocampus glucocorticoid receptor mRNA. We conclude that hypoxia spanning the period just before and for several weeks after parturition programmes the hypothalamic-pituitary-adrenal axis to hyper-respond to acute stress in adulthood, probably as a result of drive from the parvocellular CRH neurones.

Voluntary Exercise Impacts on the Rat Hypothalamic-Pituitary-Adrenocortical Axis Mainly at the Adrenal Level

Introduction: Evidence is accumulating that the regular performance of exercise is beneficial for stress coping. However, the hypothalamic-pituitary-adrenocortical (HPA) axis of voluntarily exercising rats has never been comprehensively investigated. Methods: Therefore, male Sprague-Dawley rats were given access to a running wheel in their home cage for 4 weeks in which they ran 4–7 km per night. Results: After 4 weeks, the exercising animals showed significantly less body weight gain, less abdominal fat tissue, decreased thymus weight, and increased adrenal weight (relative to body weight). Furthermore, tyrosine hydroxylase (TH) mRNA levels were selectively increased in the right adrenal me- dulla indicating an increase in sympathoadrenomedullary capacity in exercising rats. No changes were observed in paraventricular corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP) and oxytocin mRNA levels. Mineralocorticoid receptor (MR) mRNA levels in hippocampus and glucocorticoid receptor (GR) mRNA levels in frontal cortex, parvocellular paraventricular nucleus and anterior pituitary were unchanged, whereas GR mRNA levels were increased in distinct hippocampal cell layers. Early morning baseline levels of plasma ACTH and corticosterone were similar in both groups. Interestingly, the response to different stressful stimuli (e.g. forced swimming, novelty) revealed that the exercising rats showed stressor-specific changes in HPA hormone responses. Forced swimming evoked a markedly enhanced response in corticosterone levels in the exercising rats. In contrast, if rats were exposed to a novel environment, exercising rats showed a much lower response in corticosterone than the control animals. However, the response in ACTH to either stressor was comparable between groups. Thus, in exercising rats physically demanding stressors evoke enhanced glucocorticoid responses whereas mild psychologically stressful stimuli such as novelty result in an attenuated glucocorticoid response. Interestingly, this attenuated hormone response corresponded with the observation that the exercising rats showed less anxious behaviour in the novelty situation. Conclusions: The differential responses in plasma corticosterone levels to different types of stress in the face of comparable responses in ACTH levels underscore the existence of critical regulatory control mechanisms at the level of the adrenal gland. We have hypothesized that changes in the sympathoadrenomedullary input may play an important role in these distinct glucocorticoid responses to stress. Our previous studies have shown similar changes in voluntarily exercising mice. Therefore, we conclude that the effects of exercise on the organism are not species-specific. Thus, our observations may have translational implications for the human situation.

Central Type 2 Corticotropin-Releasing Hormone Receptor Mediates Hypothalamic-Pituitary-Adrenocortical Axis Activation in the Rat

In an attempt to clarify the role of the type 2 corticotropin-releasing hormone (CRH) receptor (CRHR-2) in the brain in activation of the hypothalamic-pituitary-adrenocortical axis, we conducted experiments using male Wistar rats. First, an injection of urocortin-2 (7.5 µg) into the lateral ventricle resulted in transient increases in CRH heteronuclear RNA (hnRNA) in parvocellular paraventricular nucleus (PVN) and in plasma adrenocorticotropic hormone (ACTH), whereas sustained increases in arginine vasopressin (AVP) hnRNA and c-fos mRNA in the parvocellular PVN were observed as compared with vehicle treatment. Pretreatment with the selective CRHR-2 antagonist antisauvagine-30 (20 µg) into the lateral ventricle 15 min prior to agonist injection attenuated the stimulatory effects of urocortin-2 on the above-mentioned hypothalamic-pituitary-adrenal axis variables. These effects were similar or rather more potent than those induced by pretreatment with 50 µg of α-helical CRH. Second, we found longer-lasting increases in CRH and AVP hnRNA and c-fos mRNA in parvocellular PVN and in plasma ACTH following central administration of urocortin-3 (7.5 µg) than following urocortin-2. Pretreatment with antisauvagine-30 antagonized the effects of urocortin-3 on the above-mentioned variables. Finally, central administration of antisauvagine-30 as well as α-helical CRH profoundly attenuated restraint-stress-induced increases in AVP hnRNA. However, α-helical CRH, but not antisauvagine-30, attenuated restraint-stress-induced increases in CRH hnRNA in the PVN. Both antagonists transiently attenuated stress responses of c-fos mRNA in PVN and plasma ACTH. These results indicate that there is a CRHR-2-mediated mechanism in the brain that stimulates CRH- and AVP-producing neurons in the PVN which results in the promotion of plasma ACTH secretion.

Differential regulation of parvocellular neuronal activity in the paraventricular nucleus of the hypothalamus following single vs. repeated episodes of water restriction-induced drinking

Acute activation of the hypothalamic-pituitary-adrenal (HPA) axis releases glucocorticoids to maintain homeostasis, whereas prolonged exposure to elevated glucocorticoids has deleterious effects. Due to the potential benefits of limiting stress-induced glucocorticoid secretion, the present study uses drinking in dehydrated rats as a model to delineate mechanisms mobilized to rapidly inhibit HPA activity during stress. Using Fos expression as an indicator of neuronal activation, the effect of a single or repeated episode of dehydration-induced drinking on the activity of magnocellular and parvocellular neurons in the paraventricular nucleus (PVN) of the hypothalamus was examined. Adult male rats underwent a single episode or repeated (six) episodes of water restriction and were sacrificed before or after drinking water in the AM. Plasma osmolality, vasopressin (AVP), adrenocorticotropic hormone (ACTH) and corticosterone were elevated by water restriction and reduced after drinking in both models. Fos expression was elevated in AVP-positive magnocellular PVN neurons and AVP- and corticotropin releasing hormone (CRH)-positive parvocellular PVN neurons after water restriction. Fos expression was reduced in magnocellular AVP neurons after both models of restriction-induced drinking. In contrast, Fos expression did not change in AVP and CRH parvocellular neurons after a single episode of restriction-induced drinking, but was reduced after repeated episodes of restriction-induced drinking. These data indicate that drinking-induced decreases in glucocorticoids in dehydrated rats involve multiple factors including reduction in magnocellular release of vasopressin and reduction in parvocellular neuronal activity. The differential inhibition of PVN parvocellular neurons after repeated rehydration may reflect a conditioned response to repeated stress reduction.

Splenic reflex modulation of central cardiovascular regulatory pathways

The splenorenal reflex induces changes in mean arterial pressure (MAP) and renal function. We hypothesized that, in addition to spinal pathways previously identified, these effects are also mediated through central pathways. We investigated the effect of elevated splenic venous pressure on central neural activation in intact, renal-denervated, and renal + splenic-denervated rats. Fos-labeled neurons were quantified in the nucleus of the tractus solitarius (NTS), paraventricular nucleus (PVN), supraoptic nucleus (SON), and subfornical organ (SFO) after 1-h partial splenic vein occlusion (SVO) in conscious rats bearing balloon occluders around the splenic vein, telemetric pressure transducers in the gastric vein (splenic venous pressure), and abdominal aorta catheters (MAP). SVO stimulated Fos expression in the PVN and SON, but not NTS or SFO of intact rats. Renal denervation abolished this response in the parvocellular PVN, while renal + splenic denervation abolished activation in the magnocellular PVN and the SON. In renal-denervated animals, SVO depressed Fos expression in the NTS and increased expression in the SFO, responses that were abolished by renal + splenic denervation. In intact rats, SVO also induced a fall in right atrial pressure, an increase in renal afferent nerve activity, and an increase in MAP. We conclude that elevated splenic venous pressure does induce hypothalamic activation and that this is mediated through both splenic and renal afferent nerves. However, in the absence of renal afferent input, SVO depressed NTS activation, probably as a result of the accompanying fall in cardiac preload and reduced afferent signaling from the cardiopulmonary receptors.

Social stress induces hypothalamo‐pituitary‐adrenal axis responses in lactating rats bred for high trait anxiety

Hypothalamo-pituitary-adrenal axis responses to various stressors are typically attenuated during lactation, including in rats selectively bred for high or low anxiety. As high-anxiety dams are more aggressive towards intruders than low-anxiety dams during maternal defence, we investigated their hypothalamo-pituitary-adrenal axis responses to this social stress. Maternal defence induced elevated stress responses in high-anxiety dams only; nerve-growth-factor-induced gene B mRNA expression in the parvocellular paraventricular nucleus and adrenocorticotropin hormone secretory responses were substantially enhanced after maternal defence. In contrast, secretory responses to a non-social stress (elevated platform) were not different between high- and low-anxiety dams. Thus, responsiveness of the stress axis in lactation is dependent upon the innate level of anxiety of the dam and, as a consequence, her reactiveness to social threat.

Effects of Adrenalectomy on the Excitability of Neurosecretory Parvocellular Neurones in the Hypothalamic Paraventricular Nucleus

Glucocorticoids are well known to inhibit the release of hypophysiotrophic hormones from neurones originating in the paraventricular nucleus (PVN), but the cellular mechanisms of the inhibition are not well understood. Here, we examined the effects of adrenalectomy (ADX) on the spontaneous firing activity in the neurosecretory parvocellular PVN neurones of rat brain slices. The neurones were identified by injecting a retrograde dye into the pituitary stalk and classified according to their electrophysiological properties. The intranuclear distribution, electrophysiological properties, and hypophysiotrophic hormone phenotype of the labelled type II PVN neurones were similar to neurosecretory parvocellular PVN neurones. In the neurones of sham-operated rats under the cell-attached recording mode, we observed three spontaneous activity patterns: tonic regular (24%), tonic irregular (36%), and silent (40%). Noradrenaline (100 microM) induced an excitatory or an inhibitory effect on the spontaneous activity. Noradrenergic excitation was blocked by prazosin (2 microM, alpha(1)-adrenoceptor antagonist), and mimicked by phenylephrine (100 microM, alpha(1)-adrenoceptor agonist), whereas noradrenergic inhibition was blocked by yohimbine (2 microM, alpha(2)-adrenoceptor antagonist) and mimicked by clonidine (50 microM, alpha(2)-adrenoceptor agonist). In the neurones of ADX rats, we found burst firing in 35% of neurones tested and an increase in the frequency of spontaneous firing. The burst firing was not observed in the neurones of the sham-operated rats. ADX caused a 1.7-fold increase in the proportion of neurones showing the noradrenergic excitation. Supplementation of the ADX rats with corticosterone (10 mg pellet) reversed the ADX-induced burst firing, and the potentiation of noradrenergic excitation. In summary, our results show that removal of corticosterone by ADX can elevate the neuronal excitability by increasing the spontaneous firing rate and by potentiating the alpha(1)-adrenoceptor-mediated noradrenergic excitation, and it can facilitate hormone release by inducing burst firing. Our results provide new insight to the cellular mechanisms of the feedback inhibition by glucocorticoids in the neurosecretory parvocellular neurones of the PVN.

Neuropeptide FF and neuropeptide VF inhibit GABAergic neurotransmission in parvocellular neurons of the rat hypothalamic paraventricular nucleus

Neuropeptide FF (NPFF) and neuropeptide VF (NPVF) are octapeptides belonging to the RFamide family of peptides that have been implicated in a wide variety of physiological functions in the brain, including central autonomic and neuroendocrine regulation. The effects of these peptides are mediated via NPFF1 and NPFF2 receptors that are abundantly expressed in the rat brain, including the hypothalamic paraventricular nucleus (PVN), an autonomic nucleus critical for the secretion of neurohormones and the regulation of sympathetic outflow. In this study, we examined, using whole cell patch-clamp recordings in the brain slice, the effects of NPFF and NPVF on inhibitory GABAergic synaptic input to parvocellular PVN neurons. Under voltage-clamp conditions, NPFF and NPVF reversibly and in a concentration-dependent manner reduced the evoked bicuculline-sensitive inhibitory postsynaptic currents (IPSCs) in parvocellular PVN neurons by 25 and 31%, respectively. RF9, a potent and selective NPFF receptor antagonist, blocked NPFF-induced reduction of IPSCs. Recordings of miniature IPSCs in these neurons following NPFF and NPVF applications showed a reduction in frequency but not amplitude, indicating a presynaptic locus of action for these peptides. Under current-clamp conditions, NPVF and NPFF caused depolarization (6-9 mV) of neurons that persisted in the presence of TTX but was abolished in the presence of bicuculline. Collectively, these data provide evidence for a disinhibitory role of NPFF and NPVF in the hypothalamic PVN via an attenuation of GABAergic inhibitory input to parvocellular neurons of this nucleus and explain the central autonomic effects of NPFF.

Changes in the Expression of Corticotrophin‐Releasing Hormone, Mineralocorticoid Receptor and Glucocorticoid Receptor mRNAs in the Hypothalamic Paraventricular Nucleus Induced by Fornix Transection and Adrenalectomy

The paraventricular nucleus (PVN) in the hypothalamus receives inputs from the hippocampus The present study explored the influence of the hippocampus on genes mediating glucocorticoid feedback in the PVN. Accordingly, the expression of mRNAs for corticotrophin-releasing hormone (CRH), the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) in the PVN was examined by in situ hybridisation in rats subjected to transection of the fornix. Significant increases in CRH, MR and GR mRNAs were observed in the parvocellular PVN after fornix transection (FT). FT-animals subjected to adrenalectomy also showed an increase in the number of cells positive for CRH and GR mRNAs. CRH, MR and GR mRNA expression was also increased by bilateral adrenalectomy, and GR mRNA expression was further enhanced in the parvocellular PVN of the FT transected animals. However, no such changes were evident in the magnocellular PVN. These results suggest that the input from the hippocampus to the PVN, particularly to its parvocellular region, has distinct and differential inhibitory effects on the expression of MR,GR and CRH mRNAs that may operate independently from the feedback actions of corticosterone.

The effect of adrenalectomy on Fos expression in vasopressinergic and oxytocinergic neurons in response to stress in the rat

This study evaluated the responses of vasopressin (AVP) and oxytocin (OT) neurons to alterations in hypothalamo–pituitary axis activity by adrenalectomy (ADX) or after restraint stress compared with basal conditions. Wistar male rats were perfuse-fixed by cardiac perfusion under anesthesia 3 h, 1, 3 and 14 days after ADX or Sham surgery. Coronal hypothalamic sections were used for evaluation of Fos, AVP and OT expression by immunohistochemistry. Under basal conditions and after stress, Fos-AVP double labeling showed no difference in the magnocellular subdivisions of the paraventricular nuclei (PVN) or in the supraoptic nuclei (SON), suggesting that the magnocellular AVP system is unlikely to contribute to ACTH secretion after restraint in both Sham and ADX rats. Fos–AVP double labeling in the parvocellular medial paraventricular nucleus (PaMP) in ADX groups was increased after 3 h in basal conditions, and in all periods after restraint stress. There were no differences between Sham and ADX groups in Fos–OT double labeling in any subdivision of the PVN; however, in the SON, the number of Fos–OT double labeled cells was increased at all time-points after stress in the ADX group. Fos expression was increased in the PaMP after 3 h and after restraint stress in the Sham and ADX groups, especially in the ADX group. In conclusion, Fos expression in different cell populations of the PVN can be differentially regulated by short- and long-term absence of glucocorticoid negative feedback and also by stress-related excitatory and/or inhibitory neural inputs. The Fos–AVP double labeling findings in the PaMP also indicate a minor participation of these vasopressinergic neurons in the regulation of the HPA axis after ADX.

Immunohistochemical distribution of the secretogranin II-derived peptide EM66 in the rat hypothalamus: A comparative study with jerboa

EM66 is a 66-amino acid peptide derived from secretogranin II, a member of granin acidic secretory protein family, by proteolytic processing. EM66 has been previously characterized in the jerboa ( Jaculus orientalis) hypothalamus and its potential implication in the neuroendocrine regulation of feeding behaviour has been demonstrated. In the present study, an immunohistochemical analysis of the localization of EM66 within hypothalamic structures of rat was performed and compared to the distribution of EM66 in the jerboa hypothalamus. In the rat hypothalamus, as in the jerboa, EM66 immunostaining was detected in the parvocellular paraventricular, preoptic and arcuate nuclei, as well as the lateral hypothalamus which displayed an important density of EM66-producing neurones. However, unlike jerboa, the suprachiasmatic and supraoptic nuclei of the rat hypothalamus were devoid of cellular EM66-immunolabeling. Thus, the novel peptide EM66 may exert common neuroendocrine activities in rat and jerboa, e.g. control of food intake, and species-specific roles in jerboa such as the regulation of biological rhythms and hydromineral homeostasis. These results suggest the existence of differences between jerboas and rats in neuroendocrine regulatory mechanisms involving EM66.

Cocaine- and amphetamine-regulated transcript (CART) expression is differentially regulated in the hypothalamic paraventricular nucleus of lactating rats exposed to suckling or cold stimulation

Neural stimuli, such as suckling or cold exposure, increase TRH mRNA in the paraventricular nucleus (PVN) of the rat hypothalamus, yet only suckling induces prolactin secretion. As TRH co-localizes with cocaine- and amphetamine-regulated transcript (CART) in hypophysiotropic neurons of the PVN, and CART inhibits TRH-induced prolactin release but not TRH-induced TSH release in adenohypophyseal cell cultures, we raised the possibility that differential regulation of CART gene expression in the PVN may explain the differences in prolactin secretion following each of the two stimuli. Primiparous female rats were mated and handled daily during the pre- and postpartum periods. After delivery, the litter was adjusted to 8 pups and at mid-lactation, dams were separated from their pups for 8 h and exposed to either 1 h of cold or 30 min of suckling. Long-term effects of suckling were studied by separating pups from their mothers for 24 h, followed by a 12 h period of continuous suckling. Serum TSH levels increased in response to cold exposure, while prolactin levels were increased by suckling and diminished by cold exposure. CART mRNA levels increased in rostral and mid parts of the medial parvocellular PVN following cold exposure but not after suckling stimulation. These data demonstrate a differential regulation of CART gene expression in hypophysiotropic neurons in response to stimuli that increase TRH mRNA levels, and suggest that CART activation in the PVN may contribute to the decrease in PRL release when the thyroid axis is activated by cold exposure.

Hypothalamic and brainstem sources of pituitary adenylate cyclase‐activating polypeptide nerve fibers innervating the hypothalamic paraventricular nucleus in the rat

The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons.

Inhibition by α‐Tetrahydrodeoxycorticosterone (THDOC) of Pre‐Sympathetic Parvocellular Neurones in the Paraventricular Nucleus of Rat Hypothalamus

alpha-tetrahydrodeoxycorticosterone (THDOC) is an endogenous neuroactive steroid which increases in plasma and brain concentration during stress. It has both positive and negative modulatory effects on GABA activated GABAA currents, dependent upon the dose. We investigated the effects of THDOC on spinally-projecting "pre-sympathetic" neurones in the parvocellular subnucleus of the hypothalamic paraventricular nucleus (PVN), to determine whether it activates or inhibits these neurones, and by what mechanism. Rat spinally-projecting (parvocellular) PVN neurones were identified by retrograde labelling and the action of THDOC investigated with three modes of patch-clamp: cell-attached action current, whole-cell voltage-clamp and cell-attached single-channel recording. In cell-attached patch mode, parvocellular neurones fired action potentials spontaneously with an average frequency of 3.6 +/- 1.1 Hz. Bath application of THDOC reduced this with an EC50 of 67 nM (95% confidence limits: 54 to 84 nM), Hill coefficient 0.8 +/- 0.04, n = 5. In whole-cell patch-clamp mode, pressure ejection of GABA evoked inward currents. These were clearly GABAA currents, since they were inhibited by the GABAA receptor antagonist bicuculline, and reversed near the chloride equilibrium potential. THDOC significantly potentiated GABAA currents (1 microM THDOC: 148 +/- 15% of control, n = 5, p < or = 0.05, ANOVA). Single-channel analysis showed no differences in conductance or corrected mean open times in the presence of 1 microM THDOC. THDOC inhibited parvocellular neuronal activity without showing any evidence of the bidirectional activity demonstrated previously with cultured hypothalamic neurones. Our data are consistent with the hypothesis that THDOC acts by potentiating the post-synaptic activity of endogenously released GABA.

Activation of Group II Metabotropic Glutamate Receptors Attenuates Both Stress and Cue-Induced Ethanol-Seeking and Modulates c-fosExpression in the Hippocampus and Amygdala

Major precipitating factors for relapse to drug use are stress and exposure to drug-related environmental stimuli. Group II (mGlu(2/3)) metabotropic glutamate receptors (mGluRs) are densely expressed within circuitries mediating the motivating effects of stress and drug cues and, therefore, may participate in regulating drug-seeking linked to both of these risk factors. Thus, we tested the hypothesis that pharmacological activation of group II mGluRs modifies both stress- and cue-induced ethanol-seeking, using reinstatement models of relapse. In parallel, brain c-fos expression was examined to identify neural substrates for the behavioral effects of group II mGluR activation. The selective mGlu(2/3) agonist LY379268 (1R,4R,5S,6R-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate) (0.3, 1.0, and 3.0 mg/kg, s.c.) dose dependently blocked the recovery of extinguished ethanol-seeking induced by either footshock stress or ethanol-associated discriminative stimuli. These effects were accompanied by modulation of c-fos expression in the hippocampus, central nucleus of the amygdala, bed nucleus of the stria terminalis, and medial parvocellular paraventricular nucleus of the hypothalamus. The results implicate group II mGluRs as a shared neuropharmacological substrate for ethanol-seeking elicited by both drug cues and stress and identify group II mGluRs as promising treatment targets for relapse prevention.

Distribution of insulin receptor substrate-2 in brain areas involved in energy homeostasis

Body weight regulation depends on neuronal signaling by adiposity-related hormones such as insulin and leptin. Activation of receptors for these hormones induces cell signaling via the insulin receptor substrate/phosphatidylinositol 3-kinase (IRS–PI3K) pathway, and growing evidence from knockout models implicates IRS-2 as a key component of this signal transduction mechanism. As a first step towards the identification of brain areas that utilize IRS–PI3K signaling in the control of energy homeostasis, we used immunohistochemical techniques to investigate the neuronal distribution of IRS-2 protein in rat brain. In the hypothalamus, strong IRS-2 staining was detected chiefly in the arcuate (ARC), ventromedial (VMN) nucleus and parvocellular paraventricular nucleus (PVN). Within the ARC, IRS-2 was co-localized with alpha melanocyte stimulating hormone (α-MSH) as well as neuropeptide Y (NPY). In the hindbrain, IRS-2 staining was detected in the area postrema (AP), medial nucleus of the solitary tract (mNTS), dorsal motor nucleus of the vagus nerve (DMV) and the hypoglossal nucleus (HN). Co-localization studies in the mNTS demonstrated the presence of IRS-2 in catecholamine neurons. IRS-2 protein was also found in the ventral tegmental area (VTA), an important area for reward perception, and was detected in dopamine neurons in this brain area. In summary, neurons containing IRS-2 immunoreactivity were identified in forebrain, midbrain and hindbrain areas and in cell types that are crucial for the control of food intake and autonomic function. An improved understanding of mechanisms underlying normal and abnormal energy homeostasis may be gained by analysis of the role played by signaling through IRS-2 in these brain areas.

Activation of spinally projecting and nitrergic neurons in the PVN following heat exposure

The present study investigated the effect of acute thermal stimulation in conscious rats on the production of Fos, a marker of increased neuronal activity, in spinally projecting and nitrergic neurons in the hypothalamic paraventricular nucleus (PVN). The PVN contains a high concentration of nitrergic neurons, as well as neurons that project to the intermediolateral cell column (IML) of the spinal cord that can directly influence sympathetic nerve activity (SNA). During thermal stimulation, the PVN is activated, but it is unknown whether spinally projecting PVN neurons and the nitrergic neurons are involved. Compared with controls, rats exposed to an environmental temperature of 39 degrees C for 1 h had a 10-fold increase in the number of cells producing Fos in the PVN (133 +/- 23 vs. 1,336 +/- 43, respectively, P < 0.0001). Of the spinally projecting neurons in the PVN of heated rats (98 +/- 10), over 20% expressed Fos. Additionally, of the nitrergic neurons (NADPH-diaphorase positive) located in the parvocellular PVN (723 +/- 17), 40% also expressed Fos (P < 0.0001 compared with controls). Finally, there was a significant increase in the number of spinally projecting neurons in the PVN that were nitrergic and expressed Fos after heat exposure (12%) compared with controls (0.1%) (P < 0.0001). These results suggest that spinally projecting and nitrergic neurons in the PVN may contribute to the central pathways activated by thermal stimulation.

Activation of NADPH-diaphorase-positive projections to the rostral ventrolateral medulla following cardiac mechanoreceptor stimulation in the conscious rat

Stimulation of cardiac mechanoreceptors during volume expansion elicits reflex compensatory changes in sympathetic nerve activity (SNA). The hypothalamic paraventricular nucleus (PVN) and nucleus of the tractus solitarius (NTS) are autonomic regions known to contribute to this reflex. Both of these nuclei project to the rostral ventrolateral medulla (RVLM), critical in the tonic generation of SNA. Recent reports from our laboratory show that these pathways 1) are activated following cardiac mechanoreceptor stimulation, and 2) produce nitric oxide, known to influence SNA. The aims of the present study were to determine whether 1) the activated neurons within the PVN and NTS were nitrergic and 2) these neurons projected to the RVLM. Animals were prepared, under general anesthesia, by microinjection of a retrogradely transported tracer into the pressor region of the RVLM and the placement of a balloon at the right venoatrial junction. In conscious rats, the balloon was inflated to stimulate the cardiac mechanoreceptors or was left uninflated. Balloon inflation elicited a significant increase in Fos-positive neurons in the parvocellular PVN (sevenfold) and NTS (fivefold). In the PVN, 51% of nitrergic neurons and 61% of RVLM-projecting nitrergic neurons were activated. In the NTS, these proportions were 8 and 18%, respectively. The data suggest that nitrergic neurons within the PVN and, to a lesser extent, in the NTS, some of which project to the RVLM, may contribute to the central pathways influencing SNA elicited by cardiac mechanoreceptor stimulation.

Influence of Dehydration on the Expression of Neuropeptide Y Y1 Receptors in Hypothalamic Magnocellular Neurons

Regulation of vasopressin (VP) and oxytocin (OT) secretion involves integration of neural signals from hypothalamic osmoreceptors, ascending catecholaminergic and peptidergic cell groups in the brain stem, and local and autoregulatory afferents. Neuropeptide Y (NPY) is one factor that stimulates the release of VP and OT from the supraoptic (SON) and paraventricular nuclei of the hypothalamus via activation of Y1 receptors (Y1R). The current studies were designed to assess the regulation and distribution of NPY Y1R expression in the SON of male rats that were either given 2% NaCl drinking water (24-72 h) or water deprived (48 h). Subjecting male rats to these conditions resulted in significant increases in both the number of cells expressing Y1R immunoreactivity (ir) and the amount of Y1R protein per cell within the SON. Y1R immunoreactivity was increased in the magnocellular but not medial parvocellular paraventricular nuclei, and Y1R mRNA levels were increased in the SON of salt-loaded rats. Subpopulations of both VP and OT cells in the hypothalamus express Y1R immunoreactivity and a greater percentage of VP-ir cells express Y1R after salt loading. To control for potential effects of dehydration-induced anorexia, a group of euhydrate animals was pair fed with animals consuming 2% NaCl. No detectable change in Y1R expression was observed in the SON of pair-fed animals, even though body weights were significantly lower than controls. These data demonstrate that NPY Y1R gene and protein expression are increased in the SON of salt-loaded and water-deprived animals and provide a mechanism whereby NPY can support VP/OT release during prolonged challenges to fluid homeostasis.