Direct and Indirect Effects of Cannabinoids on in vitro GABA Release in the Rat Arcuate Nucleus

Within the hypothalamic arcuate nucleus, two neuronal subpopulations play particularly important roles in energy balance; neurones expressing neuropeptide Y (NPY), agouti‐related peptide (AgRP) and GABA are orexigenic, whereas neurones expressing pro‐opiomelanocortin and CART are anorexigenic. The pivotal role of these neuropeptides in energy homeostasis is well‐known, although GABA may also be an important signal because targeted knockout of the GABA transporter in NPY/AgRP/GABA neurones results in a lean, obesity‐resistant phenotype. In the present study, we describe an in vitro model of K⁺‐evoked GABA release from the hypothalamus and determine the effects of cannabinoid receptor activation. K⁺‐evoked GABA release was sensitive to leptin, insulin and PYY(3‐36), indicating that GABA was released by arcuate NPY/AgRP/GABA neurones. In the presence of tetrodotoxin (TTX), the cannabinoid CB1 receptor agonist WIN 55,212‐2 inhibited K⁺‐evoked GABA release. This was prevented by the CB1 receptor inverse agonist rimonabant. Rimonabant had no effect when applied alone. In the absence of TTX, however, the opposite effects were observed: WIN 55,212‐2 had no effect while rimonabant inhibited GABA release. This indicates that GABA release can involve an indirect, TTX‐sensitive mechanism. The most parsimonious explanation for the inhibition of GABA release by a CB receptor inverse agonist is via the disinhibition of an cannabinoid‐sensitive inhibitory input onto GABAergic neurones. One local source of an inhibitory neurotransmitter is the opioidergic arcuate neurones. In our in vitro model, K⁺‐evoked GABA release was inhibited by the endogenous opioid peptide β‐endorphin in a naloxone‐sensitive manner. The inhibitory effect of rimonabant was also prevented by naloxone and a κ‐opioid receptor selective antagonist, suggesting that GABA release from arcuate NPY/AgRP/GABA neurones can be inhibited by endogenous opioid peptides, and that the release of opioid peptides is sensitive to cannabinoids.     

Estrous Cycle Variations in Gonadotropin-Releasing Hormone, Substance P and Beta-Endorphin Contents in the Median Eminence, the Arcuate Nucleus and the Medial Preoptic Nucleus in the Rat: A Detailed Analysis of Proestrus Changes

The concentrations of gonadotropin-releasing hormone, substance P and β-endorphin were measured in the median eminence, the arcuate nucleus and the medial preoptic nucleus of 4-day cycling female rats. Very well marked estrous cycle-related fluctuations were registered for these neuropeptides in these areas. The largest variations in concentrations of peptide levels were observed in the median eminence. Substance P concentration was highest throughout the day of proestrus as compared to the three others days of the cycle. At the time of the preovulatory luteinizing hormone surge, on the afternoon of proestrus, there was a marked increase in gonadotropin-releasing hormone concentration and a marked decrease in β-endorphin concentration.     

Melanocortin-3 receptor mRNA expression in pro-opiomelanocortin neurones of the rat arcuate nucleus

The melanocortins alpha- and gamma-melanocyte-stimulating hormones (alpha- and gamma-MSH) derive from the pro-opiomelanocortin (POMC) precursor. Melanocortins exert a wide range of biological activities in the brain through activation of at least three distinct melanocortin receptor (MC-R) subtypes. In order to determine whether POMC neurones can modulate their own activity, we looked for the possible expression of the MC3-R gene in POMC-positive cell bodies in the rat hypothalamus. In situ hybridization experiments revealed that the density of MC3-R mRNA is particularly high in the arcuate nucleus which contains the main population of POMC neurones in the brain. The occurrence of MC3-R mRNA in POMC-positive cell bodies was demonstrated using a double-labelling in situ hybridization technique. The proportion of POMC neurones expressing MC3-R mRNA was significantly higher in the most rostral (43.5%) than in the most posterior part of the arcuate nucleus (8.2%). These results indicate that melanocortins likely exert a direct regulatory feedback on POMC neurones through activation of MC3-R receptors. Our data also suggest that MC3-R may be involved in the neuroendocrine responses induced by centrally administered melanocortins.     

Proopiomelanocortin, Preproenkephalin-A and Preprocholecystokinin mRNA in the Hypothalamus of CCI4-Treated Rats

It has previously been shown that β-endorphin (BE) and cholecystokinin (CCK) concentrations decrease in brain areas of rats or humans affected by severe liver disease (SLD). We now report the increase of proopiomelanocortin (POMC) and preprocholecystokinin mRNA in the hypothalamus and cortex of rats with experimental SLD, suggesting an increased post-translational turnover of BE and CCK in this experimental condition.     

Fine Structural Changes in the Rat Arcuate Nucleus by Forced Running Stress

Abstract: The forced running stress in male rats induced some fine structural changes in the arcuate nucleus. By inducing stress for 2 days, the number of multilamellar astrocytic wrappings and endoplasmic reticular (ER) whorls increased significantly. After a stress of 12 days, half of the rats remained inactive for several weeks, and the other half regained their normal activity. In the inactive group, the ER whorls were partially degenerated at 2 weeks after the stress. These degenerative changes remained for 14 weeks after the stress with the increased lysosomes and the disorganization of r-ER. These findings may suggest that long-term stress induces degenerative changes in the arcuate nucleus neurons and that some of these changes persist with some aging-like morphological changes.     

Effects of Dehydroepiandrosterone (DHEA) on Pituitary Prolactin and Arcuate Nucleus Neuron Tyrosine Hydroxylase mRNA Levels in the Rat

It is well documented that dehydroepiandrosterone (DHEA), an adrenal androgen, is converted into potent androgens and/or estrogens in peripheral tissues. Since sex steroids are involved in the regulation of prolactin (PRL) secretion, we have studied the effect of DHEA administration on PRL mRNA levels in both adult male and female rats. Since tuberoinfundibular dopaminergic (TIDA) neurons are involved in the negative regulation of PRL, we have also evaluated the effects of DHEA on the genetic expression of tyrosine hydroxylase (TH), the limiting enzyme in catecholamine biosynthesis in TIDA neurons. Sham-operated and castrated animals of both sexes received during 2 days DHEA at the dose of 6 mg/kg/day, starting on the first day after castration. PRL and TH mRNA levels were measured by quantitative in situ hybridization. In the male rat, orchiectomy performed 3 days earlier did not modify PRL mRNA levels. DHEA administration increased the hybridization signal in both sham-operated and orchiectomized animals. In the female, ovariectomy decreased PRL mRNA levels and, as observed in the male, DHEA treatment induced an increase in the hybridization signal in both control and ovariectomized rats. In TIDA neurons, castration increased TH mRNA levels as evaluated by number of grains over labelled neurons and the number of TH-labelled cells per section in both male and female animals. In both sham-operated male rats and orchiectomized animals, DHEA decreased the hybridization signal. In the female, DHEA administration completely prevented the increase in TH mRNA levels due to ovariectomy. In sham-operated female rats, the treatment had no effect. These data clearly indicate that in both male and female rats DHEA exerts an estrogenic influence on both PRL and TH gene expression. Although these in vivo experiments do not allow to establish whether the stimulation of PRL gene expression is due to an action of the steroid on the pituitary or at the hypothalamic level or alternatively at both sites, it is likely that one of the mechanisms of action of DHEA might be related to a decrease in dopamine release following a depression of TIDA neuron activity.     

Electrical Stimulation of the Rat Periventricular Nucleus Influences the Activity of Hypothalamic Arcuate Neurones

In rats, the release of growth hormone (GH) is inhibited during electrical stimulation of the periventricular nucleus but after the end of stimulation, there is a rebound ‘hypersecretion’ of GH. We examined the responses of arcuate neurones in pentobarbitone-anaesthetized male rats, following electrical stimulation of the periventricular nucleus to test the hypothesis that the effects of periventricular nucleus stimulation on GH secretion are mediated via effects upon GH-releasing hormone (GRF) neurones in the arcuate nucleus. The electrical activity of 2 groups of arcuate neurones were analysed before, during and after periventricular nucleus stimulation (10 Hz, 5 min, 0.5 mA biphasic, 0.5/1.0 ms): a) putative neurosecretory cells which were antidromically identified (AD) as projecting to the median eminence (n = 53) and b) non-neurosecretory cells, identified by their spontaneous ‘bursting’ pattern of activity (n = 29). During stimulation predominantly inhibitory responses were observed in both AD and bursting cell groups. Of the 39 AD cells which were spontaneously active, 25 were inhibited during the periventricular nucleus stimulation, and 10 of these showed a rebound hyperactivation following the end of stimulation. Fifteen bursting cells were inhibited during stimulation and 4 of these displayed a rebound hyperactivation following the end of stimulation. Additional evidence was sought for the identity of these cells by testing their response to electrical stimulation of the basolateral amygdala (which has previously been shown to increase plasma GH concentration without influencing the release of other pituitary hormones). Six of the 10 AD cells which displayed the inhibition/rebound response to periventricular nucleus stimulation were also excited following electrical stimulation of the basolateral amygdala. We conclude that 1) electrical stimulation of the periventricular nucleus and the basolateral amygdala exert predominantly inhibitory and excitatory effects respectively upon the activity of arcuate neurones but for neither site were the effects of stimulation exclusively upon GRF neurones, and 2) the rebound hypersecretion of GH following PeN stimulation is likely to involve the rebound activation of arcuate neurones.     

Effects of Long-Term Treatment with Antidepressant Drugs on Proopiomelanocortin and Neuropeptide Y mRNA Expression in the Hypothalamic Arcuate Nucleus of Rats

Antidepressant drugs have in common a delayed onset of clinical efficacy. In rats, long-term, daily administration of four different types of clinically effective antidepressant drugs results in decreased corticotropin releasing hormone (CRH) mRNA expression levels in the hypothalamic paraventricular nucleus (PVN). Because a subpopulation of neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (Arc) projects to the PVN, we measured NPY and POMC mRNA expression in the Arc using in situ hybridization histochemistry at several time points following daily administration of four different antidepressant drugs. After 14 and 56 days of imipramine treatment, Arc NPY mRNA levels are decreased to 85% and 75% of control levels, but are unchanged compared to control after one or five days of treatment. Arc POMC mRNA levels are unchanged compared to controls at 1, 5, 14, or 56 days following imipramine treatment. Unlike after imipramine, Arc NPY and POMC mRNA levels are increased significantly to 134–172% of control following 56-day treatment with the antidepressant drugs fluoxetine, phenelzine, or idazoxan. The divergent effects of imipramine vs the other 3 antidepressant drugs on Arc NPY mRNA expression are similar to the pattern of changes in tyrosine hydroxylase (TH) mRNA expression levels in the locus coeruleus (LC) using the same experimental paradigm, but are different from the unidirectional depressive effects of all four drugs on CRH mRNA expression in the PVN. Thus, the Arc NPY and LC noradrenergic systems may act coordinately in mediating antidepressant effects. The present data are consistent with the delayed onset of clinical efficacy for antidepressant drugs, and suggest that Arc NPY and POMC neurotransmitter systems play a role in the pathophysiology of depression.     

Role of Neuropeptide Y in the Regulation of Tyrosine Hydroxylase Messenger Ribonucleic Acid Levels in the Male Rat Arcuate Nucleus as Evaluated by in situ Hybridization

Neuroanatomical data have clearly demonstrated the existence of synaptic contacts between neuropeptide Y (NPY) endings and tuberoinfundibular dopaminergic (TIDA) neurons in the rat arcuate nucleus. In order to determine the influence of NPY in the biosynthesis of dopamine, we have studied the effects of NPY and some NPY analogs on tyrosine hydroxylase (TH) gene expression in TlDA neurons in the male rat. The following peptides: NPY, PYY, [Leu³¹, Pro³⁴]-NPY (a Y, receptor agonist) and NPY_13–36 (a Y₂ receptor agonist) were injected into the left lateral ventricle of adult male rats. All the animals were perfused with 4% paraformaldehyde 4 h after injection. Cryostat sections through the arcuate nucleus were processed for quantitative in situ hybridization. The intracerebroventricular injection of NPY, PYY and [Leu³¹, Pro³⁴]-NPY induced an increased of 43, 33 and 42%, respectively, in the number of grains overlying TH neurons. On the other hand, the Y₂ receptor agonist NPY_13–36 did not influence mRNA levels. These data then strongly suggest that NPY positively regulates the genetic expression of TH in rat TlDA neurons via the Y, NPY receptor subtype.     

Chronic Administration of Glucocorticoids Directly Upregulates Prepro-Neuropeptide Y and Y1-Receptor mRNA Levels in the Arcuate Nucleus of the Rat

The complete sequence of the cDNA encoding the neuropeptide Y (NPY) Y₁-receptor has recently been deduced from a rat brain library, and the presence of messenger ribonucleic acid (mRNA) encoding Y₁-receptor protein has been demonstrated within the brain. Using quantitative in situ hybridization histochemistry, the content and distribution of Y₁receptor and preproNPY mRNAs have been investigated in the hypothalamic arcuate nucleus of adrenalectomized rats receiving glucocorticoid replacement therapy for 12 days by means of either high doses of dexamethasone in their drinking water or by subcutaneous corticosterone pellets. Basal metabolic parameters such as weight gain or loss, blood glucose and plasma insulin were monitored: Dexamethasone treatment induced weight loss and a state of hyperinsulinemia with normoglycemia, while corticosterone treated animals displayed metabolic parameters identical to sham ADX animals. Within the arcuate nucleus of glucocorticoid treated animals, levels of Y₁receptor and preproNPY mRNAs were increased. In contrast, adrenalectomy itself had no effect upon Y₁-receptor mRNA levels or preproNPY mRNA levels in the arcuate nucleus. These studies demonstrate that glucocorticoids exert a stimulatory action on levels of Y₁-receptor mRNA and preproNPY mRNA levels in the hypothalamic arcuate nucleus. This is the first evidence to suggest that the expression of a neuropeptide-receptor gene in the central nervous system may be directly sensitive to peripheral hormonal signals.     

Arcuate Nucleus and Preoptic Area Involvement in Beta-Endorphin-lnduced Release of Prolactin in Conscious Ovariectomized Rats†

The ability of ß-endorphin (ß-END) to release prolactin and the ability of naloxone to block prolactin's release when delivered to specific hypothalamic areas via push-pull perfusion was studied in unrestrained, conscious, Ovariectomized rats. Perfusion of either the arcuate nucleus or the preoptic area with ß-END for 15 to 30 min caused a large, brief increase in plasma prolactin levels. Perfusion for a longer time period (120 min) resulted in peak prolactin levels at 60 min, with a return to baseline by 120 min, suggesting that ß-END primarily acts to induce a sequence of events that culminates in prolactin release, but other factors are needed to maintain this release over long periods of time. Perfusion of the arcuate nucleus for two 15-min periods 90 min apart resulted in two surges of prolactin. When naloxone, the opiate receptor antagonist, was added to the perfusate, ß-END was not capable of stimulating prolactin release. These results provide a model to answer whether endogenous ß-END has a role in the neuroendocrine regulation of prolactin surges and what the location is of the opiate neurons involved in this neuronal pathway.     

Influence of Testosterone on LHRH Release,LHRH mRNA and Proopiomelanocortin mRNA in Male Sheep1

The mechanism whereby testosterone (T) reduces pulsatile LHRH and LH release is unknown. We tested the hypothesis that hypothalamic levels of LHRH mRNA decrease and proopiomelanocortin (POMC) mRNA increase coincident with reduced LHRH release induced by either long-term or short-term T treatment in male sheep. Experiment 1 examined the effect of long-term T exposure on LHRH and LH release and LHRH and POMC mRNA levels. Yearling Suffolk rams were castrated and assigned to one of four treatments: 1) castrated (n = 4); 2) castrated, portal cannula (n = 5); 3) castrated +T (n = 4) and 4) castrated+T, portal cannula (n = 4). T-treated males received ten 10-cm silastic T-implants immediately after castration. Surgical placement of devices for collecting hypophyseal-portal blood occurred 2 to 3 months after castration. Seven to 10 days after surgery, blood samples were collected at 10-min intervals for 8h from portal cannulated males or for 5 h from non-cannulated males to assess pulsatile LHRH and/or LH release. Immediately after blood sample collection, hypothalamic tissue was collected for in situ measurement of LHRH or POMC mRNA. T-treatment decreased (P < 0.01) mean LHRH and LH and decreased (P < 0.01) LHRH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron, but decreased (P < 0.01) silver grain area per POMC neuron. Portal cannulation tended to decrease (P= 0.057) silver grain area per LHRH neuron without significantly affecting (P > 0.10) LHRH cell numbers while reducing (P < 0.01) silver grain area per POMC neuron and POMC cell numbers. A second experiment examined the effect of 72 h of T-infusion on LHRH and POMC mRNA levels. Castrated yearling males were assigned to receive either vehicle (n = 4) or T (768 ug/kg/day;n=4). Blood samples were collected at 10 min intervals for 4h prior to and during the final 4 h of infusion. Infusion of T decreased (P < 0.01) mean LH and LH pulse frequency. T did not significantly affect (P > 0.10) silver grain area per LHRH neuron or LHRH cell numbers. T reduced (P < 0.01) silver grain area per POMC neuron without affecting (P > 0.10) POMC cell number. We reject our hypothesis and conclude that reduced LHRH or heightened POMC gene expression are not mechanisms whereby T reduces pulsatile LHRH release in male sheep.     

Combined Autoradiographic and Immunohistochemical Evidence for an Association of Somatostatin Binding Sites with Growth Hormone-Releasing Factor-Containing Nerve Cell Bodies in the Rat Arcuate Nucleus

The regulation of growth hormone secretion depends upon the complex interplay between two hypothalamic hypophysiotropic factors: growth hormone-releasing factor and somatotropin release inhibiting factor or somatostatin. Interactions between these two neurohormones appear to be exerted both distally, at the level of pituitary somatotropes, and proximally, within the hypothalamus. In an attempt to detect a possible anatomical substrate for central interactions between the two neurohormones, we compared the autoradiographic distribution of specifically labeled somatostatin binding sites with the immunohistochemical distribution of growth hormone-releasing factor-containing neurons in the hypothalamus of adult rats. Somatostatin binding sites were labeled in vitro by incubating serial brain sections with [¹²⁵l]TyrO-DTrp8-somatostatin. Growth hormone-releasing factor-immunoreactive neurons were visualized in a second set of animals, using an antiserum raised against synthetic rat growth hormone-releasing factor (1–29) NH₂. In light microscopic autoradiograms of sections incubated with [¹²⁵l]somatostatin the label was found to be concentrated over small, round or oval neuronal perikarya clustered within the ventrolateral aspect of the arcuate nucleus. The topographic distribution of these [¹²⁵l]somatostatin-labeled cells was similar to that of growth hormone-releasing factor-immunoreactive neurons detected within the same region. Moreover, the number of [¹²⁵l]somatostatin-labeled cells was found to vary in parallel with that of growth hormone-releasing factor-immunoreactive neurons throughout the rostro-caudal extent of the arcuate nucleus (coefficient of correlation r = 0.80). These results suggest that somatostatin binding sites may be directly associated with the perikarya of arcuate growth hormone-releasing factor neurons. Such an association would provide an anatomical substrate for a direct regulation of growth hormone-releasing factor secretion by somatostatin at the hypothalamic level.     

The Diurnal Variations of Glutamic Acid Decarboxylase Activity in Some Discrete Nuclei of Rat Brain

Abstract: The diurnal rhythmicity of glutamic acid decarboxylase (GAD) activity in the nucleus level of the rat brain was examined by the radiochemical micromethod in a light-dark condition. GAD activity showed a biphasic variation in globus pallidus and substantia nigra (zona reticulata and compacta), but a monophasic variation, i. e. high in the dark, in colliculus superior (stratum griseum superficiale and stratum opticum). As for hypothalamus GAD activity of some nuclei (N. arcuatus, N. periventricularis, N. preopticus, etc.) it indicated a monophasic variation, while that of some nuclei (N. suprachiasmaticus, N. ventromedialis, A. lateralis, etc.) it showed no significant variations despite their proved functional activity with rhythmicity.     

N-terminal ACTH fragments increase the CSF β-EP content in Alzheimer type dementia

Eleven patients with presenile Alzheimer type dementia (ATD) were treated with N-terminal ACTH fragments for 14 days. No change in cognitive functions was observed during the treatment. A significant increase in CSF beta-endorphin (beta-EP) levels was found, while ACTH and beta-lipoprotein remain unaffected. The possibility that ACTH and its moieties could interfere with beta-EP activities in CNS is discussed.     

Diurnal Rhythm in Appearance of Frontal Midline Theta Activity

Abstract: It has been reported that there is a certain rhythm in the appearance of Fmθ. In the present study, round-the-clock changes in the appearance of Fmθ were analyzed. The Uchida-Kraepelin test was conducted for 5 minutes a day over a period of 3 days on 18 university students. The span of time from 08:00 to 24:00 was divided into 9 temporal points and each subject was tested at 4 different points. EEGs were recorded and the changes in the appearance of Fmθ were investigated. As a result, it was found that there were signs of a frequent appearance of Fmθ in the morning, particularly between 10:00 and 12:00. Accordingly, it was evident that Fmθ appears at a certain diurnal rhythm.     

Nutritional Programming of Accelerated Puberty in Heifers: Involvement of Pro‐Opiomelanocortin Neurones in the Arcuate Nucleus

The timing of puberty and subsequent fertility in female mammals are dependent on the integration of metabolic signals by the hypothalamus. Pro‐opiomelanocortin (POMC) neurones in the arcuate nucleus (ARC) comprise a critical metabolic‐sensing pathway controlling the reproductive neuroendocrine axis. α‐Melanocyte‐stimulating hormone (αMSH), a product of the POMC gene, has excitatory effects on gonadotrophin‐releasing hormone (GnRH) neurones and fibres containing αMSH project to GnRH and kisspeptin neurones. Because kisspeptin is a potent stimulator of GnRH release, αMSH may also stimulate GnRH secretion indirectly via kisspeptin neurones. In the present work, we report studies conducted in young female cattle (heifers) aiming to determine whether increased nutrient intake during the juvenile period (4–8 months of age), a strategy previously shown to advance puberty, alters POMC and KISS1 mRNA expression, as well as αMSH close contacts on GnRH and kisspeptin neurones. In Experiment 1, POMC mRNA expression, detected by in situ hybridisation, was greater (P < 0.05) in the ARC in heifers that gained 1 kg/day of body weight (high‐gain, HG; n = 6) compared to heifers that gained 0.5 kg/day (low‐gain, LG; n = 5). The number of KISS1‐expressing cells in the middle ARC was reduced (P < 0.05) in HG compared to LG heifers. In Experiment 2, double‐immunofluorescence showed limited αMSH‐positive close contacts on GnRH neurones, and the magnitude of these inputs was not influenced by nutritional status. Conversely, a large number of kisspeptin‐immunoreactive cells in the ARC were observed in close proximity to αMSH‐containing varicosities. Furthermore, HG heifers (n = 5) exhibited a greater (P < 0.05) percentage of kisspeptin neurones in direct apposition to αMSH fibres and an increased (P < 0.05) number of αMSH close contacts per kisspeptin cell compared to LG heifers (n = 6). These results indicate that the POMC‐kisspeptin pathway may be important in mediating the nutritional acceleration of puberty in heifers.     

Nitric Oxide Inhibits Neuronal Activity in the Supraoptic Nucleus of the Rat Hypothalamic Slices

The presence of abundant nitric oxide synthase (NOS) in magnocellular neurons of the rat hypothalamus suggests that nitric oxide (NO) may be involved in controlling the release of oxytocin and vasopressin. To test this possibility, we examined the effect of NO-related drugs on extracellular discharges of 124 supraoptic nucleus (SON) neurons from slices of rat hypothalamus in vitro. Twenty-three (43%) of 53 neurons were inhibited by sodium nitroprusside (SNP), a spontaneous releaser of NO, at 1–3 mM. This inhibition was prevented by preincubation of the slices with 1

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hemoglobin, an inactivator of NO (

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), whereas hemoglobin alone enhanced neuronal activity in seven (35%) of 20 neurons.

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-Arginine (1 mM), a precursor of NO, inhibited neuronal activity in five (36%) of 14 neurons, while

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-arginine (1 mM), the inactive counterpart of

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-arginine, was ineffective (

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). N-

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-nitro-

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-arginine methyl ester (

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-NAME, 10

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), an inhibitor of NOS, also enhanced neuronal activity in five (29%) of 17 neurons, while N-

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-nitro-

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-arginine methyl ester (DNAME, 10

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), the inactive enantiomer of

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-NAME, was without effect (

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). Together, our data show that NO exerts predominantly an inhibitory effect on SON neurons and may serve as a negative feedback loop in controlling release of oxytocin and vasopressin.     

Effects of Restraint Stress and Spontaneous Hypertension on Neuropeptide Y Neurones in the Brainstem and Arcuate Nucleus

Neuropeptide Y (NPY) is found in autonomic neurones and participates in regulation of autonomic functions. To investigate the role of NPY in the stress response in normo- and hypertensive rats, activation of brainstem and arcuate nucleus (ARC) NPY neurones and levels of NPY mRNA in the ARC were measured in response to restraint stress in adult spontaneously hypertensive rats (SHRs) and two strains of normotensive rats. Controls from each strain were not restrained. Sections of the brain were prepared for Fos immunohistochemistry and NPY in-situ hybridization to identify activated NPY neurones in the nucleus of the tractus solitarii (NTS), ventrolateral medulla (VLM), and ARC. NPY mRNA levels were quantified in the ARC. In the NTS and VLM of restrained rats, approximately 33% and 75%, respectively, of NPY neurones were activated. No differences among strains were found. In the ARC, about 36% of neurones activated by restraint contained NPY mRNA with no differences found among strains. In unrestrained rats, NPY mRNA levels were significantly elevated in SHRs compared to the normotensive rats. Restraint led to significant decreases in mRNA levels in all strains and mRNA levels among strains were no longer different from one another. These data show that NPY likely participates as a neurotransmitter in the autonomic pathways utilized during stress and originating in the NTS, VLM, and ARC. On the other hand, the decreased gene expression of NPY in the ARC in response to restraint stress argues against a role for activation of autonomic pathways or the hypothalamo-pituitary-adrenal (HPA) axis by NPY from the ARC of stressed rats. The elevated NPY gene expression in resting SHRs compared to normotensive rats is abrogated after restraint, suggesting that this gene is differentially regulated in SHRs compared to normotensive rats.