Adrenalectomy decreases neuropeptide Y mRNA levels in the arcuate nucleus

Recent studies suggest that glucocorticoids may increase NPY and NPY mRNA levels. To determine if endogenous corticosterone affects the level of NPY mRNA in areas that control NPY levels in the paraventricular nucleus, we examined the effects of adrenalectomy and corticosterone replacement on NPY mRNA levels in the arcuate nucleus and brainstem. Rats were either adrenalectomized, adrenalectomized and corticosterone replaced, or sham-operated. The arcuate nucleus, hypothalamus (excluding arcuate nucleus), and brainstem were collected and the RNA isolated. Dot blots were made of each tissue and the NPY mRNA quantitated by densitometry. Adrenalectomy significantly reduced NPY mRNA levels in the arcuate nucleus, while corticosterone replacement restored the NPY mRNA levels. NPY mRNA levels in the remainder of the hypothalamus were not affected by adrenalectomy. Adrenalectomy also had no affect on NPY mRNA levels in the brainstem. These data suggest that the paraventricular nucleus may be affected by glucocorticoids via an NPY pathway and that the two major afferent pathways of NPY-containing neurons to the paraventricular nucleus may be regulated by different mechanisms.     

The effects of acute phencyclidine treatment on neuropeptide Y (NPY) neuronal system in the rat arcuate nucleus studied by immunocytochemistry and in situ hybridization

Summary Phencyclidine (PCP) is a dissociative drug and an antagonist of N-methyl-D-aspartate (NMDA) receptor. The effects of PCP treatment on neuropeptide Y (NPY) system in the arcuate nucleus of the rat hypothalamus were examined both by immunocytochemistry and in situ hybridization. In acute PCP-treated rats, the NPY-immunoreactive perikarya appeared in the arcuate nucleus but no perikarya were detected in controls, without colchicine pretreatment. The signals of NPY mRNA by in situ hybridization increased in the PCP-treated rats than those of controls. These results suggest that the NPY system in the arcuate nucleus might be partly controlled by glutamatergic neurons.     

Regulation of proopiomelanocortin gene expression by neuropeptide Y in the rat arcuate nucleus

In the arcuate nucleus which is richly innervated by both proopiomelanocortin (POMC) and neuropeptide Y (NPY) neurons, it has been shown that NPY fibers are in synaptic contact with POMC cell bodies. In order to determine whether NPY could influence POMC neuronal activity, we have studied the effects of NPY and some NPY analogs on POMC gene expression using quantitative in situ hybridization. The following peptides NPY, [Leu³¹, Pro³⁴]-NPY (a Y₁ receptor agonist), and NPY_13–36 (a Y₂ receptor agonist) were injected into the left lateral cerebral ventricle of adult male rats 4 h before being perfused for histological procedures. The intracerebroventricular injection of NPY and NPY_13–36 induced a significant decrease in the number of grains overlying the labelled neurons. On the other hand, the Y₁ receptor agonist [Leu31, Pro34]-NPY did not modify POMC mRNA levels. These data then strongly suggest that NPY negatively regulates the genetic expression of POMC neurons via the Y₂ NPY receptor subtype.     

Participation of the nucleus locus coeruleus in DOCA-salt hypertensive rats

The locus coeruleus was subjected to biphasic electrical stimulation, and a group of deoxycorticosterone acetate (DOCA)-salt hypertensive rats revealed a greater pressor response than a group of normotensive control rats. The pressor threshold current (i.e. minimum current to raise the arterial pressure by 10 mm Hg) of DOCA-salt hypertensive rats was also lower. The threshold current was low even in the prehypertensive stage of DOCA-salt treated rats. During arterial pressure fall, the pressurethreshold current lowered only in normotensive rats and neither group underwent a change during pressure rise. These data indicated that the locus coeruleus had a pressor role and it had been accelerated in DOCA-salt treated rats before hypertension was evident. Following bilateral electrical lesions of the locus coeruleus no significant differences were observed in the arterial pressure changes, while vascular reactivity to norepinephrine did not differ between the two groups. The results suggest that the locus coeruleus may be involved in the development of hypertension but may not be important in the maintenance of it.     

Inhibition from locus coeruleus of nucleus accumbens neurons activated by hippocampal stimulation

Electrophysiological studies using rats were performed to examine the influence of locus coeruleus (LC) on nucleus accumbens (Acc) neurons. Spike generation by hippocampal stimulation was inhibited by both LC conditioning stimulation and iontophoretic application of noradrenaline, but spikes elicited by stimulation of parafascicular nucleus of thalamus were rarely affected by LC conditioning stimulation or noradrenaline. The LC-induced inhibition was antagonized by iontophoretic sotatol, but not by phentolamine, suggesting that noradrenaline derived from the LC inhibits the Acc neurons receiving input from the hippocampus, probably acting on a β-adrenergic receptor.     

Gene expression of neuropeptide Y in the nucleus of the solitary tract is activated in rats under restricted daily feeding but not under 48-h food deprivation

The two neuropeptide Y (NPY) systems innervating the hypothalamic paraventrivular nucleus were examined regarding their roles in the prefeeding corticosterone peak developed under restricted daily feeding (RF). Protein and mRNA levels of NPY were measured in the arcuate nucleus (ARC) and the nucleus of the solitary tract (NST) in rats under 48-h food deprivation (48-hFD), RF, and 72-h food deprivation imposed after RF (post-RF 72-hFD) with 7 days of ad libitum feeding in between. NPY protein and mRNA levels in the ARC significantly increased with 48-hFD and decreased with re-feeding, whereas those in the NST were not changed by 48-hFD. When rats had RF imposed with free access to food from 10.00 to 12.00 h (lights on from 06.00 to 18.00 h) for 3 weeks, NPY concentrations in the ARC increased at 10.00 h, just prior to the daily meal, but those in the NST did not change significantly throughout the period examined. On the other hand, NPY mRNA levels in both the ARC and NST increased before the meal supply and remained high for 4 h after feeding. Under post-RF 72-hFD, the prefeeding peak of NPY mRNA was detected in the NST, but NPY mRNA levels in the ARC were continuously high throughout the 24-h period. These findings indicate that the NPY neurons from the NST are specifically activated by RF, whereas those from the ARC are generally stimulated by an increased food demand.     

Adrenal steroid regulation of neuropeptide Y (NPY) mRNA: differences between dentate hilus and locus coeruleus and arcuate nucleus

Regulation by adrenal steroids of neuropeptide Y (NPY) mRNA was investigated in hilus of the dentate gyrus, arcuate nucleus of hypothalamus and locus coeruleus (LC) of the adult rat brain. Adrenalectomy (ADX) increased NPY mRNA in hilus but decreased NPY mRNA levels in arcuate nucleus and LC. Using a steroid replacement paradigm previously shown to discriminate between Type I and Type II adrenal steroid receptor mediated effects, it was shown that Type I receptor stimulation by aldosterone prevented the ADX-induced increase of NPY mRNA in hilus, whereas Type II receptor stimulation by Ru28362 prevented the ADX-induced decrease in NPY mRNA in arcuate nucleus and LC. The results for hilus are consistent with evidence for a role of Type I receptors in maintaining levels of a number of gene products associated with neurotransmission. The different regulation in hilus from that in arcuate and LC indicate, along with evidence for regulation of NPY expression by insulin, NGF and cyclic AMP and phorbol esters, that the adrenal steroid regulation of NPY gene expression is part of a complex set of regulatory mechanisms that depend on the brain region and cell type.     

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.     

A circulating ghrelin mimetic attenuates light-induced phase delay of mice and light-induced Fos expression in the suprachiasmatic nucleus of rats

Anatomical evidence suggests that the ventromedial arcuate nucleus (vmARC) is a route for circulating hormonal communications to the suprachiasmatic nucleus (SCN). Whether this vmARC-SCN connection is involved in the modulation of circadian activity of the SCN is not yet known. We recently demonstrated, in rats, that intravenous (i.v.) injection of a ghrelin mimetic, GHRP-6, during the daytime activated neurons in the vmARC and reduced the normal endogenous daytime Fos expression in the SCN. In the present study we show that i.v. administration of GHRP-6 decreases light-induced Fos expression at ZT13 in the rat SCN by 50%, indicating that light-induced changes in the SCN Fos expression can also be reduced by GHRP-6. Because it is difficult to study light-induced phase changes in rats, we examined the functional effects of GHRP-6 on light-induced phase shifts in mice and demonstrated that peripherally injected GHRP-6 attenuates light-induced phase delays at ZT13 by 45%. However, light-induced Fos expression in the mice SCN was not blocked by GHRP-6. These results illustrate that acute stimulation of the ghrelinergic system may modulate SCN activity, but that its effect on light-induced phase shifts and Fos expression in the SCN might be species related.     

Effects of adrenalectomy on CART expression in the rat arcuate nucleus

In order to test for glucocorticoid regulation of CART in the arcuate nucleus, adrenalectomies (ADX) and hormone replacements (HRs) were carried out in groups of rats. CART mRNA levels were determined by in situ hybridization and CART peptide levels by immunocytochemistry. ADX caused a lowering of CART mRNA and peptides levels in the arcuate and this was reversed by HR. These results indicate a glucocorticoid regulation of CART in the arcuate. The regulation could be direct through an action of glucocorticoid receptors or indirectly through ADX-induced changes in leptin levels. These findings suggest a role for CART in the stress response.     

Inhibition of sexual maturation in male rats by melatonin: evidence linking the mechanism of action to changes in the regulation of hypothalamic neuropeptide y

Activation of gonadotrophin-releasing hormone (GnRHJ pathways is a pivotal event in the process of sexual maturation, however the regulatory influences that precipitate this change and lead to the onset of puberty remain poorly understood. Recent studies indicate that neuropeptide Y (NPY) may participate in the regulation of luteinizing hormone secretion by modulating the pattern of GnRH secretion and by directly altering the pituitary responsiveness to GnRH stimulation. To determine whether NPY plays a role in puberty-associated changes in hypothalamic function, levels of NPY-like immunoreactivity (NPY-IR) were measured in a fragment of the hypothalamus encompassing the median eminence and medial portion of the arcuate nucleus (ME-AN), and also in the remainder of the hypothalamus from male rats of different ages. To identify changes in hypothaiamic NPY linked to the process of sexual development, the effect of delaying sexual maturation by daily afternoon administration of 100 μg melatonin (MT) from 20 to 40 days was investigated. In the hypothalamus and ME-AN, total NPY content increased progressively with age. Expressed as a concentration (fmol/μg extracted protein), peak values for the ME-AN (55.4 ± 7.0) were observed at 30 days of age followed by a decline to lower levels (30.2 ± 1.9) at 40 days. Daily afternoon administration of MT from 20 days of age resulted in significant increases (P<0.01) in the levels of NPY-IR in the ME-AN compared to control values at 30 and 40 days of age. MT was without effect on NPY-IR levels in the remainder of the hypothalamus. When MT was administered in the early morning, a procedure which does not delay sexual maturation, NPY-IR values for the ME-AN region were not different from control rats indicating that the MT-induced changes in NPY were related to the effects on sexual maturation. Using pituitary luteinizing hormone content and seminal vesicle weight as indices of sexual development, significant inverse correlation coefficients (P<0.001) between these parameters and the NPY concentration in the ME-AN were observed (r =-0.79 and -0.70, respectively). From published data it is not possible to conclude whether the main effects of NPY are exerted at the hypothalamic or pituitary level. However, the changes in the NPY content of the ME-AN observed during the onset of puberty, and the influence of MT on these changes, support assertions that NPY is involved in the regulation of sexual maturation.     

Prenatal stress alters Fos protein expression in hippocampus and locus coeruleus stress-related brain structures

Prenatal stress (PS) durably influences responses of rats from birth throughout life by inducing deficits of the hypothalamo-pituitary-adrenal (HPA) axis feedback. The neuronal mechanisms sustaining such alterations are still unknown. The purpose of the present study was to determine whether in PS and control rats, the exposure to a mild stressor differentially induces Fos protein in hippocampus and locus coeruleus, brain areas involved in the feedback control of the HPA axis. Moreover, Fos protein expression was also evaluated in the hypothalamic paraventricular nucleus (PVN) that reflect the magnitude of the hormonal response to stress. Basal plasma corticosterone levels were not different between the groups, while, PS rats exhibited higher number of Fos-immunoreactive neurons than controls, in the hippocampus and locus coeruleus in basal condition. A higher basal expression of a marker of GABAergic synapses, the vGAT, was also observed in the hypothalamus of PS rats. Fifteen minutes after the end of the exposure to the open arm of the elevated plus-maze (mild stress) a similar increased plasma corticosterone levels was observed in both groups in parallel with an increased number of Fos-immunoreactive neurons in the PVN. Return to basal plasma corticosterone values was delayed only in the PS rats. On the contrary, after stress, no changes in Fos-immunoreactivity were observed in the hippocampus and locus coeruleus of PS rats compared to basal condition. After stress, only PS rats presented an elevation of the number of activated catecholaminergic neurons in the locus coeruleus. In conclusion, these results suggest for the first time that PS alters the neuronal activation of hippocampus and locus coeruleus implicated in the feedback mechanism of the HPA axis. These data give anatomical substrates to sustain the HPA axis hyperactivity classically described in PS rats after stress exposure.     

Corticotropin-releasing factor mRNA and substance P receptor binding in the paraventricular hypothalamic nucleus, central nucleus of the amygdala, and locus coeruleus of sprague-dawley rats following restraint-induced stress

The central mechanism of stress is poorly understood. This study was designed to examine how corticotropin-releasing factor (CRF) neurons, together with substance P (SP) receptors in the paraventricular hypothalamic nucleus (PVN), central nucleus of the amygdala (CeA), and locus coeruleus (LC), are affected by stress. Sprague-Dawley rats were restrained for 2 h. Animals were sacrificed by decapitation immediately after the 2-h restraint (the 0-h group) and 4, 24, or 48 h after restraint. Tissue sections were cut and collected on two sets of slides. Tissue sections of the first set were processed for studying CRF mRNA using ³³P-labeled 60-mer oligonucleotide probe. Immediately adjacent tissue sections were processed for studying SP receptor-binding capacity using ¹²⁵I-SP ligand. Quantitative results showed that CRF mRNAs in the PVN were significantly up-regulated at the 4- and 24-h stages, and they seemed not to be regulated by SP receptors. In addition, SP receptors in the CeA were up-regulated at the 24- and 48-h stages, whereas SP receptors were down-regulated in the LC at the same stages. In concert with the literature indicating SP antagonist’s antidepressive effects, up-regulated SP receptors in the CeA might contribute to the development of stress-related depression. Parts of the results have been presented at the summer neuropeptide conference (June 8–12, 2003, Montauk, NY) as P-37 (Neuropeptides [2003] 37, 192).     

Direct inhibition of arcuate kisspeptin neurones by neuropeptide Y in the male and female mouse

Adverse energy states exert a potent suppressive influence on the reproductive axis by inhibiting the pulsatile release of gonadotrophin‐releasing hormone and luteinising hormone. One potential mechanism underlying this involves the metabolic‐sensing pro‐opiomelanocortin and agouti‐related peptide/neuropeptide Y (AgRP/NPY) neuronal populations directly controlling the activity of the arcuate nucleus kisspeptin neurones comprising the gonadotrophin‐releasing hormone pulse generator. Using acute brain slice electrophysiology and calcium imaging approaches in Kiss1‐GFP and Kiss1‐GCaMP6 mice, we investigated whether NPY and α‐melanocyte‐stimulating hormone provide a direct modulatory influence on the activity of arcuate kisspeptin neurones in the adult mouse. NPY was found to exert a potent suppressive influence upon the neurokinin B‐evoked firing of approximately one‐half of arcuate kisspeptin neurones in both sexes. This effect was blocked partially by the NPY1R antagonist BIBO 3304, whereas the NPY5R antagonist L152,804 was ineffective. NPY also suppressed the neurokinin B‐evoked increase in intracellular calcium levels in the presence of tetrodotoxin and amino acid receptor antagonists, indicating that the inhibitory effects of NPY are direct on kisspeptin neurones. By contrast, no effects of α‐melanocyte‐stimulating hormone were found on the excitability of arcuate kisspeptin neurones. These studies provide further evidence supporting the hypothesis that AgRP/NPY neurones link energy status and luteinising hormone pulsatility by demonstrating that NPY has a direct suppressive influence upon the activity of a subpopulation of arcuate kisspeptin neurones.     

Differentiated cardiovascular afferent regulation of locus coeruleus neurons and sympathetic nerves

The activity of brain norepinephrine (NE) neurons in the locus coeruleus (LC) and peripheral sympathetic nerve activity (NE-SNA) in the splanchnic/renal nerve were recorded simultaneously during alterations of arterial blood pressure and circulating blood volume. Utilizing this experimental procedure we have previously found that both central and peripheral NE neurons are inhibited during blood pressure elevation. Furthermore, both neuronal systems were found to be inhibited during blood volume load, an effect apparently mediated by vagal afferents. In the present study both brain NE-LC activity and NE-SNA were increased during blood volume depletion. However, during prolonged hemorrhage the initial excitation of NE-SNA was followed by a marked inhibition. In contrast, the increase in NE-LC activity remained throughout the volume depletion period. The responses of central and peripheral NE neurons during hemorrhage were abolished in animals subjected to bilateral cervical vagotomy. Nitroprusside- or phenylephrine-induced blood pressure variations were associated with reciprocal changes in both central and peripheral NE neuronal activity. The NE-LC responses to blood pressure variations were abolished after bilateral vagotomy. NE-SNA responses, on the other hand, persisted after bilateral vagotomy. Our present and previous findings show that brain NE-LC neurons, similarly to peripheral NE neurons in the splanchnic/renal nerve, are regulated by tonically active cardiovascular afferents. Whereas peripheral NE-SNA is regulated by both arterial (high pressure) baroreceptors and cardiac volume (low pressure) receptors, the NE-LC neurons seem exclusively regulated by cardiac volume (low pressure) receptors.     

Enhancement of learning four weeks after stimulation of the nucleus locus coeruleus in the rat: Differential effects of dorsal noradrenergic bundle lesion and lesion of the locus coeruleus proper

In previous studies we showed that electrical stimulation of the nucleus locus coeruleus produced, four weeks later, a significant improvement in performance in acquisition of food-reinforced operant conditioning. In the two experiments reported here, we tested the role of the dorsal noradrenergic bundle and of the locus coeruleus proper in this long-term effect. Lesioning the dorsal noradrenergic bundle did not have a clear and consistent effect, whereas lesion of the nucleus coeruleus proper suppressed almost totally the beneficial effect of the stimulation. In the first experiment, the dorsal noradrenergic bundle was lesioned by local bilateral injection of 6-hydroxydopamine, 8 days before stimulation on the locus coeruleus. Four weeks after the stimulation, the rats were tested for acquisition of the operant task. Three control groups were used: not lesioned but stimulated, lesioned but not stimulated, and not lesioned/not stimulated. The locus coeruleus stimulation produced the same improvement of performance at the beginning of the acquisition, whether or not the dorsal noradrenergic bundle had been lesioned. However, a significant decrement of performance was observed in lesioned and stimulated rats during the last 40 min of the acquisition. In the second experiment, the locus coeruleus proper was destroyed by bilateral local injection of 6-hydroxydopamine and the locus coeruleus region was stimulated 15 days later. Three control groups were used, as in the first experiment. All the rats were tested 4 weeks later for acquisition of the operant task. The locus coeruleus lesion significantly attenuated the beneficial effect of the stimulation; however, the performance of the lesioned and stimulated rats was still significantly superior to that of the lesioned but not stimulated rats. These results suggest that the noradrenergic locus coeruleus system is involved in the long-term effect, but that the rostral projections passing through the dorsal bundle, in front of the lesion, are not critically involved in the observed effect.     

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.     

Corticotropin releasing hormone neurons in the paraventricular nucleus are direct targets for neuropeptide Y neurons in the arcuate nucleus: an anterograde tracing study

In the present study, anterograde tracing combined with triple label immunofluorescent staining was conducted to examine the possible anatomical interactions between Neuropeptide Y (NPY) neurons in the arcuate nucleus of the hypothalamus (ARH) and the corticotropin releasing hormone (CRH) system in the paraventricular nucleus of the hypothalamus (PVH). The anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), was iontophresed into the ARH of female rats and triple label immunofluorescence staining with three different fluorophores was performed to visualize PHA-L, NPY and CRH, with the aid of confocal microscopy. In PVH, NPY and PHA-L double-labeled fibers were found mainly in the parvocellular part of the PVH (PVHp). Confocal analysis demonstrated that NPY/PHA-L double-labeled fibers came in close apposition to CRH perikarya. In the median eminence, NPY/PHA-L double-labeled fibers were found both in the inner and the outer zones of the median eminence. However, very few double-labeled fibers were found in the proximity of CRH neuronal fibers in the median eminence. Double label staining was also performed to determine if NPY Y1 receptors were expressed in CRH neurons. Two different fluorophores were used to visualize CRH neurons and Y1 receptor. No convincing Y1-positive staining was found in CRH cell bodies in the PVH, even though Y1-positive staining in numerous fibers and cell bodies was observed throughout the region. However, Y1-positive fibers were shown to make close contact with CRH cell bodies in the PVH. In the ME, the majority of the Y1-positive fibers were located in the lateral portion of the ME, whereas the CRH fibers were found mainly in the medial portion of the external zone of the ME. The results of the present study suggest that ARH NPY neurons provide direct input into CRH cell bodies in the PVH region. However, the direct effects of NPY must be mediated by some receptor subtype other than Y1. Y1 receptor involvement in NPY modulation of CRH neuronal function in the PVH appears to be indirect through modulation of neuronal afferents making contact with CRH neurons.