Food deprivation and hypothalamic neuropeptide gene expression: effects of strain background and the diabetes mutation.

We have used a novel method to identify genes expressed in the hypothalamus which may be potentially involved in controlling food intake and energy metabolism. We assumed that food deprivation, a powerful stimulus of food intake, would stimulate the activity of neural pathways involved in feeding behavior which should be reflected in an increase in the synthesis of any relevant neuropeptide and its messenger RNA. A study of 5 neuropeptides in 5 strains of mice has identified neuropeptide Y (NPY) as a gene whose expression in the hypothalamus is controlled by nutritional status, suggesting that hypothalamic NPY neurons are a link in the neural network regulating feeding behavior and energy metabolism. In addition, we have studied the effect of the diabetes mutation on neuropeptide gene expression during fasting and refeeding. Our findings suggest that abnormal NPY and enkephalin gene expression in the hypothalamus may be two important determinants of the expression of the diabetes mutation.     

The responses of hypothalamic NPY and OBRb mRNA expression to food deprivation develop during the neonatal-prepubertal period and exhibit gender differences in rats

Neuropeptide Y (NPY) is an important hypothalamic orexigenic neuropeptide that acts in the brain. It has been established that the fasting-induced up-regulation of NPY expression is mainly caused by a reduction in the activity of leptin, which is a hormone secreted by adipose tissue. We have reported that in female rats hypothalamic NPY mRNA expression does not respond to fasting during the early neonatal period, but subsequently becomes sensitive to it later in the neonatal period. In this study, we compared the developmental changes in the responses of NPY and leptin expression to fasting between male and female rats during the neonatal to pre-pubertal period. Fasting was induced by maternal deprivation during the pre-weaning period (postnatal days 10 and 20) and by food deprivation during the post-weaning period (postnatal day 30). Hypothalamic NPY mRNA expression was not affected by fasting on postnatal day 10, whereas it was increased by fasting on postnatal day 20 and 30 in both males and females. On the other hand, the serum leptin level was decreased by fasting at all examined ages in both sexes. Namely, hypothalamic NPY mRNA expression was not correlated with the reduction in the serum leptin level at postnatal day 10 in either sex. Under the fasted conditions, the hypothalamic NPY mRNA levels of the males were higher than those of the females on postnatal days 20 and 30, whereas no such differences were observed under the normal nourishment conditions. The serum leptin levels observed under the fasted conditions did not differ between males and females at any examined age. These results suggest that some hypothalamic NPY functions develop during the neonatal period and that there is no major difference between the sexes with regard to the time when NPY neurons become sensitive to fasting. They also indicate that hypothalamic NPY expression is more sensitive to under-nutrition in male rats than in female rats, at least during the pre-pubertal period.     

Preproneuropeptide Y Messenger Ribonucleic Acid in the Hypothalamic Arcuate Nucleus of the Rat is Increased by Food Deprivation or Dehydration

The distribution of messenger ribonucleic acid (mRNA) encoding preproneuropeptide Y (prepro-NPY) in the hypothalamus of rats subjected to food deprivation or dehydration has been investigated by quantitative in situ hybridization. Levels of prepro-NPY mRNA in the arcuate nucleus (ARC) were selectively increased by both treatments. The very high concentration of prepro-NPY mRNA seen following 96 h of food deprivation had returned towards control levels after 24 h of refeeding. Levels of preprogalanin (prepro-GAL) mRNA throughout the hypothalamus were essentially unaffected by both regimes. These results demonstrate that hypothalamic NPY gene expression is regulated by peripheral metabolic status (and osmolality), and confirm the key physiological role of NPY in controlling ingestive behaviour.     

Environmental light conditions alter gene expression of rat catecholamine biosynthetic enzymes and Neuropeptide Y: differential effect in superior cervical ganglia and adrenal gland

The hypothalamic suprachiasmatic nuclei (SCN) comprise the main site in the brain involved in the control of the homeostatic mechanism which respond to environmental daily light changes. The sympathetic nervous system and hypothalamic releasing or inhibiting factors mediate the SCN control of a number of peripheral organs and tissues. In this work we analyzed the involvement of two environmental light conditions, constant light (LL) and constant dark (DD) for 20 days, on the expression of mRNAs for catecholamines biosynthetic enzymes and neuropeptide Y (NPY) genes in rat superior cervical ganglia (SCG) and adrenal gland. The results of Northern blot analysis show that LL exposure reduces mRNA levels for tyrosine hydroxylase (TH) the rate limiting catecholamine biosynthetic enzyme and also of dopamine beta-hydroxylase (DBH) as well as for NPY in SCG to about half the levels in control animals. In contrast, exposure of the rats to DD did not elicit any change in the SCG. In the adrenal gland, both, LL and DD conditions increased the TH, DBH as well as phenylethanolamine N-methyltransferase (PNMT) mRNA levels. Under the same conditions, adrenal NPY mRNA levels were decreased by either LL or DD. The results show, for the first time, that prolonged changes in environmental light can alter the gene expression of catecholamine biosynthetic enzymes and of NPY. There was differential response in SCG and adrenal gland.     

Localization of neuropeptide Y mRNA and peptide in the chicken hypothalamus and their alterations after food deprivation, dehydration, and castration.

Localization of neuropeptide Y (NPY) mRNA in the hypothalamus of chickens was studied by in situ hybridization with digoxigenin-labeled chicken NPY cRNA probe. The largest number of perikarya-expressing NPY mRNA was found within the mediobasal hypothalamus, including the infundibular nucleus, inferior hypothalamic nucleus, and median eminence. Many NPY perikarya were noted to surround the nucleus rotundus and to be present in the supraoptic nucleus. Moreover, some perikarya were detected in the nucleus of basal optic root, bed nucleus pallial commissure, and nucleus striae terminalis close to the lateral forebrain bundle. NPY-immunoreactive nerve fibers were densely distributed in these regions containing the NPY mRNA-expressing perikarya. Following food deprivation for four days, perikarya-expressing NPY mRNA and peptide were markedly increased in the mediobasal hypothalamus and particularly so in the infundibular nucleus. No changes, however, were detected in other regions containing NPY-positive perikarya. Water deprivation induced less increase in NPY-positive perikarya in the mediobasal hypothalamus compared to food deprivation. After gonadectomy, the number of NPY-positive perikarya in the mediobasal hypothalamus was unaltered. Northern blot analysis with (32)P-labeled chicken NPY cDNA probe demonstrated that a 2.7-fold increase of NPY mRNA was induced by starvation and a 1.5-fold increase was induced by dehydration, whereas the NPY mRNA band remained unchanged after gonadectomy. Thus, it seems that NPY neurons located in the mediobasal hypothalamus are involved in feeding behavior but not reproductive activity.     

Neuropeptide Y mRNA and serotonin innervation in the arcuate nucleus of anorexia mutant mice

The anorexia (anx) mutation causes reduced food intake in preweanling mice, resulting in death from starvation within 3–4 weeks. In wild-type rodents, starvation induces increased neuropeptide Y (NPY) mRNA levels in the arcuate nucleus that promotes compensatory hyperphagia. Despite severely decreased body weight and food intake at 3-weeks age, anx/anx mice do not show elevated NPY mRNA levels in the hypothalamic arcuate nucleus compared to wild-type/heterozygous littermates. The NPY mRNA levels can be upregulated in normal mice at this chronological age, because 24-h food deprivation increased arcuate NPY mRNA in wild-type littermates. The unresponsiveness of NPY expression in the arcuate of anx/anx mice was paralleled by serotonergic hyperinnervation of the arcuate nucleus, comparable to the serotonergic hyperinnervation previously reported in the rest of the anx/anx brain. This result is consistent with the hypothesis that wasting disorders are accompanied by disregulation of NPY mRNA expression in the arcuate nucleus, and suggests that reduced food intake, the primary behavioral phenotype of the anx/anx mouse, may be the result of altered hypothalamic mechanisms that normally regulate feeding.     

Regulation of Activin Type-II Receptor mRNA Levels in Rat Hypothalamus by Estradiol in vivo

A solution-hybridization S1-nuclease protection assay was used to evaluate the expression of messenger RNAs for the activin β _A subunit and type II activin receptor in adult rat brain. Results indicate the presence of β _A subunit mRNA in both hypothalamus and brainstem, with approximately two-fold higher levels in brainstem. Levels of activin type II receptor mRNA were similar in the hypothalamus of young virgin and 15-day lactating females, and in females in which pups were removed after a 5-day lactation period. Male rats castrated prepubertally (30  days p.n.) had approximately 220% higher ( P <0.05) hypothalamic activin type II receptor mRNA levels than postpubertal, 3-month old age-matched sham controls. Two month treatment of castrate rats with estradiol (200  ng/g, i.p. every 2 days) reduced hypothalamic activin type II receptor mRNA expression to control levels; the same dose of testosterone had no effect. The expression of the hypothalamic activin type II receptor gene may be estrogen-regulated in vivo .     

Neuropeptide Y and lipid increase apolipoprotein AIV gene expression in rat hypothalamus

Apolipoprotein AIV (apo AIV) is a circulating signal released from intestinal cells in response to lipid feeding and contributes to the anorectic effect of a lipid meal. We have demonstrated that apo AIV is also synthesized in the hypothalamus, and that hypothalamic apo AIV gene expression is regulated physiologically. Neuropeptide Y (NPY) is a hypothalamic neuropeptide with broad regulatory actions in the central nervous system. In the present studies, the effects of intracerebroventricular (i.v.t.) administration of NPY and of intraduodenal lipid infusion on hypothalamic apo AIV gene expression were determined using competitive RT-PCR in fasted rats. I.v.t. injection of NPY alone significantly increased apo AIV mRNA levels in the hypothalamus in a dose-dependent manner. Intraduodenal infusion of lipid also stimulated the gene expression of hypothalamic apo AIV, but no further significant increment occurred when i.v.t. injection of NPY was combined with lipid infusion. These results suggest that NPY and lipid may regulate apo AIV gene expression in the rat hypothalamus.     

Changes in the responsiveness of serum leptin and hypothalamic neuropeptide Y mRNA levels to food deprivation in developing rats

Neuropeptide Y (NPY) is an important orexigenic peptide that acts in the brain. The increase in hypothalamic NPY mRNA expression induced by fasting is mainly caused by a decrease in the effects of leptin. We investigated the developmental changes in the sensitivities of leptin and hypothalamic neuropeptide Y to fasting. Hypothalamic NPY mRNA levels were increased by fasting in postnatal days 15 and 25 rats, but not in postnatal day 5 rats. Serum leptin levels were decreased by fasting in rats at all ages (days 5, 15, and 25). In addition, hypothalamic OB-Rb mRNA levels were decreased by fasting in postnatal day 25 rats, but not in postnatal day 5 or 15 rats. Although the percentage of fating-induced decrease in the serum leptin level was larger in the postnatal day 15 rats than in the postnatal day 25 rats, the percentage of increase in the hypothalamic NPY mRNA level in the postnatal day 15 rats was smaller than that in the postnatal day 25 rats. There was a strong inverse correlation between serum leptin levels and hypothalamic NPY mRNA levels in the postnatal day 25 rats, whereas no significant correlation was found between these parameters in the postnatal day 5 or 15 rats. These findings indicate that the sensitivity of hypothalamic NPY mRNA expression to food deprivation and hypoleptinemia has developed by postnatal day 25.     

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.     

Hypothalamic neuropeptide Y gene expression in rats on scheduled feeding regimen.

Recent evidence suggests that neuropeptide Y (NPY) is an important signal in the neural circuitry that controls feeding behavior. Previously we observed that in rats entrained to 4 h daily scheduled feeding regimen (SFR), NPY content and release in the paraventricular nucleus (PVN) was elevated but decreased rapidly in association with food consumption. In the present study, we investigated the pattern of hypothalamic NPY gene expression in SFR rats before and after food consumption by measuring the content of preproNPY mRNA in the medial basal hypothalamus (MBH). Adult male rats were maintained on either ad libitum diet (control) or on SFR. Rats were killed before food presentation at 11.00 h and at the end of 4 h food consumption at 15.00 h. The levels of preproNPY mRNA in the MBH were determined by solution hybridization/RNase protection assay using a cRNA probe complementary to rat NPY precursor mRNA. We observed that, as compared to that in control rats on ad libitum diet, preproNPY mRNA levels in the MBH were increased two-fold in the SFR rat at 11.00 h and remained elevated even after 4 h of food consumption. These results show a simultaneous enhancement in PVN NPY release and hypothalamic gene expression in advance of scheduled feeding time, but food intake rapidly decreases PVN NPY release and content, with little impact on hypothalamic gene expression.     

Characterization of neurochemical phenotypes in cultured hypothalamic neurons with immunohistochemistry and in situ hybridization

The expression of neurochemical phenotypes was studied in long-term cultures of dissociated embryonic neurons from rat hypothalamus. With time in culture, these neurons establish a complex dendritic and axonal network, as indicated by staining with antibodies against microtubulin-associated protein (MAP2) and neurofilaments (SM132 and SM133) as well as GABA and glutamate decarboxylase mRNA immunoreactivity. Neurons expressing neuropeptide Y (NPY) mRNA and NPY peptide and opioid-like peptides as well as vasopressin were observed. Further, weakly acetylcholinesterase- and NADPH diaphorase (nitric-oxide synthase)-labelled neurons were present. In conclusion, the neurochemical phenotypes reported for hypothalamic neurons in vivo can be observed in these cultures. This indicates that the culture conditions allow morphological and molecular differentiation. These findings support the view that long-term hypothalamic cultures provide a valuable model for studying mechanisms of neurosecretion in hypothalamic networks.     

Location and electrophysiological characterization of rostral medullary adrenergic neurons that contain neuropeptide Y mRNA in rat medulla

The objective of this study was to characterize the projection pattern and electrophysiological properties of the rostral medullary adrenergic neurons (C(1)) that express neuropeptide Y (NPY) mRNA in rat. NPY mRNA was found in a variable fraction of tyrosine hydroxylase immunoreactive (TH-IR) neurons depending on the medullary level. By retrograde labeling (Fast Blue, FluoroGold), NPY mRNA was detected in virtually all C(1) cells (96%) and C(3) cells (100%) with hypothalamic projections but in only 9% of C(1) cells and 58% of C(3) cells projecting to thoracic segment 3 (T(3)) or T(6) of the spinal cord. To identify the electrophysiological properties of the C(1) cells that express NPY mRNA, we recorded from baroinhibited neurons within the C(1) region of the ventrolateral medulla (RVLM) and tested for projections to segment T(3), the hypothalamus, or both. By using the juxtacellular method, we labeled these cells with biotinamide and determined whether the recorded neurons were TH-IR and contained NPY mRNA. At rostral levels (Bregma -11.8 mm), barosensitive neurons had a wide range of conduction velocities (0.4-6.0 m/second) and discharge rates (2-28 spikes/second). Most projected to T(3) only (27 of 31 cells), and 4 projected to both the hypothalamus and the spinal cord. Most of the baroinhibited cells with spinal projections but with no hypothalamic projections had TH-IR but no NPY mRNA (11 of 17 cells). Only 1 cell had both (1 of 17 cells), and 5 cells had neither (5 of 17 cells). Both TH-IR and NPY mRNA were found in neurons with dual projections (2 of 2 cells). At level Bregma -12.5 mm, baroinhibited neurons had projections to the hypothalamus only (13 of 13 cells) and had unmyelinated axons and a low discharge rate. Four of five neurons contained both TH-IR and NPY mRNA, and 1 neuron contained neither. In short, NPY is expressed mostly by C(1) cells with projection to the hypothalamus. NPY-positive C(1) neurons are barosensitive, have unmyelinated axons, and have a very low rate of discharge. Most bulbospinal C(1) cells with a putative sympathoexcitatory role do not make NPY.     

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.     

Distribution of prodynorphin mRNA and its interaction with the NPY system in the mouse brain

Using radioactive in situ hybridisation, the distribution of prodynorphin mRNA in the brains of C57Bl/6 mice was systemically investigated, and double-labelling in situ hybridisation was used to determine the extent to which neuropeptide Y (NPY) and prodynorphin mRNAs were co-expressed. Our results demonstrate that prodynorphin mRNA expression in the mouse brain is localised at specific subregions of the olfactory bulb, cortex, hippocampus, amygdala, basal ganglia, thalamus, hypothalamus, mesencephalon and myelencephalon. Among the regions displaying the most intense labelling were the olfactory tubercle, lateral septum (LS), caudate putamen (Cpu), central amygdaloid nucleus (Ce), paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), lateral reticular nucleus (LRt) and solitary tract nucleus (NTS). In the arcuate nucleus of the hypothalamus (Arc), double-labelling in situ hybridisation revealed that prodynorphin expressing neurons also contained NPY mRNA, with a co-localisation rate of approximately 88% in the lateral part of the Arc, and 79% in the dorsal part of the Arc, respectively, suggesting potential overlapping functions of these two neurotransmitters in feeding type behaviour.     

Fasting-induced increases of arcuate NPY mRNA and plasma corticosterone are blunted in the rat experienced neonatal maternal separation

This study was conducted to examine the effects of neonatal maternal separation on the hypothalamic expression of feeding peptides in later life. Pups in maternal separation (MS) groups were separated from their dam for 3 h daily from postnatal day (PND) 1-14, while pups in non-handled (NH) groups were left undisturbed. Rats were sacrificed on PND 60 to examine the gene expression of neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) in the hypothalamic arcuate nucleus by mRNA in situ hybridization. Half of the rats from each group were food-deprived for 48 h before sacrifice. POMC mRNA expression increased in the free fed MS group compared with the free fed NH group. Food deprivation significantly decreased the arcuate POMC mRNA level in both groups. Body weight gain, basal levels of plasma corticosterone, leptin, and arcuate NPY mRNA were not modulated by experience of neonatal maternal separation. However, fasting-induced increases of plasma corticosterone and arcuate NPY expression were blunted in MS rats. These results suggest that neonatal maternal separation may increase the basal expression level of arcuate POMC mRNA, while inhibit the fasting-induced expression of arcuate NPY mRNA, later in life. Lastly, the altered expression of arcuate NPY mRNA, but not of arcuate POMC mRNA, appeared to be related with altered activity of the hypothalamic-pituitary-adrenal gland axis in offspring by neonatal maternal separation.     

Distribution of NPY receptors in the hypothalamus

Neuropeptide Y (NPY) neurons abundantly innervate the hypothalamus, where NPY is involved in the regulation and integration of a broad range of homeostatic functions. In order to understand NPY-mediated behavioral, autonomic and neuroendocrine effects, it is important to characterize in detail the distribution of the hypothalamic NPY receptors. In this review, we briefly summarize the origin of NPY and its two related peptides, peptide YY and pancreatic polypeptide in the hypothalamus. Moreover, based on the results obtained with histological techniques such as in situ hybridization, immunohistochemistry and ligand binding, we summarize data on the hypothalamic distribution of the known NPY receptors, the Y1 Y2, Y4 and Y5 receptors as best characterized to date. These NPY receptors are found with individual distribution patterns in many hypothalamic neurons including neuroendocrine motoneurons, magnocellular neurosecretory neurons and numerous neurons connecting the hypothalamus with the limbic and the autonomic nervous systems. The histochemical analyses allow characterization of coexisting molecules and in this way definition of the neurochemistry of NPY circuitries. By showing coexistence of various NPY receptors they provide a morphological basis for in vitro studies showing heterodimerization of NPY receptors. The NPY neurons and their circuitries underlie the integrative role of NPY as a pleiotropic neuropeptide in the regulation of homeostasis.