Shuye Pu

Interactions between Neuropeptide Y and γ -Aminobutyric Acid in Stimulation of Feeding: A Morphological and Pharmacological Analysis1

Neuropeptide Y (NPY) produced in neurons in the arcuate nucleus and brain stem and released in the paraventricular nucleus (PVN) and surrounding areas is involved in stimulation of feeding in rats. We recently reported that γ-aminobutyric acid (GABA) is coexpressed in a subpopulation of NPY neurons in the arcuate nucleus. To determine whether GABA is colocalized in NPY terminals in the PVN, the site of NPY action, light and electron microscopic double staining for NPY and GABA using pre- and postembedding immunolabeling was performed on rat brain sections. GABA was detected in NPY-immunopositive axons and axon terminals within both the parvocellular and magnocellular divisions of the PVN. These morphological findings suggested a NPY-GABA interaction in the hypothalamic control of feeding. Therefore, the effects of muscimol (MUS), a GABAA receptor agonist, on NPY-induced food intake were examined in sated rats. When injected intracerebroventricularly, both NPY and MUS elicited dose-dependent feeding respon...

The Interactive Language of the Hypothalamus for the Gonadotropin Releasing Hormone (GNRH) System

The enormous diversity in neurochemical signals employed within the network of afferents to GnRH neurons is well‐documented. An examination of novel and accumulating knowledge on the operation of these messengers indicates the presence of an interactive language governing GnRH secretion. The basic operational structures identified to date to affirm this interactive form of communication summarized in this review are the following: (i) the demonstration of interconnections within various components of the afferent network; (ii) coexistence and possible co‐release of excitatory and inhibitory neurotransmitters/ neuromodulators; (iii) co‐action of various messengers at synaptic targets, and (4) modulation by gonadal steroids of the synthesis and release of signals and their receptors, and induction of synaptic plasticity for the timely relay of signals for GnRH secretion. Unraveling the molecular sequelae that promote this interactive communication to elicit periodic GnRH secretion is now a new challenge.

Evidence that stimulation of two modalities of pituitary luteinizing hormone release in ovarian steroid-primed ovariectomized rats may involve neuropeptide Y Y1 and Y4 receptors.

A large body of evidence indicates that neuropeptide Y (NPY) is involved in stimulation of basal and cyclic release of hypothalamic LHRH and pituitary LH. To identify the NPY receptor subtypes that mediate the excitatory effects of NPY in these two modalities of LH release, we studied the effects of 1229U91, a selective Y1 receptor antagonist and Y4 receptor agonist, in two experimental paradigms that reproduce the two modalities of LH secretion in steroid-primed ovariectomized (OVX) rats. Rats were ovariectomized and implanted with a permanent cannula into the lateral cerebroventricle. In the first experiment, rats received estradiol benzoate (EB, 30 μg/rat) on day 5, followed 2 days later with progesterone (2 mg/rat) at 1000 h to induce an afternoon LH surge. 1229U91 (30 μg/3 μl) or vehicle (control) was injected intracerebroventricularly into these rats either once at 1300 h or twice (15 μg/injection) at 1100 and 1200 h. Blood samples were collected before progesterone injection at 1000 h and at hourly...

Neural substrates for leptin and neuropeptide Y (NPY) interaction: hypothalamic sites associated with inhibition of NPY-induced food intake

Intracerebroventricular (i.c.v.) injection of leptin, the adipocyte hormone, inhibits neuropeptide Y (NPY)-induced feeding in the rat. To identify the neural substrate for leptin and NPY interaction in the hypothalamus, we evaluated the expression of c-fos-like immunoreactivity (FLI), an early marker of neuronal activation, in response to icv administration of leptin, NPY and leptin plus NPY. As expected, leptin significantly decreased NPY-induced feeding in leptin plus NPY-treated rats. A comparative evaluation of the number of FLI-positive neurons in hypothalamic sites showed that both leptin and NPY activated FLI in the parvocellular subdivision of the paraventricular nucleus (pPVN), dorsomedial nucleus (DMN) and ventromedial nucleus (VMN). NPY also augmented the FLI response in the magnocellular PVN (mPVN) and supraoptic nucleus (SON), two sites where leptin alone was ineffective. Combined leptin and NPY treatment significantly decreased the number of FLI-positive neurons in the magnocellular PVN but increased their number in the dorsomedial nucleus as compared to the number of FLI-expressing neurons in these sites after NPY and leptin alone. Because there is morphologic evidence of a link between magnocellular PVN and dorsomedial nucleus, these results suggest the functional involvement of leptin plus NPY responsive elements in these sites in reduction of NPY-induced feeding by leptin.

Neuropeptide Y Counteracts the Anorectic and Weight Reducing Effects of Ciliary Neurotropic Factor

Ciliary neurotrophic factor (CNTF), a cytokine of the interleukin‐6 superfamily, has been shown to induce hypophagia and weight loss. Neuropeptide Y (NPY) and orexin are potent orexigenic signals in the hypothalamus. Anorexia, normally seen in response to infection, injury and inflammation, may result from diminished hypothalamic orexigenic signalling caused by persistently elevated cytokines, including CNTF. To test this hypothesis, we first examined the effects of chronic intracerebroventricular (i.c.v.) infusion of CNTF for 6–7 days on food intake and body weight as well as hypothalamic NPY and orexin gene expression in male rats. Subsequently, the effectiveness of NPY replacement to counteract the effects of CNTF by coinfusion of NPY and CNTF was evaluated. Chronic i.c.v. infusion of CNTF (2.5 µg/day) reduced body weight (14.3% vs control) at the end of 7 days. Food intake remained suppressed for 5 days postinfusion and subsequently gradually returned to the control range by day 7. Serum leptin concentrations in these rats were in the same range seen in control rats. Chronic i.c.v. infusion of higher doses of CNTF (5.0 µg/day) produced sustained anorexia and body weight loss (29% vs controls) through the entire duration of the experiment. This severe anorexia was accompanied by markedly suppressed serum leptin concentrations. Furthermore, CNTF infusion alone significantly reduced hypothalamic NPY gene expression (P < 0.05) without affecting orexin gene expression. As expected, in fusion of NPY alone (18 µg/day) augmented food intake (191.6% over the initial control, P < 0.05) and produced a 25.1% weight gain in conjunction with a 10‐fold increase in serum leptin concentrations at the end of the 7‐day period. Interestingly, coinfusion of this regimen of NPY with the highly effective anorectic and body reducing effects of CNTF (5.0 µg/day) not only prevented the CNTF‐induced anorexia and weight loss, but also normalized serum leptin concentrations and hypothalamic NPY gene expression. These results demonstrate that chronic central infusion to produce a persistent elevation of the cytokine at pathophysiological levels (a situation that may normally manifest during infection, injury and inflammation) produced severe anorexia and weight loss in conjunction with reduction in both serum leptin concentrations and hypothalamic NPY gene expression. Reinstatement of hypothalamic NPY signalling by coinfusion of NPY counteracted these CNTF‐induced responses.

Evidence Showing That β-Endorphin Regulates Cyclic Guanosine 3′,5′-Monophosphate (cGMP) Efflux: Anatomical and Functional Support for an Interaction between Opiates and Nitric Oxide1

Nitric oxide (NO) is now recognized as a diffusible messenger molecule that normally augments intercellular communication in the central nervous system, but is neurotoxic if released in excessive amounts. NO is synthesized from l-arginine by the Ca2+/calmodulin-dependent neuronal isoform NO synthase (NOS) localized in subpopulations of neurons throughout the brain, including the hypothalamus. In the hypothalamus, NO stimulates the release of GnRH, the primary neurohormone governing reproduction in mammals. Although the excitatory amino acid, glutamate, acting through the N-methyl-d-aspartate (NMDA) receptor is believed to be responsible for stimulation of NO release, the neuronal system(s) that inhibits NO efflux is unknown. As the endogenous opioids, primarily β-endorphin (βEND), exert a tonic restraint on GnRH secretion, we sought evidence for a possible functional link between βEND and NOS pathways in the hypothalamus. We observed that restraining the opioid influence with the opiate receptor antagonis...

Ovarian Steroid-Independent Diurnal Rhythm in Cyclic GMP/Nitric Oxide Efflux in the Medial Preoptic Area: Possible Role in Preovulatory and Ovarian Steroid-Induced LH Surge*

The aim of this study was to evaluate the relationship between cyclic LH hypersecretion and nitric oxide (NO) release in the medial preoptic area (MPOA), the hypothalamic site implicated in induction of LH hypersecretion. The MPOA extracellular cyclic GMP (cGMP) efflux (an index of NO release), was monitored by microdialysis. Quite unexpectedly, we observed a daily afternoon rise in the MPOA cGMP efflux in cycling female rats on proestrus and diestrus II, in ovariectomized (ovx) rats and in ovx rats treated with ovarian steroids to induce the LH surge. The daily rise in cGMP efflux occurred earlier in diestrous and in estradiol benzoate (EB)‐treated ovx rats than in ovx rats. Progesterone (P) injection to estrogen‐primed ovx rats further advanced the onset of the rise close to the earliest time of rise as seen on proestrus. The afternoon increase in the cGMP efflux in proestrous rats was abolished by pentobarbital treatment that blocked the LH surge. Intracerebroventricular (icv) injection of 1H‐[1,2,4]oxadiazo[4,3‐a]quinoxalin‐one (ODQ), a selective inhibitor of soluble guanylyl cyclase, suppressed the P‐induced LH surge in EB‐primed ovx rats, but not basal LH secretion in unprimed ovx rats. Analysis of brain NOS (bNOS) levels in the POA by Western blotting showed that the morning bNOS levels were higher in the POA of EB‐treated rats than in unprimed ovx rats. Further, with the exception of ovx rats treated with sequential EB and P treatment, the POA bNOS levels rose significantly in the afternoon in unprimed ovx and EB‐treated ovx rats. Collectively, these findings reveal a diurnal rhythm in the MPOA cGMP/NO efflux that is ovarian steroid‐independent. Ovarian steroids apparently shift the timing of the afternoon rise in cGMP/NO efflux to synchronize with the activation of steroid‐dependent neuronal systems responsible for the LH surge.

Diurnal rhythm in cyclic GMP/nitric oxide efflux in the medial preoptic area of male rats

Since nitric oxide (NO) is implicated in the neuroendocrine control of luteinizing hormone-releasing hormone (LHRH) secretion and sexual behavior which show diurnal variations, we monitored cGMP levels (an index of NO activity) in the extracellular compartment of the medial preoptic area (MPOA) using microdialysis. It was observed that MPOA cGMP levels rose significantly in the afternoon in both castrated and intact male rats, thereby suggesting the existence of a diurnal rhythm in MPOA cGMP/NO efflux which may participate in eliciting the well-known diurnal variations in LHRH neuronal activity and male sexual behavior.

Melanocortin signaling is decreased during neurotoxin-induced transient hyperphagia and increased body-weight gain

Hypothalamic neuropeptides play critical roles in the regulation of feeding behavior and body weight (BW). Disruption of signaling in the ventromedial nucleus by microinjection of the neurotoxin, colchicine (COL), produces transient hyperphagia with corresponding BW gain lasting for 4 days. Because the melanocortin system exerts an inhibitory control on food intake, we hypothesized that hyperphagia in COL-treated rats is due to decreased melanocortin-induced restraint on feeding. Melanocortin restraint is exerted through alpha-melanocortin-stimulating hormone derived from proopiomelanocortin (POMC) and is antagonized by agouti-related peptide produced in neurons located in the arcuate nucleus (ARC). COL (4 microg/0.5 microl saline) or saline was microinjected bilaterally into the ventromedial nucleus of adult male rats. In conjunction with BW gain, blood leptin levels were elevated, whereas POMC mRNA in the ARC was significantly decreased in COL-injected rats. Levels of alpha-melanocortin-stimulating hormone were also decreased in the micropunched paraventricular nucleus, dorsomedial nucleus, and perifornical hypothalamus, sites implicated in the control of food intake. That diminution in melanocortin signaling underlies hyperphagia was supported by the observation that intracerebroventricular injection of the MC3/MC4 melanocortin receptor agonist, MTII, prevented the hyperphagia and BW gain. Surprisingly, however, mRNA levels of the orexigenic peptide agouti-related peptide in the ARC were decreased perhaps due to the action of elevated leptin. These results show that transient hyperphagia and BW gain induced by disruption of signaling in the ventromedial nucleus results from two neurochemical rearrangements: development of leptin resistance in POMC neurons and diminution in melanocortin signaling as reflected by decreased POMC gene expression in the ARC and decreased availability of alpha-melanocortin-stimulating hormone for release in feeding relevant sites.

Disruption of neural signaling within the hypothalamic ventromedial nucleus upregulates galanin gene expression in association with hyperphagia: an in situ hybridization analysis

Hypothalamic neuropeptides play critical roles in the regulation of appetite and body weight. We recently reported that disruption of neural signaling in the ventromedial nucleus (VMN) by microinjection of the neurotoxin, colchicine (COL), produced transient hyperphagia with attendant body weight gain lasting for 4 days. The neural mechanism(s) underlying this temporary shift in energy homeostasis is still unknown. Galanin (GAL) is produced in several hypothalamic nuclei and since microinjection of GAL into these sites stimulates feeding, we tested the hypothesis that galaninergic signaling is upregulated in COL-treated rats. COL (4 microgram in 0.5 microliter saline) or saline alone was microinjected into the VMN of adult male rats and GAL mRNA was evaluated in the basal hypothalamus by ribonuclease protection assay on day 1, day 2 and day 4 after injection. Whereas in saline-treated rats body weight and GAL mRNA remained unaffected, they were significantly increased in COL-injected rats through the period of observation. To identify the specific neuronal subpopulations involved, GAL mRNA levels were analyzed in feeding-related hypothalamic nuclei using semiquantitative in situ hybridization histochemistry on day 4 after microinjection of COL or saline into the VMN. In COL-treated rats, GAL mRNA levels increased dramatically over controls in the supraoptic nucleus, paraventricular nucleus (PVN), dorsomedial nucleus (DMN), arcuate nucleus (ARC) and lateral hypothalamic area (LHA); no significant change was observed in the central nucleus of amygdala. These results indicated that disruption of neurotransmission in the VMN upregulated GAL gene expression in those hypothalamic sites (PVN, DMN, LHA and ARC) that are implicated in regulation of feeding, and since GAL stimulates feeding, this neurochemical rearrangement may contribute to the over-eating in these animals. These results also suggest that, normally, neurons in the VMN may suppress GAL neurotransmission in feeding-regulating hypothalamic neural circuits.