Induction of the galanin-like peptide gene expression in the posterior pituitary gland after acute osmotic stimulus in rats

Galanin-like peptide (GALP) is a 60 amino-acid peptide, and the GALP mRNA is restricted to pituicytes in the posterior pituitary gland (PP) and neurons in the hypothalamic arcuate nucleus (Arc). We examined whether the GALP gene expression in the PP and Arc would be induced after intraperitoneal (i.p.) administration of hypertonic saline, that is, acute osmotic stimulus, in rats. The dose-response (2.8, 4.5, 6.0 and 9.0% NaCl) and time-course (6.0% NaCl, 1, 3, 6, 12 and 24h) effects of acute osmotic stimulus on GALP mRNA levels in the PP and Arc were examined in rats by using in situ hybridization histochemistry. Plasma osmolality and plasma sodium concentration increased significantly at 1h, and returned to control level at 6h after i.p. administration of hypertonic saline (6.0% NaCl). The GALP mRNA level in the PP increased significantly 3 and 6h after i.p. administration of hypertonic saline (6.0% NaCl), but the level in the Arc did not change. These results showed that acute osmotic stimulus-induced GALP gene expression in the pituicyte of the PP, but not in the neurons in the Arc, and the gene expression in the pituicyte might be regulated by plasma osmolality and/or plasma sodium concentration.     

Immunocytochemical localization of a galanin-like peptide (GALP) in pituicytes of the rat posterior pituitary gland.

A galanin-like peptide (GALP) was recently isolated as a ligand of GalR2, a galanin receptor subtype. The GALP mRNA is expressed in the arcuate nucleus of the hypothalamus and the posterior pituitary (PP). In this study, we demonstrated the localization of GALP-immunoreactive (-ir) cells in the rat PP. In normal conditions, a few GALP-ir cells were detected in the PP, and these cells increased on dehydration for 4 days. The GALP-immunopositive reaction was dramatically enhanced by the intraperitoneal injection of colchicine. For the identification of GALP-ir cells in the PP, we performed electron microscopic observation, and also double immunocytochemical staining for GALP and S-100 protein. Both studies clearly indicated that the GALP-ir cells in the PP are pituicytes.     

On-line feedback control of human visually guided slow ramp tracking: effects of spatial separation of visual cues

Galanin-like peptide (GALP) is a newly discovered 60 amino acid peptide from the porcine hypothalamus. GALP has been shown to be expressed predominantly in the arcuate nucleus (ARC) of the rat hypothalamus, a region considered to be one of the most important feeding-regulating centers in the brain. GALP-containing neurons in the ARC express leptin receptors, but relationships between GALP and other feeding-regulating neurons have not yet been fully elucidated. Given that Orexin (OX)-containing neurons make synaptic inputs to the ARC, we thus examined the relationship between GALP and OX in the ARC by use of a dual immunostaining technique. OX-immunoreactive fibers appeared to be closely apposed to GALP-immunoreactive cell bodies and their processes. We also examined whether the OX receptor, OX₁-R was expressed in the GALP-containing neurons. Immunoreactivity for both OX₁-R and GALP was detectable in 9.6 % neurons (range 4.2–14.6%) in the ARC. These findings strongly suggest that GALP may participate in the regulation of feeding behavior under the influence of leptin and OX.     

Galanin and galanin-like peptide differentially modulate neuronal activities in rat arcuate nucleus neurons

Neuropeptides galanin and galanin-like peptide (GALP) share similar amino acid sequence and presumably interact with the same group of receptors, but they differentially regulate a variety of physiological and pathophysiological processes including metabolism and reproduction. Here we explored the neurophysiological basis of the in vivo differential effect between galanin and GALP by examining galanin and GALP modulation of neuronal activities of neurons in the arcuate nucleus (Arc), a brain region critically involved in energy homeostasis and reproductive function. We demonstrated that galanin and GALP inhibited excitatory and inhibitory postsynaptic currents in a similar way. In contrast, galanin and GALP differentially affected the intrinsic membrane property. In most recorded Arc neurons, galanin perfusion induced significant hyperpolarization of the resting membrane potential, which was not affected by GALP perfusion. In addition, galanin perfusion substantially suppressed the spontaneous spike firing in most Arc neurons, whereas in response to GALP perfusion, about half of the Arc neurons exhibited mild reduction in spontaneous spike firing and the other half showed enhancement. Furthermore, the Arc neurons that had been previously responsive to galanin perfusion no longer responded to galanin if co-applied with GALP, indicating that GALP can physiologically antagonize galanin effect. This differential effect appears to be mediated by G protein within the recorded cell, as the galanin effect on firing rate was abolished when the recorded cell was loaded with GDP-betaS, an agent that blocks G protein activity. Taken together, these differential effects of galanin and GALP may provide a neurophysiological mechanism through which galanin and GALP differentially regulate energy balance, reproductive function, and other physiological processes.     

Effects of stress and adrenalectomy on activity-regulated cytoskeleton protein (Arc) gene expression

Activity-regulated cytoskeletal-associated protein (Arc) is an effector immediate early gene induced by novelty and involved in consolidation of long-term memory. Since activation of glucocorticoid receptors is a prerequisite for memory consolidation, we therefore aimed to study the effect of acute restraint stress on Arc gene expression in adrenalectomized rats. Acute stress produced a significant increase in Arc gene expression in the medial prefrontal cortex, but not in the parietal cortex or in the pyramidal cell layer of the hippocampus. The basal level of Arc mRNA in adrenalectomized animals was high in the medial prefrontal cortex and unaffected by acute stress in these animals. These data are consistent with the role of Arc as an integrative modulator of synaptic plasticity by emphasizing the potential role of stress and glucocorticoids in the control of Arc gene expression.     

Galanin-like peptide stimulates feeding and sexual behavior via dopaminergic fibers within the medial preoptic area of adult male rats

Galanin-like peptide (GALP) is located in the arcuate nucleus (Arc) of the hypothalamus and is known to regulate both food intake and sexual behaviors in adult male rats. We have previously demonstrated that ICV GALP administration elicits a significant fos response within the medial preoptic area (mPOA). GALP is known to stimulate both food intake and male-typical sex behavior, presumably by direct actions within the mPOA. Recent data from our and other labs have led us to suspect that GALP effects on sex behaviors are due to activation of incertohypothalamic dopaminergic neurons that terminate within the mPOA. To test the hypothesis that GALP activates mPOA dopaminergic systems, we utilized an immunolesion technique to eliminate dopaminergic fiber input to the mPOA via a dopamine transporter-specific toxin (DATSAP, n = 8) and compared to control injections (SAP, n = 8). All animals were sexually experienced adult male Long-Evans rats. DATSAP-treated male rats showed a significant (p < 0.001) reduction in male sexual behaviors compared to SAP controls. We found that elimination of dopaminergic fibers within the mPOA significantly (p < 0.001) eliminated all aspects of male sexual behavior under normal mating paradigms. Injections of GALP (5.0 nmol) significantly increased (p < 0.01) male sex behavior and food intake in SAP control male rats but GALP did not stimulate the expression of these behaviors in DATSAP-treated rats. The orexigenic and anorexigenic effects of GALP were significantly (p < 0.001) attenuated in DATSAP-treated male rats compared to SAP controls; however, ICV GALP was still able to significantly (p < 0.05) reduce 24 h body weight in both DATSAP and SAP rats. ICV GALP significantly (p < 0.05) stimulated fos within the mPOA of SAP rats but not in DATSAP-treated male rats. These data suggest that GALP activates feeding and sexual behaviors in male rats by stimulating dopaminergic neurons that terminate within the mPOA.     

Chronic food restriction differentially affects NPY mRNA level in neurons of the hypothalamus and in neurons that innervate liver

Neuropeptide Y (NPY) is found in neurons of the brain and in the neurons that innervate abdominal organs including liver. Major biological function of hypothalamic NPY is regulation of appetite and body weight homeostasis. In the periphery, biological function of NPY varies, depending on the organ/tissue. Increased hypothalamic NPY mRNA level in response to chronic caloric restriction is a well documented phenomenon. The effect of food restriction on NPY mRNA level in neurons that innervate liver has not been published so far. To evaluate how chronic food restriction affects liver (and other abdominal organs) NPY mRNA level, we compared NPY mRNA abundance in liver, kidney cortex, perirenal white adipose tissue and in hypothalamus of rats maintained on chronic restricted diet. Data presented in this paper indicate that chronic food restriction: (a) caused the increase of NPY mRNA level in the hypothalamus, (b) caused the decrease of NPY mRNA level in the liver, and (c) was without effect on NPY mRNA level in kidney cortex and perirenal white adipose tissues. Moreover, rats maintained on restricted diet displayed lower serum NPY, leptin and insulin concentrations and higher serum corticosterone concentration. Together, these data suggest that hypothalamus and liver (and other abdominal organs) NPY gene expression is differentially regulated by caloric restriction. It seems that liver NPY gene expression in contrast to the hypothalamus NPY gene expression is not suppressed by leptin.     

Agouti-related peptide-expressing neurons are mandatory for feeding

Multiple hormones controlling energy homeostasis regulate the expression of neuropeptide Y (NPY) and agouti-related peptide (AgRP) in the arcuate nucleus of the hypothalamus. Nevertheless, inactivation of the genes encoding NPY and/or AgRP has no impact on food intake in mice. Here we demonstrate that induced selective ablation of AgRP-expressing neurons in adult mice results in acute reduction of feeding, demonstrating direct evidence for a critical role of these neurons in the regulation of energy homeostasis.     

Sex differences in the effect of prepubertal GALP infusion on growth, metabolism and LH secretion

The hypothalamic neuropeptide, galanin-like peptide (GALP), is known to have an effect on energy expenditure and reproduction in adult male rats, but little work has been done on prepubertal rats. We hypothesized that hypothalamic GALP is involved in physiological changes associated with the onset of puberty. To test this hypothesis, we first determined the postnatal ontogeny of GALP gene expression via in situ hybridization of developing male and female rat pups through adulthood. GALP gene expression was not observed in either male or female rat pups until after postnatal day (PND) 10 and did not reach adult-like levels until after weaning (PND25). To determine if exogenous GALP could induce the onset of puberty, PND25 male and female rats were implanted with lateral ventricular cannulas connected to an osmotic minipump that delivered either GALP or vehicle. GALP infusion significantly (p<0.05) increased body weight, food intake, and metabolic rate in male but not female rats compared to control infusion. After 2 weeks, GALP infusion had no significant effect on the onset of puberty, percent body fat, nor plasma levels of insulin, FSH or gonadal steroids in either sex; however, GALP did significantly (p<0.05) increase plasma levels of LH and leptin in male but not female rats and increased plasma growth hormone (GH) in both sexes. Our observations further demonstrate a sex difference in GALP responsiveness in prepubertal rats. These data suggest that GALP may be involved with the prepubertal increase in circulating leptin, LH, and GH resulting in an increase in metabolic rate and lean growth associated with puberty.     

Preprotachykinin gene expression in the mediobasal hypothalamus of estrogen-treated and ovariectomized control rats

Summary We have determined the localization of preprotachykinin (PPT) mRNA-containing neurons in the mediobasal hypothalamus of the rat. PPT mRNA-containing neurons are present in the ventromedial nucleus (with a concentration in the ventrolateral aspect (VL-VM)), the dorsomedial nucleus, the lateral hypothalamus and the arcuate nucleus. This distribution is consistent with the findings of immunocytochemical studies of substance P-immunoreactive neurons in the hypothalamus. We have also examined whether PPT gene expression is regulated by estrogen in the VL-VM by comparing the levels of PPT mRNA in the VL-VM of ovariectomized rats and ovariectomized, estrogen-replaced rats. Both in situ hybridization and slot blot hybridization analysis revealed no changes in PPT mRNA content in the VL-VM following estrogen treatment. These results suggest that estrogen does not regulate lordosis behavior by affecting PPT gene expression in VL-VM neurons.     

Amygdalar vasopressin mRNA increases in acute cocaine withdrawal: evidence for opioid receptor modulation

In humans, stress is recognized as a major factor contributing to relapse to drug abuse in abstinent individuals; drugs of abuse themselves or withdrawal from such drugs act as stressors. In the animals, evidence suggests that centrally released arginine vasopressin in both amygdala and hypothalamus plays an important role in stress-related anxiogenic behaviors. The stress responsive hypothalamic–pituitary–adrenal axis is under tonic inhibition via endogenous opioids, and cocaine withdrawal stimulates hypothalamic–pituitary–adrenal activity. The present studies were undertaken to determine whether: (1) 14-day (chronic) “binge” pattern cocaine administration (45mg/kg/day) or its withdrawal for 3 h (acute), 1 day (subacute) or 10 days (chronic) alters arginine vasopressin mRNA levels in amygdala or hypothalamus; (2) the opioid receptor antagonist naloxone (1mg/kg) alters arginine vasopressin mRNA or hypothalamic–pituitary–adrenal hormonal responses in acute cocaine withdrawal; and (3) there are associated changes of mu opioid receptor or proopiomelanocortin mRNA levels. In amygdala, arginine vasopressin mRNA levels were unchanged after chronic “binge” cocaine, but were increased during acute cocaine withdrawal. Naloxone completely blocked this increase. Neither chronic cocaine nor its acute withdrawal altered amygdalar mu opioid receptor mRNA levels. The increase in amygdalar arginine vasopressin mRNA levels was still observed after subacute withdrawal, but not after chronic withdrawal. Although hypothalamic–pituitary–adrenal tolerance developed with chronic “binge” cocaine, there were modestly elevated plasma adrenocorticotropin hormone levels during acute withdrawal. While naloxone produced modest adrenocorticotropin hormone elevations in cocaine-naïve rats, naloxone failed to elicit an adrenocorticotropin hormone response in cocaine-withdrawn rats. In hypothalamus, neither chronic cocaine nor acute withdrawal altered arginine vasopressin, proopiomelanocortin or mu opioid receptor mRNA levels. These results show that: (1) opioid receptors mediate increased amygdalar arginine vasopressin gene expression during acute cocaine withdrawal, and (2) cocaine withdrawal renders the hypothalamic–pituitary–adrenal axis insensitive to naloxone. Our findings suggest a potential role for amygdalar arginine vasopressin in the aversive consequences of early cocaine withdrawal.     

中晚期孕期束缚应激对子代下丘脑多巴胺能神经元发育的影响

目的　研究中晚期孕期束缚应激对子代下丘脑多巴胺能神经元发育的影响。 方法　使用怀孕SD大鼠,实验组在孕期第15~21 d进行束缚应激,对照组则不受任何干扰。子代大鼠出生的当天为出生后的第0 天。在子代大鼠出生后的第1天（P1）,第7天（P7）和第30天（P30）,分别测子代大鼠的体重,通过免疫组织化学染色和Western Blotting,观察和比较下丘脑内合成多巴胺的关键酶酪氨酸羟化酶（TH）染色像素密度及TH蛋白水平变化。 结果　与正常对照组相比,中晚期孕期束缚应激子代大鼠在P1体重较低,下丘脑TH染色像素密度和TH蛋白水平上调, 这种异常在P7和P30消失。 结论　孕期应激使子代下丘脑多巴胺神经元早期发育异常, 可能是造成子代神经系统行为学异常的原因之一。     

Signaling pathways that mediate nerve growth factor-induced increase in expression and release of calcitonin gene-related peptide from sensory neurons

Nerve growth factor (NGF) can augment transmitter release in sensory neurons by acutely sensitizing sensory neurons and by increasing the expression of calcitonin gene-related peptide (CGRP) over time. The current study examined the intracellular signaling pathways that mediate these two temporally distinct effects of NGF to augment CGRP release from sensory neurons. Growing sensory neurons in 30 or 100 ng/mL of NGF for 7 days increases CGRP content and this increase augments the amount of CGRP that is released by high extracellular potassium. Overexpressing a dominant negative Ras, Ras(17N) or treatment with a farnesyltransferase inhibitor attenuates the NGF-induced increase in CGRP content. Conversely, overexpressing a constitutively active Ras augments the NGF-induced increase in content of CGRP. Inhibiting mitogen activated protein kinase (MEK) activity also blocks the ability of NGF to increase CGRP expression. In contrast to the ability of chronic NGF to increase peptide content, acute exposure of sensory neurons to 100 ng/mL NGF augments capsaicin-evoked release of CGRP without affecting the content of CGRP. This sensitizing action of NGF is not affected by inhibiting Ras, MEK, or PI3 kinases. In contrast, the NGF-induced increase in capsaicin-evoked release of CGRP is blocked by the protein kinase C (PKC) inhibitor, BIM and the Src family kinases inhibitor, PP2. These data demonstrate that different signaling pathways mediate the alterations in expression of CGRP by chronic NGF and the acute actions of the neurotrophin to augment capsaicin-evoked release of CGRP in the absence of a change in the content of the peptide.     

Galanin-like peptide modulates energy balance by affecting inflammatory mediators?

Galanin-like peptide (GALP) is a neuropeptide that is thought to play a role in the regulation of energy balance. However, the effects of GALP on food intake and body weight appear to be complex. In rats, central administration of GALP initially stimulates food intake, an effect that is followed by a reduction in food intake and body weight, whereas in mice, GALP has an anorectic action only. In rats and mice, GALP also causes a prostaglandin-dependent increase in core body temperature. These anorectic effects of GALP are similar to those observed after central administration of the pro-inflammatory cytokine interleukin-1 (IL-1). This review will discuss the evidence for the dichotomous actions of GALP on energy balance, and the potential mechanisms involved. I also describe a role for IL-1 in mediating the anorectic and febrile actions of GALP, and consider the possibility that GALP may act like an inflammatory mediator.     

Widespread release of peptides in the central nervous system: quantitation of tannic acid-captured exocytoses.

Tannic acid methods have been applied to capture the exocytosis of peptide-containing granules from peptidergic neurons. The captured exocytoses have been quantitated to assess the proportion and amount of peptide released at different parts of the neuronal membrane. Examination of hypothalamic synaptic boutons shows that only about one-half of the peptidergic vesicles is exocytosed into the synaptic cleft and also that exocytosis also occurs from undilated peptidergic axons. Study of the magnocellular neurosecretory system reveals that all parts of their extensive terminal arborization appear to be equally capable to exocytose peptide. Only about one-half of their peptide is released from their nerve endings, which about the capillaries. The remainder is released much deeper in the lobules of secretory tissue where its principal target(s) could be the pituicytes and/or neurosecretory axons. Dendrites of magnocellular neurons are also capable of releasing peptide by exocytosis and dendrites could release sufficient oxytocin and vasopressin to account for the peptide known to be released into the hypothalamus. We conclude that peptidergic neurons release substantial amounts of peptides from all of their processes and that this must be taken into account when considering what functions those peptides might serve.     

Association of interleukin-1 beta polymorphisms with idiopathic Parkinson's disease

We have isolated a novel hypothalamic peptide, Galanin-like peptide (GALP), as a ligand for galanin receptor subtype GalR2. To investigate the physiological role of GALP, we examined the effect of the intracerebroventricular administration of GALP and found that GALP induced food intakes. GALP had ten-fold the orexigenic activity of galanin. We also observed the anxiogenic-like behavior after the administration of 1 nmol GALP. These results suggest that GALP is a novel orexigenic and anxiogenic peptide.