Fumiko Takenoya

Pleiotropic Functions of PACAP in the CNS

Abstract:  Pituitary adenylate cyclase‐activating polypeptide (PACAP) is a pleiotropic neuropeptide that belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. PACAP prevents ischemic delayed neuronal cell death (apoptosis) in the hippocampus. PACAP inhibits the activity of the mitogen‐activated protein kinase (MAPK) family, especially JNK/SAPK and p38, thereby protecting against apoptotic cell death. After the ischemia‐reperfusion, both pyramidal cells and astrocytes increased their expression of the PACAP receptor (PAC1‐R). Reactive astrocytes increased their expression of PAC1‐R, released interleukin‐6 (IL‐6) that is a proinflammatory cytokine with both differentiation and growth‐promoting effects for a variety of target cell types, and thereby protected neurons from apoptosis. These results suggest that PACAP itself and PACAP‐stimulated secretion of IL‐6 synergistically inhibit apoptotic cell death in the hippocampus. The PAC1‐R is expressed in the neuroepithelial cells from early developmental stages and in various brain regions during development. We have recently found that PACAP, at physiological concentrations, induces differentiation of mouse neural stem cells into astrocytes. Neural stem cells were prepared from the telencephalon of mouse embryos and cultured with basic fibroblast growth factor. The PAC1‐R immunoreactivity was demonstrated in the neural stem cells. When neural stem cells were exposed to PACAP, about half of these cells showed glial fibrillary acidic protein (GFAP) immunoreactivity. This phenomenon was significantly antagonized by a PAC1‐R antagonist (PACAP6‐38), indicating that PACAP induces differentiation of neural stem cell into astrocytes. Other our physiological studies have demonstrated that PACAP acts on PAC1‐R in mouse neural stem cells and its signal is transmitted to the PAC1‐R‐coupled G protein Gq but not to Gs. These findings strongly suggest that PACAP plays very important roles in neuroprotection in adult brain as well as astrocyte differentiation during development.

Morphological analysis of ghrelin and its receptor distribution in the rat pancreas.

Ghrelin, a novel peptide isolated from stomach tissue of rats and humans, has been identified as the endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to its secretion from the stomach, ghrelin is also expressed in the hypothalamic arcuate nucleus, intestine, kidney, placenta, and pancreas. GHS-R mRNA, on the other hand, is expressed in the hypothalamus, pituitary, heart, lung, liver, pancreas, stomach, intestine, and adipose tissue. Ghrelin is considered to have important roles in feeding regulation and energy metabolism as well as in the release of growth hormone (GH). Recent physiological experiments on the pancreas have shown that ghrelin regulates insulin secretion. However, sites of action of ghrelin in the pancreas are yet to be identified. In this study, to gain insight into the role of ghrelin in rat pancreatic islets, we used immunohistochemistry to determine the localization of ghrelin and GHS-R in islet cells. Double fluorescence immunohistochemistry revealed that weak GHS-R-like immunoreactivity was found in B cells containing insulin. GHS-R immunoreactivity overlapped that of glucagon-like immunoreactive cells. Moreover, both ghrelin and GHS-R-like immunoreactivities were detected mostly in the same cells in the periphery of the islets of Langerhans. These observations suggest that ghrelin is synthesized and secreted from A cells, and acts back on A cells in an autocrine and/or paracrine manner. In addition, ghrelin may act on B cells via GHS-R to regulate insulin secretion.

Neuronal circuits involving ghrelin in the hypothalamus-mediated regulation of feeding

Ghrelin, an n-octanoylated 28-amino acid brain-gut peptide, was first isolated from extracts of porcine stomach. Ghrelin is an endogenous ligand for the growth hormone secretagogue type 1a receptor (GHS-R1a), the functionally active form of GHS-R, and stimulates feeding and growth hormone secretion. Ghrelin is mainly produced in the A/X-like cells of the oxyntic glands of the stomach and is the main orexigenic circulating hormone that acts on the hypothalamus to affect feeding behavior and energy metabolism. Ghrelin-containing neuronal cell bodies are localized in the hypothalamic arcuate nucleus, a center that integrates signals for energy homeostasis. Ghrelin-containing nerve fibers are widely distributed in the brain. Accumulated evidence shows that hypothalamic neuropeptides such as neuropeptide Y (NPY), orexin and proopiomelanocortin (POMC) are involved in the regulation of feeding behavior and energy homeostasis via neuronal circuits in the hypothalamus. Ghrelin also forms part of the feeding-regulating neuronal circuitry in conjunction with other feeding-regulating peptide-containing neurons within the hypothalamus. In view of the fact that one decade has now passed since ghrelin was first discovered, we review advances that have been made in ghrelin research during that time and how this has impacted on our knowledge of feeding regulation in the hypothalamus. We also summarize our current understanding of the neuronal interactions between ghrelin and the different kinds of feeding-regulating peptide-containing neurons in the hypothalamus based on evidence at the ultrastructural level.

Synaptic interactions between ghrelin- and neuropeptide Y-containing neurons in the rat arcuate nucleus.

Morphological relationships between neuropeptide Y- (NPY) like and ghrelin-like immunoreactive neurons in the arcuate nucleus (ARC) were examined using light and electron microscopy techniques. At the light microscope level, both neuron types were found distributed in the ARC and could be observed making contact with each other. Using a preembedding double immunostaining technique, some NPY-immunoreactive axon terminals were observed at the electron microscope level to make synapses on ghrelin-immunoreactive cell bodies and dendrites. While the axo-somatic synapses were mostly symmetric in nature, the axo-dendritic synapses were both symmetric and asymmetric. In contrast, ghrelin-like immunoreactive (ghrelin-LI) axon terminals were found to make synapses on NPY-like immunoreactive (NPY-LI) dendrites although no NPY-like immunoreactive perikarya were identified receiving synapses from ghrelin-LI axon terminals. NPY-like axon terminals were also found making synapses on NPY-like neurons. Axo-axonic synapses were also identified between NPY- and ghrelin-like axon terminals. The present study shows that NPY- and ghrelin-LI neurons could influence each other by synaptic transmission through axo-somatic, axo-dendritic and even axo-axonic synapses, and suggests that they participate in a common effort to regulate the food-intake behavior through complex synaptic relationships.

Galanin-like peptide is co-localized with α-melanocyte stimulating hormone but not with neuropeptide Y in the rat brain

Galanin-like peptide (GALP), recently isolated from the hypothalamus, is a novel peptide of 60 amino acid residues. GALP is an endogenous ligand of the orphan receptor and shows a high affinity to its specific receptor GalR2. GALP mRNA was 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. According to recent reports of morphological and physiological experiments, GALP-containing neurons express leptin receptors and respond to leptin treatment by increasing mRNA expression. However, the relationships between GALP and other feeding-regulating neurons have not yet been proven. In this study, we examined the relationships between GALP- and neuropeptide Y (NPY)- or alpha-melanocyte stimulating hormone ( MSH)-containing neurons by using a dual immunostaining technique. We found that many NPY-immunoreactive fibers were in close apposition with GALP-immunoreactive cell bodies. Furthermore, immunoreactivity for GALP and alpha-MSH was detectable in the same neurons (3.3-11.8%) in the ARC. However, the co-existence of GALP and NPY was never demonstrated. These findings strongly suggest that GALP may participate in the regulation of feeding behavior in harmony with alpha-MSH.

Galanin-like peptide in the brain: effects on feeding, energy metabolism and reproduction

The hypothalamus plays an important role in the regulation of feeding behavior, energy metabolism and reproduction. A novel peptide containing 60 amino acid peptide and a non-amidated C-terminus is produced in the hypothalamic arcuate nucleus (ARC) and has been named galanin-like peptide (GALP) on the basis of a portion of this peptide being homologous with galanin. It acts in the central nervous system (CNS), where it is involved in the regulation of feeding behavior. GALP-producing neurons make neuronal networks with several feeding related peptide-producing neurons. Since GALP is involved in the control of food intake and energy balance, it is possible that it plays an important role in the development of obesity. Furthermore, GALP regulates plasma lateral hypothalamus (LH) levels via the activation of gonadotropin-releasing hormone (GnRH)-producing neurons, suggesting that GALP is active in the reproductive system. Thus, interesting findings on the roles of GALP have made across a number of physiological systems. This review will attempt to summarize the research carried out to date on these areas. Because GALP may be involved in feeding behavior, energy metabolism and reproduction, further studies on the morphology and function of GALP-containing neurons in the CNS should increase our understanding of the role of GALP in brain function.

Co-existence of leptin- and orexin-receptors in feeding-regulating neurons in the hypothalamic arcuate nucleus: a triple labeling study

The arcuate nucleus (ARC) of the hypothalamus has been identified as a prime feeding regulating center in the brain. Several feeding regulating peptides, such as neuropeptide Y (NPY) and proopiomelanocortin (POMC), are present in neurons of the ARC, which also serves as a primary targeting site for leptin, a feeding inhibiting hormone secreted predominantly by adipose tissues, and for orexin (OX)-containing neurons. OX is expressed exclusively around the lateral hypothalamus, an area also established as a feeding regulating center. Some recent physiological analyses have shown that NPY- and POMC-containing neurons are activated or inactivated by leptin and OX. Moreover, we have already shown, using double immunohistochemical staining techniques, that NPY- and POMC-containing neurons express leptin receptors (LR) and orexin type 1 receptors (OX-1R). However, no morphological study has yet described the possibility of whether or not these arcuate neurons are influenced by both leptin and OX simultaneously. In order to address this issue, we performed histochemistry on ARC neurons using a triple immunofluorescence method. We found that 77 out of 213 NPY- and 99 out of 165 POMC-immunoreactive neurons co-localized with both LR- and OX-1R-immunoreactivities. These findings strongly suggest that both NPY- and POMC-containing neurons are regulated simultaneously by both leptin and OX.

Visualization of ghrelin-producing neurons in the hypothalamic arcuate nucleus using ghrelin-EGFP transgenic mice.

The gut-brain hormone ghrelin is known to stimulate growth hormone release from the pituitary gland, and to regulate appetite and energy metabolism. Ghrelin-containing neurons have been shown to form neuronal network with several types of appetite-regulating neurons in the hypothalamus. Although ghrelin-containing cell bodies have been reported to localize in the hypothalamic arcuate nucleus, the published results present large discrepancies regarding the localization of ghrelin-positive cell bodies in the brain. In order to address this issue, we have generated a transgenic mouse model by microinjecting a DNA construct in which the transcription regulatory regions of ghrelin drive the enhanced green fluorescent protein (EGFP) gene. These transgenic mice expressed EGFP and ghrelin mRNA in the stomach and hypothalamus. Double immunostaining revealed that GFP-like immunoreactivity was co-localized with ghrelin-like immunoreactivity in the stomach of these animals, while EGFP fluorescence was clearly demonstrated in the hypothalamic arcuate nucleus by confocal laser microscopy. The ghrelin-EGFP transgenic mouse model described in this study therefore provides a powerful tool with which to analyze ghrelin neuronal circuits in the brain and should contribute to our understanding of the functional significance of ghrelin in the central nervous system.

Neuronal interactions between galanin-like-peptide- and orexin- or melanin-concentrating hormone-containing neurons

Galanin-like peptide (GALP) is a novel orexigenic neuropeptide that is recently isolated from the porcine hypothalamus. GALP-containing neurons predominantly locate in the hypothalamic arcuate nucleus (ARC). The expression of GALP mRNA within the ARC is increased after the administration of leptin. GALP-containing neurons express leptin receptor and contain alpha-melanocyte-stimulating hormone. We have recently reported that neuropeptide Y (NPY)- and orexin-containing axon terminals are in close apposition with GALP-containing neurons in the ARC. In addition, GALP-containing neurons express orexin-1 receptor (OX1-R). Thus, GALP may function under the influence of leptin and orexin. However, the target neurons of GALP have not yet been clarified. To clarify the neuronal interaction between GALP-containing and other feeding regulating neurons, double-immunostaining method using antibodies against GALP- and orexin- or melanin-concentrating hormone (MCH) was performed in the rat lateral hypothalamus (LH). GALP-immunoreactive fibers appeared to project to the LH around the fornix. They were also found from the rostral to the caudal part of the ARC, paraventricular nucleus (PVH), stria terminalis (BST), medial preoptic area (MPA), and lateral septal nucleus (LSV). Moreover, GALP-like immunoreactive nerve fibers were directly contacted with orexin- and melanin-concentrating hormone (MCH)-like immunoreactive neurons in the LH. Our findings strongly suggest that GALP-containing neurons interact with orexin- and/or MCH-containing neurons in the lateral hypothalamus and that it participates in the regulation of feeding behavior in harmony with other feeding-regulating neurons in the hypothalamus.

Galanin-Like Peptide Promotes Feeding Behaviour Via Activation of Orexinergic Neurones in the Rat Lateral Hypothalamus

Galanin‐like peptide (GALP) is produced in neurones in the hypothalamic arcuate nucleus and is implicated in the neural control of feeding behaviour. Previously, we have reported that GALP immunoreactive fibres were in direct contact with orexin/hypocretin immunoreactive neurones in the rat lateral hypothalamus using double‐immunofluorescence. Centrally administered GALP is known to stimulate feeding behaviour. However, the target neurones of this action have not been clarified. The present study aimed to determine features of the GALP‐mediated neuronal feeding pathway in rat. Accordingly, at the ultrastructural level, GALP‐immunoreactive axon terminals were found to make synapses on orexin/hypocretin immunoreactive cell bodies and dendritic processes in the lateral hypothalamus. c‐Fos immunoreactivity was expressed in orexin/hypocretin‐immunoreactive neurones but not in melanin concentrating hormone‐immunoreactive neurones in the lateral hypothalamus at 90 min after the application of GALP by i.c.v. infusion. Furthermore, to determine whether GALP regulates feeding behaviour via orexin/hypocretin neurones, the feeding behaviour of rats was studied following GALP i.c.v. injection with or without anti‐orexin A and B immunoglobulin (IgG) pretreatment. The anti‐orexin IgGs markedly inhibited GALP‐induced hyperphagia. These results suggest that orexin/hypocretin‐containing neurones in the lateral hypothalamus are targeted by GALP, and that GALP‐induced hyperphagia is mediated via orexin/hypocretin neurones in the rat hypothalamus.