Research ReportElectrophysiologically identified presynaptic mechanisms underlying amylinergic modulation of area postrema neuronal excitability in rat brain slices
Highlights
► First evidence for the presynaptic effects of amylin on the area postrema neruons. ► Possible role for the area postrema neurons expressing H channels in food intake. ► Possible contribution of amylinergic system in the CNS on the obesity treatment.
Introduction
The area postrema is one of the circumventricular organs, located on the dorsal surface of the medulla oblongata at the caudal end of the fourth ventricle. Blood vessels in the area postrema lack a blood brain barrier, offering specific central neural components unique access to circulating substances (Borison, 1989). A close relation between area postrema neuronal activity and autonomic functions has been suggested by many electrophysiological and behavioral studies, including the autonomic control of food intake (Contreras et al., 1984, van der Kooy, 1984, Ritter and Edwards, 1984), body fluid homeostasis (Miselis et al., 1984, Iovino et al., 1988), cardiovascular control (Ferguson and Smith, 1991), and emesis (Borison and Wang, 1953).
Amylin is a peptide hormone that is co-secreted with insulin from the pancreatic beta-cell in response to food intake. Previous studies have demonstrated the physiological effects of amylin, such as a reduction of food intake (Chance et al., 1991, Lutz et al., 1995b), an inhibition of gastric emptying (Young et al., 1995, Young, 2005, Clementi et al., 1996), and a suppression of glucagon secretion (Gedulin et al., 1997). The anorectic effect has attracted much attention because of the potential for obesity treatment.
Physiological and histological studies have demonstrated the multiple mechanisms for the anorectic effects of amylin. The anorectic actions of amylin occurred after peripheral administration (e.g. intraperitoneal) or central administration (e.g. intrahypothalamic or intraventricular) (Chance et al., 1991, Lutz et al., 1995b, Rushing et al., 2002). Behavioral experiments identified the area postrema as the primary site for circulating amylin since peripherally applied amylin did not show anorectic actions in area postrema-lesioned rats (Lutz et al., 1998, Barth et al., 2004). Area postrema lesions also blocked the control of gastric emptying by amylin (Edwards et al., 1998). The amylin antagonist, AC-187, injected into the area postrema blocked the anorectic effect induced by peripherally-administered amylin, furthermore, AC187 alone increased food intake in rats (Mollet et al., 2004). These studies suggest that the area postrema is the primary amylin target site, while dense amylin binding sites were found in many brain areas including the area postrema (D’Este et al., 2000, Sexton et al., 1994). Although further study is necessary to determine other target sites of amylin, the action of amylin in the area postrema is much better characterized than in any other brain regions at present.
Taking these various studies together, the anorectic effects of amylin could be mediated by the activation of a neural network from the area postrema to the rostro-dorsal lateral hypothalamic area (dLHA) through the nucleus tractus solitarius (NTS), the lateral parabrachial nucleus (LPB), the central nucleus of amygdala (Ce) and the lateral bed nucleus of stria terminalis (BSTL), i.e. AP–NTS–LPB–Ce–BSTL–dLHA axis (Becskei et al., 2007, Cline et al., 2008, Lutz et al., 1995a, Potes and Lutz, 2010, Potes et al., 2010, Riediger et al., 2004). It was also suggested that amylin-induced excitation of area postrema neurons resulted in the reduction of orexinergic neuropeptides, such as orexin and melanin concentrating hormone, in the lateral hypothalamic area (LHA) (Barth et al., 2003).
While the LHA contains neurons playing an important role in the control of feeding, it is unlikely that amylin affects LHA neurons directly, even when amylin was injected into the LHA because amylin receptors have not been detected anywhere in the LHA (Beaumont et al., 1993, Sexton et al., 1994, van Rossum et al., 1994). Rather, the effects of amylin in the hypothalamic area are more likely mediated by neurons in the satiety center and its surrounding nuclei since amylin-immunoreactive neurons have been detected in these hypothalamic nuclei, e.g. periventricular, ventromedial, arcuate, and tuberomammillary nuclei (D'Este et al., 2001). Interestingly, some studies have suggested a role for the histaminergic system in amylin-induced anorexia (D’Este et al., 2001, Lutz et al., 1996, MolletPlease provide the page range for the bibliography in Ref. [Mollet et al., 2001]. et al., 2001, Mollet et al., 2003). Finally, functional amylin receptors can be derived when calcitonin receptors and receptor activity modifying protein (RAMP) are co-expressed (Christopoulos et al., 1995, Christopoulos et al., 1999).
The effects of amylin on area postrema neuronal activity have been studied by using electrophysiological and immunohistochemical techniques (Riediger et al., 2001, Riediger et al., 2004). The responses of arcuate neurons to amylin also have been studied by using extracellular recording technique in the brain slices (Davidowa et al., 2004). Although these studies demonstrated the presence of amylin responding neurons in the area postrema and the arcuate nucleus, the mechanisms of amylinergic modulation of neuronal excitability have not been established.
In the present study, we investigated the responses of area postrema neurons to amylin using patch-clamp recording methods in rat brain slices. We also assessed whether cells display Ih or not (Funahashi et al., 2002, Funahashi et al., 2003). The aim of the study was to clarify the intrinsic membrane properties of cells responsive to amylin, and to identify the mechanism(s) by which amylin modulates neuronal excitability.
Section snippets
Results
The voltage-clamp recording was performed in 66 neurons in the presence of TTX to test whether they respond to amylin or not, when amylin was applied into the bath solution or was focally applied with a micropipette using a pressure injection system. All cells were classified based on the presence or absence of electrophysiological evidence of Ih (Funahashi et al., 2002, Funahashi et al., 2003). Half of the total 66 neurons recorded displayed Ih and half did not. Excitatory responses to amylin
Discussion
The present study used a perforated patch-clamp recording technique to provide the first evidence for a presynaptic mechanism of amylinergic modulation of glutamate synapses in the area postrema. Our data indicate that amylin receptors appear to be presynaptically located on glutamatergic inputs to a subclass of area postrema cells, i.e. the cells not displaying Ih. Furthermore, the generation of action potentials in these area postrema cells was shown to be induced by a facilitation of the
Animals and brain slice preparations
The experimental protocols were approved by the Hokkaido University Animal Committee. Brain slices were prepared from Sprague-Dawley albino rats (7–21days post-natal) as previously described (Funahashi et al., 2002, Funahashi et al., 2003). Briefly, animals were decapitated under halothane anesthesia, and each brain was rapidly removed from the skull. Brains were maintained for 1 min in ice cold, oxygenated sucrose artificial cerebrospinal fluid containing (in mM) 234 sucrose, 2.5 KCl, 0.5 CaCl2
Acknowledgment
We thank Dr. Mark Stewart (SUNY Downstate Medical Center, Brooklyn, NY) for his helpful comments on the manuscript. This research was supported by the Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
References (49)
- et al.
Differential effects of amylin and salmon calcitonin on neuropeptide gene expression in the lateral hypothalamic area and the arcuate nucleus of the rat
Neurosci. Lett.
(2003) - et al.
Peripheral amylin activates circumventricular organs expressing calcitonin receptor a/b subtypes and receptor-activity modifying proteins in the rat
Brain Res.
(2004) - et al.
Lesion of the lateral parabrachial nucleus attenuates the anorectic effect of peripheral amylin and CCK
Brain Res.
(2007) Area postrema: chemoreceptor circumventricular organ of the medulla oblongata
Prog. Neurobiol.
(1989)- et al.
Anorexia following the intrahypothalamic administration of amylin
Brain Res.
(1991) - et al.
Amylin causes anorexigenic effects via the hypothalamus and brain stem in chicks
Regul. Pept.
(2008) - et al.
Immunohistochemical localization of amylin in rat brainstem
Peptides
(2000) - et al.
Area postrema (AP) lesions block the regulation of gastric emptying by amylin (Abstract)
Gastroenterology
(1998) - et al.
Electrophysiological properties of the rat area postrema neurons displaying both the transient outward current and the hyperpolarization-activated inward current
Brain Res. Bull.
(2002) - et al.
Dose-response for glucagonostatic effect of amylin in rats
Metabolism
(1997)
Neuroanatomical and biochemical evidence for the involvement of the area postrema in the regulation of vasopressin release in rats
Brain Res.
Subdiaphragmatic vagotomy does not influence the anorectic effect of amylin
Peptides
Amylin decreases meal size in rats
Physiol. Behav.
The histaminergic, but not the serotoninergic, system mediates amylin's anorectic effect
Peptides
Lesion of the area postrema/nucleus of the solitary tract (AP/NTS) attenuates the anorectic effects of amylin and calcitonin gene-related peptide (CGRP) in rats
Peptides
Infusion of the amylin antagonist AC 187 into the area postrema increases food intake in rats
Physiol. Behav.
Histamine H1 receptors in the ventromedial hypothalamus mediate the anorectic action of the pancreatic hormone amylin
Peptides
Brainstem mechanisms of amylin-induced anorexia
Physiol. Behav.
Identification of central projections from amylin-activated neurons to the lateral hypothalamus
Brain Res.
Low access resistance perforated patch recordings using amphotericin B
J. Neurosci. Methods
Area postrema lesions cause overconsumption of palatable foods but not calories
Physiol. Behav.
Acute 3rd-ventricular amylin infusion potently reduces food intake but does not produce aversive consequences
Peptides
In vitro autoradiographic localization of amylin binding sites in rat brain
Neuroscience
The role of area postrema neurons expressing H-channels in the induction mechanism of nausea and vomiting
Physiol. Behav
Cited by (17)
Creating the amylin story
2022, AppetiteEffects of methyl methacrylate on the excitability of the area postrema neurons in rats
2020, Journal of Oral BiosciencesCitation Excerpt :Whether the MMA in the blood directly excites the AP neurons remains unexplained. To answer this question, we will perform another experiment using electrophysiological methods [17–24]. Considering the functional role of AP in triggering nausea and/or emesis, animals may feel nauseous during exposure to MMA in the present study.
Amylin brain circuitry
2020, PeptidesCitation Excerpt :While more than approximately half of amylin activated neurons are noradrenergic, further studies are needed to characterize the neurochemical nature of the remaining non-noradrenergic amylin activated AP neurons and their possible function. A recent electrophysiological study suggested that glutamatergic neurotransmission in the AP may mediate or rather modulate amylin's effect; indeed, AMYs appear to be mainly located on presynaptic glutamatergic terminals connecting to AP neurons, and amylin has been shown to increase glutamate release and cause cell firing [85]. In addition, vesicular glutamate transporters (VGLUT2) boutons were shown to be apposed to amylin activated noradrenergic neurons in AP [83].
Amylin – Its role in the homeostatic and hedonic control of eating and recent developments of amylin analogs to treat obesity
2018, Molecular MetabolismCitation Excerpt :Indeed using anterograde and retrograde tracing, the AP was shown to project to the NTS, LPB, CeA, and the bed nucleus of the stria terminalis [37]. Furthermore, a recent study suggested that glutamatergic neurotransmission in the AP seems to play a role in mediating amylin effects on eating, and that the amylin receptors appear to be located mainly on presynaptic glutamatergic terminals synapsing with AP neurons [38]; interestingly, our own studies also showed a close apposition of amylin-activated neurons that expressed DBH with VGLUT2-positive boutons [39]. How these effects may be linked mechanistically, and whether this mechanism is physiologically relevant, is currently unknown.
Amylin and its G-protein-coupled receptor: A probable pathological process and drug target for Alzheimer's disease
2017, NeuroscienceCitation Excerpt :Additionally, amylin activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway by inducing the phosphorylation of ERK1/2 (pERK) (Potes et al., 2012). A study showed that amylin treatment increased glutamate release to levels sufficient to cause cell firing (Fukuda et al., 2013); this may be necessary for restoring learning and memory in AD (Danysz and Parsons, 2012). Amylin has been proposed to play a role as a trophic factor (Potes and Lutz, 2010), for example, for osteoblast growth (Cornish et al., 1998).
Presynaptically mediated effects of cholecystokinin-8 on the excitability of area postrema neurons in rat brain slices
2015, Brain ResearchCitation Excerpt :Based on our results, we believe that CCK-A, not CCK-B, receptors acting presynaptically modulate glutamate release in area postrema. In our previous study, we demonstrated the responsiveness of area postrema neurons to the satiety peptide, amylin (Fukuda et al., 2013). Amylin is well known as a feeding suppressive peptide, which targets cells of the area postrema.