A high density of galanin binding sites was found by using 1251-labeled galanin, iodinated by chloramine-T, followed by autoradiography in the ventral, but not in the dorsal, hippocampus of the rat. Lesions of the runbria and of the septum caused disappearance of a major population of these binding sites, suggesting that a large proportion of them is localized on cholinergic nerve terminals of septal afferents. Amersham. Scopolamine hydrobromide was purchased from Aldrich; physostigmine sulfate was from Sigma. All the other chemicals used were of analytical grade. Galanin antiserum was purchased from Peninsula Laboratories (San Carlos, CA). Secondary antisera were purchased from Boehringer Mannheim (Stockholm).Surgical Procedures. Four rats were anesthetized with chloral hydrate. Lesions of the dorsal hippocampal afferents were performed by the use of a retractable knife. The knife was lowered into the brain (coordinates in relation to bregma: AP, -1.4; ML, 3.0; DV, -4.0), and the blade was pushed out to the midline and retracted through the brain.Immunohistochemical, Histochemical, and Autoradiographic Analyses. Two rats were perfused through the ascending aorta with a picric acid/formalin mixture (14) for 6 min. The brains were postfixed in the same fixative, rinsed, and sectioned on a cryostat. Sections were incubated overnight at 4°C with galanin antiserum (1:400 dilution), rinsed, incubated with fluorescein isothiocyanate-conjugated secondary antibody (1:40 dilution) at 37°C for 30 min (15), rinsed, mounted, and examined with a fluorescence microscope. Some sections were processed for demonstration of AcChoE, either by the method of Karnovsky and Roots (16) or by immunohistochemistry using rabbit polyclonal antibody to AcChoE (17). For the autoradiographic analysis of 1251_ labeled galanin binding sites, the procedure of Young and Kuhar (18) was used. Briefly, pig galanin was iodinated with Na[1251] by the chloramine-T method and purified on an ion-exchange column. Rats were perfused with ice-cold Tyrode's solution, and the brain was sectioned on a cryostat, followed by incubation with 1251I-labeled galanin for 45 min at room temperature. Sections were rinsed, dried by a stream of cold air, exposed to formalin vapors, and covered by tritiumsensitive film (Ultrofilm, LKB, Stockholm). For control of nonspecific binding, unlabeled galanin (1 ,uM) was added to the incubation medium.In Vivo Experiments. In the in vivo release experiments, a thin dialysis fiber was implanted, essentially as described by Ungerstedt (19) and Benveniste et al. (20), in the hippocampi of anesthetized rats. A looped dialysis probe was implanted vertically into the ventral hippocampus of one side, while a straight dialysis probe was inserted through both dorsal hippocampi. The day after implantation, the dialysis tube was Abbreviations: AcCho, acetylcholine; AcChoE, acetylcholinesterase. §To whom reprint requests should be addressed. 7339The publication costs of this article were defrayed in part by page charge payment. This a...
The glanin-receptor Hlgand M40 [galanin-(1-12)-Pro3-(Ala-Leu)2-AIa amide] binds with high affinity to galanin-binding sites in hippocampal, hypothalamic, and spinal cord membranes and in membranes from Rin m5F rat insulinoma cells . Receptor autoradlographic studies show that M40 (1 "M) displaces galanin from binding sites in the hippocampus, hypothalamus, and spinal cord. In the brain, M40 acts as a potent galanin-receptor antagonist: M40, in doses comparable to that of galanin, antagonizes the stimulatory effects of glanin on feeding, and it blocks the galaninergic inhibition of the scopolamine-induced acetylcholne release in the ventral hippocampus in vivo. In contrast, M40 completely fails to antagonize both the galanin-mediated inhibition of the glucoseinduced insulin release in isolated mouse pancreatic islets and the inhibitory effects of galanin on the forskolin-stimulated accumulation of 3',5'-cAMP in Rin mSF cells; instead M40 is a weak agonist at the galanin receptors in these two systems. M40 acts as a weak antagonist of galanin in the spinal flexor reflex model. These results suggest that at least two subtypes of the glanin receptor may exist. Hypothalamic and hippocampal galanin receptors represent a putative central galaninreceptor subtpe (GL-1-receptor) that is blocked by M40. The pancreatic galanin receptor may represent another subtype (GL-2-receptor) that recognizes M40, but as a weak agonist. The galanin receptors in the spinal cord occupy an intermediate position between these two putative subtypes.Galanin is an important neuroendocrine peptide with multiple biological and pharmacological actions (1). It is a potent inhibitor of glucose-induced insulin release (2), it inhibits hippocampal acetylcholine release (3) induced by systemic administration of scopolamine (4), it impairs cognitive performance (5, 6), it stimulates feeding behavior upon hypothalamic or intracerebroventricular injection (7,8), it stimulates growth hormone secretion (9), and it has a biphasic effect on the spinal flexor reflex (10). Galanin hyperpolarizes noradrenergic cell bodies in the locus coeruleus (11) Accordingly, a galanin-receptor subtype has been suggested, which is composed of nervous tissue and pancreatic galanin receptors that recognize the N-terminal 1-to 15-aa or 1-to 16-aa fragment of galanin as high-affinity agonists, whereas another putative galanin-receptor subtype in smooth muscle requires both the N and C terminus of galanin for binding and biological action (25). On the basis of the differential affinity ofgalanin (3-29) and ofa galanin-receptor antagonist M15 (17), existence of an additional galaninreceptor subtype has been suggested in the rat anterior pituitary (26), which differs from other CNS galanin receptors. Finally, galanin-(1-15)-binding sites have been demonstrated in the dorsal hippocampus, neocortex, and neostriatum, areas that seem to lack galanin-(1-29)-binding sites (27).
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