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.
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