Brain Research Bulletin, Vol. 32, pp. 601-609, 1993 Printed in the USA. All rights reserved. 0361-9230/93 $6.00 + .OO Copyright 0 1993 Pergamon Press Ltd. GABAergic Neurons of the Rat Dorsal Hippocampus Express Muscarinic Acetylcholine Receptors E. A. VAN DER ZEE AND P. G. M. LUITEN’ Department zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA of Animal Physiology, University of Groningen, P.O. Box 14, 9750 AA Haren, The Netherlands Received 25 January 1993; Accepted 7 May 1993 VAN DER ZEE, E. A. AND P. G. M. LUITEN. GABAergic neurons ofthe rat dorsal hippocampus expressmuscarinie acety lcholine receptors. BRAIN RES BULL 32(6) 60 l-609,1993.-The expression of muscarinic acetylchohne receptors (mAChRs) in glutamic acid decarboxylase (GAD)-positive cells in the different strata ofCA 1, CA3, and the dentate gyrus (DG) of the dorsal hippocampus is examined by way of quantitative immunofluorescent double labeling employing M35, the monoclonal antibody raised against purified mAChR protein. Of all GAD-positive neurons, 97.5% express mAChI&. Conversely, 92.9% of the muscarinic cholinoceptive nonpyramidal neurons express GAD. These results indicate that the vast majority of the y-aminobutyric acid (GABA)ergic neurons express mAChRs. In addition to GAD, parvalbumin (PARV) and somatostatin (SOM) are two neurochemical substances notably expressed in GABAergic neurons. In order to examine whether the entire muscarinic cholinoceptive nonpyramidal cell group can be characterized by these three GABAergic markers, a cocktail of GAD, PARV, and SOM was used in a fluorescent double-labeling experiment with M35. These results show that 97.2% of ah muscarinic cholinoceptive nonpyramidal neurons can be neurochemically characterized by the content of GAD, PARV, and SOM. In conclusion, nearly all GABAergic cells express mAChRs and, conversely, virtually the entire muscarinic cholinoceptive nonpyramidal cell group belongs to the GABAergic cell population. This study, therefore, provides anatomical evidence for an extensive neuronal connectivity of the hippocampal muscarinic cholinoceptive nonpyramidal system and the inhibitory GABAergic circuitry. GAD Parvalbumin Somatostatin Muscarinic acetylcholine receptor Double-labeling immunocytochemistry Dorsal hippocampus Rat brain THE hippocampus and its afferent cholinergic projection from the medial septum and diagonal band (MSDB) are intimately involved in a variety of learning and memory processes (28). The projection of the cholinergic neurons of the MSDB termi- nates upon granule cells, pyramidal cells, and nonpyramidal neurons (8,14,15,27). In the hippocampus, muscarinic acetyl- choline receptors (mAChRs) are considered to be the primary receptors in choline& synaptic transmission involved in leam- ing and memory (6,7). Previously, we have extensively employed the monoclonal antibody M35, raised against purified mAChR protein (2-4), to visualize the muscarinic cholinoceptive target structures of the cholinergic systems in the central and peripheral nervous system (40,44). In the hippocampus, half of all mus- carinic cholinoceptive nonpyramidal neurons are parvalbumin (PARV) positive (42), while approximately one-third of all mus- carinic cholinoceptive nonpyramidal neurons react positively for presence of somatostatin (SOM) (39). Both PARVergic and SOMergic neurons belong to the larger y-aminobutyric acid (GABA)ergic cell group (20,21,25,26,34). GABAergic neurons constitute various inhibitory circuits in the hippocampus and consist of several distinct, largely nonoverlapping subpopulations (17,20,2 1,25,26,3 1,32,34,38). The interaction between the cho- linergic and the GABAergic system in the septo-hippocampal circuitry is of particular importance to, for example, learning and memory functions and the pathophysiology of disorders such as epilepsy (28,3 1). Therefore, a more detailed knowledge of the GABA-related neurochemical identity of the muscarinic cholinoceptive nonpyramidal neurons and the neuronal con- nectivity between the choline& and the GABAergic system will contribute to the basic understanding of the neuronal cir- cuitry involved in hippocampal functioning. The present study is aimed at a further definition of the neurochemical character- istics of the GABAergic and muscarinic cholinoceptive non- pyramidal cells in the dorsal hippocampus. The degree of colo- calization of glutamic acid decarboxylase (GAD), the GABA- synthesizing enzyme, and mAChRs is determined by means of immunofluorescent double labeling. In addition, the relationship between neurons immunoreactive for one or more GABAergic markers (GAD, PARV, and SOM) and the expression of mAChRs in the nonpyramidal cells is described. i To whom requests for reprints should be addressed. 601