~ Pergamon Neuroscience Vol. 73, No. 2, pp. 299-315, 1996 Copyright © 1966 IBRO. Published by ElsevierScienceLtd Printed in Great Britain 0306-4522(95)00610-9 0306-4522/96 $15.00 + 0.00 CORRELATED MORPHOLOGICAL AND NEUROCHEMICAL FEATURES IDENTIFY DIFFERENT SUBSETS OF VASOACTIVE INTESTINAL POLYPEPTIDE-IMMUNOREACTIVE INTERNEURONS IN RAT HIPPOCAMPUS L. ACS,ADY, D. ARABADZISZ and T. F. FREUND* Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest P.O.B. 67, H-1450, Hungary A~tract--Vasoactive intestinal polypeptide-immunoreactive interneurons have been classified according to their axonal and dendritic patterns and neurochemical features in the hippocampus of the rat. A correlation of these characteristics unravelled three distinct types of vasoactive intestinal polypeptide- containing cells. Interneurons forming a dense axonal plexus at the border of stratum oriens and alveus always contain the calcium binding protein, calretinin, but lack the neuropeptide cholecystokinin. The axon of another type of vasoactive intestinal polypeptide-positive interneuron surrounds pyramidal cell bodies in a basket-like manner, and co-localizes cholecystokinin but not calretinin. Vasoactive intestinal polypeptide-containing cells projecting to stratum radiatum form two subsets distinguished by dendritic morphology. Those with dendrites restricted to stratum lacunosum-moleculare lack both calretinin and cholecystokinin, whereas the other subtype with dendrites spanning all layers contains calretinin in 40% of the cases and occasionally also cholecystokin. GABA was shown to be present, and the calcium binding proteins calbindin D-28k and parvalbumin absent from all three types of vasoactive intestinal polypeptide- positive interneurons. The specific dendritic and axonal arbours imply different input and output properties for the three interneuron types. The correlation of these features with the content of neurochemical markers strongly suggests that they are specialized for distinct inhibitory functions in the hippocampal network. Copyright © 1996 IBRO. Published by Elsevier Science Ltd. Key words: hippocampus, interneuron, GABA, VIP, co-localization, neuropeptides. In the hippocampus local inhibitory neurons play fundamental roles in the control of synaptic plasticity and network activity patterns of principal c e l l s . 5'7'9'16'41'48'5° In contrast to the rather homo- geneous population of principal cells, interneurons show a remarkable variation in their morphological, neurochemical and electrophysiological character- istics suggesting that diverse inhibitory processes o p e r a t e w i t h i n the h i p p o c a m p u s , sA9,21'24'25`29'as'39 From the large number of studies dealing with the functional classification of hippocampal interneurons two basic classes seem to emerge, each including several subpopulations. Neurons of both classes in- nervate pyramidal cells, but at different locations. Basket and axo-axonic cells contact the perisomatic *To whom correspondence should be addressed. Abbreviations: ABC, avidin biotinylated-horseradish per- oxidase complex; CA, cornu Ammonis; CCK, chole- cystokinin; DAB, diaminobenzidine; FITC, fluorescein isothiocyanate; GAD: glutamate decarboxylase; LRSC, Lissamine rhodamine sulfonyl chloride; NGS, normal goat serum; O/A border, border of strata oriens and alveus; PB, phosphate buffer; TBS, Tris-buffered saline; VIP, vasoactive intestinal polypeptide. region, and are likely to control the output (genesis of sodium spikes), whereas the other types contact specific dendritic domains, 8,15,17,23,39 and are likely to influence electrogenesis and synaptic plasticity in the dendrites) ~ Golgi impregnation and intracellular labelling techniques were the first to reveal these cell types with nearly complete dendritic trees and various pro- portions of their axonal arborizations. While the inputs and outputs of neurons visualized this way could be determined, these techniques provided no information about the frequency of a particular cell type, nor about the consistency of certain features within a morphologically established cell class. An ideal tool to study these questions is immunostaining for neurochemical markers, such as calcium-binding proteins 2'1°:4'3° and neuropeptides, 35,4°,43 which selec- tively label large populations of interneurons. These markers, either individually or in combination with other markers, are often specific for a well defined population of interneurons. For example, the cal- cium-binding protein, parvalbumin, is present exclu- sively in basket and axo-axonic cells. 23 The neuropeptide somatostatin is present in interneurons 299