~ Pergamon Neuroscience Vol. 73, No. 3, pp. 831-844, 1996 Copyright © 1996 IBRO. Published by ElsevierScienceLtd Printed in Great Britain PII: S0306-4522(96)00064-4 0306-4522/96 $15.00+0.00 CHOLINOCEPTIVE NEURONS WITHOUT ACETYLCHOLINESTERASE ACTIVITY AND ENZYME-POSITIVE NEURONS WITHOUT CHOLINERGIC SYNAPTIC INNERVATION ARE PRESENT IN THE MAIN OLFACTORY BULB OF ADULT RAT P. KASA,*t S. KARCSU,~ I. KOVACSt and J. R. WOLFF§ tDepartment of Neurology and Psychiatry, Division of Alzheimer's Disease Research Laboratory, Hungary $1st Department of Internal Medicine, Albert Szent-Gy6rgyi Medical University, H-6720 Szeged, Hungary §Department of Anatomy, University of G6ttingen, D-37075 G6ttingen, Germany Abstract--Light and electron microscopic histochemistry revealed acetylcholinesterase-positive and acetylcholinesterase-negative neurons in the main olfactory bulb of adult rat. Their distribution patterns on various neuron types have been analysed in detail. (1) No acetylcholinesterase staining could be demonstrated in the granule cells which receive a large number of the cholinergic synapses. (2) In contrast, enzyme activity was present in the soma and dendrites in most of the non-cholinergic and non-cholinoceptive relay ceils (mitral cells and tufted cells) and in a subset of short-axon interneurons, where cholinergic synapses could not be detected. (3) Within the neuropil of glomeruli, two compartments were present, one of which was free of acetylcholinesterase-positive structures, while many enzyme-positive neuronal elements were seen in the other. (4) Characteristically, cholinergic and non-cholinergic neuronal structures showed triadic arrangements. (5) The axonal release of acetylcholinesterase from cholinergic axons is probable. It is suggested that, in the olfactory bulb, acetylcholinesterase is released by cholinergic afferent axons, and it is the cholinergic synapses that determine which postsynaptic neurons are cholinoceptive rather than the intraneuronal presence of acetylcholinesterase. In the main olfactory bulb, the acetylcholinesterase present in the relay cells therefore appears to have functions other than the hydrolysis of acetylcholine. Copyright © 1996 IBRO. Published by Elsevier Science Ltd. Key words: acetylcholinesterase, histochemistry, olfactory bulb, rat, release, triad. On the basis of the histochemical visualization of acetylcholinesterase (ACHE), Shute and Lewis 44 suggested that some of the nerve fibres projecting from the basal forebrain to the main olfactory bulb (MOB) of the rat are cholinergic. Light microscopic AChE histochemistry suggested that the cholinergic fibres terminate on the juxtaglomerular short-axon cells and on the inframitrally located AChE-positive interneurons. 39 Neurons of origin were found in the medial part of the horizontal limb of the diagonal band.41,53.sa Choline acetyltransferase (CHAT) immunohisto- chemistry has been used to determine the fine struc- tural distribution of cholinergic fibres and their synapses in atypical glomeruli 3~ and in typical ones, as well as on neurons in various layers of the MOB. 26 Cholinergic projections were shown to terminate on *To whom correspondence should be addressed. Abbrev&tions: ACHE, acetylcholinesterase; CHAT, choline acetyltransferase; EPL, external plexiform layer; GCL, granule cell layer; GL, glomerular layer; IPL, internal plexiform layer; MOB, main olfactory bulb. the periglomerular cell dendrites and dendritic spines and on the soma and dendrites of the short-axon cells within the periglomerular region of the MOB. In the external plexiform layer (EPL), the internal plexiform layer (IPL) and the granule cell layer (GCL), the fibres are in synaptic contact with the granule cell dendritic gemmules, and in non-synaptic contact with the mitral cell and/or tufted cell primary and secondary dendrites in the glomerular layer (GL) and EPL. In contrast, light microscopic histochemistry suggested that the juxtaglomerular short-axon cells and the inframitral AChE-positive neurons are the cholinoceptive structures. 39 The divergence of results relating to the light microscopic histochemistry of AChE and the ultrastructural localization of ChAT prompted us to reinvestigate the presence of AChE activity in the MOB at the electron microscopy level in an attempt to establish whether or not the neurons innervated by cholinergic synapses possess AChE activity in their perikarya, dendrites and related intercellular clefts. In addition, we tried to determine the origin of extracellular AChE between the different cellular structures. 831