Cholinoceptive neurons without acetylcholinesterase activity and enzyme-positive neurons without cholinergic synaptic innervation are present in the main olfactory bulb of adult rat

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Pergamon

Neuroscience Vol. 73, No. 3, pp. 831-844, 1996 Copyright © 1996 IBRO. Published by Elsevier ScienceLtd Printed in Great Britain S0306-4522(96)00064-4 0306-4522/96 $15.00+0.00

PII:

CHOLINOCEPTIVE NEURONS WITHOUT ACETYLCHOLINESTERASE ACTIVITY AND ENZYME-POSITIVE NEURONS WITHOUT CHOLINERGIC S Y N A P T I C I N N E R V A T I O N A R E P R E S E N T IN THE M A I N O L F A C T O R Y BULB OF A D U L T R A T P. K A S A , * t S. K A R C S U , ~ I. K O V A C S t and J. R. W O L F F § 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 A C h E 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) immunohistochemistry has been used to determine the fine structural 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

*Towhom 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. 831

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 A C h E and the ultrastructural localization of C h A T prompted us to reinvestigate the presence of A C h E activity in the M O B at the electron microscopy level in an attempt to establish whether or not the neurons innervated by cholinergic synapses possess A C h E activity in their perikarya, dendrites and related intercellular clefts. In addition, we tried to determine the origin of extracellular A C h E between the different cellular structures.

P. Kasa et al.

832 EXPERIMENTAL PROCEDURES

Tissue preparation Anaesthetized Wistar rats (in-house bred) (weighing 200 250 g) were perfused with 2% glutaraldehyde and 4% formaldehyde solution (freshly prepared from paraformaldehyde), buffered with 0.1 M sodium cacodylate (pH 7.4) and containing 2 m M calcium acetate and 4% sucrose. The MOB was removed immediately and fixed for 12 h at 4'~C in the same fixative. Tissue samples were then washed at least overnight in a solution consisting of 0.1 M sodium cacodylate, 0.2 M calcium acetate and isotonic sodium sulphate. Coronal slices were made either with a freezing microtome (20~40/~m) for light microscopic studies or with a Vibratome (100-120/~m) for ultrastructural studies, and kept chilled. Sections were processed with the freefloating technique for AChE histochemistry. Two different techniques were used for the visualization of AChE activity.

Light microscopic histochemistry For light microscopy, a sensitive metho& 9 was used in a slightly modified form. After fixation, tissue samples were rinsed several times in 0.1 M sodium maleate buffer (pH 6.0). Sections were pretreated for 30 rain at 25-'C in ethopropazine hydrochloride (2 × 10-4M), rinsed in 0.1 M sodium maleate (pH 6.0), incubated for 4 0 4 5 min in a medium consisting of acetylthiocholine chloride (1.8 raM), sodium citrate (0.1 M), copper sulphate (0.03 M), potassium ferricyanide (5.0 ~M) and sodium maleate (0.1 M; pH 6.0),

and rinsed in Tris HCI buffer (Sigma) solution (pH 7.6). Finally, a solution consisting of 0.05% 3,3'-diaminobenzidine tetrahydrochloride, 0.15% nickel ammonium sulphate in 50 m M Tris-HCl buffer (pH 7.6) and 0.005% hydrogen peroxide was applied for 5 10min. This was followed by washing in Tris-HC1 buffer (5 mM, pH 7.6). Sections were mounted on glass slides, dehydrated in a series of alcohol, cleared in xylene and coverslipped with Entellan. Non-specific cholinesterase activity was selectively inhibited continuously with ethopropazine hydrochloride (2× 10-4M) introduced directly into the incubation medium.

Electron microscopic histochemist~v For electron microscopy, 100 120-#m-thick Vibratome sections were treated according to Lewis and Shute. 33'34 Briefly, the samples were preincubated for 30 min at 25~C in ethopropazine hydrochloride (2 × 10 4 M) and thereafter incubated in the following solution for 2 h: 13 mM acetyIthiocholine, 18mM copper sulphate, 33 mM glycine and 2 x I0 4 M ethopropazine hydrochloride in 25 mM sodium succinate buffer. Thereafter, the sections were washed in 0.5 M sodium succinate buffer (pH 5.3) for 60 min, then treated in 2% sodium sulphide solution (pH 5.3) for 30 rain, washed in sodium succinate buffer (0.5 M, pH 5.3), and postfixed at room temperature in a solution containing 1% potassium dichromate and 1% osmium tetroxide (pH 7.2). The samples were dehydrated and embedded in Durcupan ACM (Fluka). Thin sections were stained with uranyl acetate and lead citrate,

Fig. 1. (A) Light microscopic arrangement of AChE-positive nerve fibres in a glomerulus (encircled by dashed line) of the MOB. Bundles of stained structures penetrate the glomerular neuropil, but are restricted to interconnected strands which subdivide the neuropil into small "empty" compartments (stars). Note that enzyme-positive nerve fibres are also absent from the olfactory nerve layer (onl). Scale bar = 40 #m. (B) AChE-positive structures form interconnected strands, but are absent from cores of the chamber-like compartments (stars). AChE-positive strands apparently form shells around sphere-like enzyme-negative neuropil compartments. At the periphery of the glomerulus, an AChE-positive shortaxon cell can be observed (arrowhead). Scale bar = 25 #m.

Cholinoceptive neurons without AChE activity in the MOB

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Fig. 2. Ultrastructural localization of AChE in glomeruli of the MOB. (A) The reaction product surrounding a small unmyelinated nerve fibre (white star) seemingly diffuses away into surrounding intercellular clefts, including those adjacent to a sensory (S) nerve fibre. Scale bar = 0.7 #m. (B) AChE positivity is present on the surface of an unmyelinated axon (white star), which adheres to an unstained dendrite (D). Scale bar = 0.7 #m. (C) AChE reaction product completely surrounds the surface of an unmyelinated axon (white star), from where it diffuses away towards different neuronal processes (large arrowheads) and along a glial cell process (small arrowheads). Scale bar = 0.7 #m. (D) AChE staining is present in intercellular clefts between unmyelinated nerve fibres and a fibrous astroglia process (G). Scale bar = 0.7/~m. (E) AChE positivity completely surrounds the surface of an unmyelinated axon (A) and is present in a tubular endoplasmic structure (arrowhead) inside the axon. Scale bar = 0.7 #m. (F) Higher magnification of the area framed in E. Note the strong AChE staining in the endoplasmic tubule (arrowhead). Scale bar = 1.4 #m. (G-I) In AChE-covered nerve terminals (G, I) and an axon varicosity (H), enzyme-stained vesicles are present (arrowheads). Scale bars = 1.0 #m. (J, K) Within AChE-labelled axon terminals, reaction product within vesicles (arrowheads) is continuous with that present in the extracellular space. Scale bar = 1.0/~m (J); 0.5 #m (K).

RESULTS

Light microscopy The general localization o f A C h E in the rat MOB, already described in detail, 22'23'43 has been largely

confirmed. Here, we draw attention to enzymepositive structures which have not been d e m o n s t r a t e d in earlier studies. In the GL, A C h E - s t a i n e d strands penetrated the periphery o f glomeruli and subdivided the glomerular

Cholinoceptive neurons without AChE activity in the MOB neuropil into small compartments (Fig. 1A). These compartments had central cores, where no ACHEpositive fibres could be detected, and shell, in which most of the stained axons appeared in bundles (Fig. 1B). In the EPL, faintly stained tufted cells and heavily stained short-axon neurons were observed; AChE activity was undetectable in the small periglomerular neurons or in the granule cells. However, large multipolar neurons scattered in the G C L were strongly stained for ACHE.

Electron microscopy Ultrastructurally, A C h E reaction product was localized in intracellular and extracellular distribution patterns of various layers. Extracellularly, the reaction product appeared around the thin unmyelinated cholinergic axons, with some leakage into intercellular clefts between neighbouring structures, irrespective of whether these were sensory nerve fibres (Fig. 2A), dendrites (Fig. 2B), glial cells (Fig. 2C, D), cholinoceptive and/or other types of neuronal and/or non-neuronal perikarya. In stained neurons (mitral cells, tufted cells and short-axon neurons), enzyme positivity was detected in the rough-surface endoplasmic reticulum. Within the cholinergic axons, enzyme staining could be seen inside the tubular structures (Fig. 2E, F), while in axon varicosities and terminals, a few synaptic vesicle-like structures bore the reaction product (Fig. 2G-J). In vesicles near to the axon membrane, reaction product was in continuity with that present in the extracellular space (Fig. 2K).

Distribution o f acetylcholinesterase-positive structures in periglomerular zones Ultrastructural details of the periglomerular zone are depicted in Fig. 3A. Different neuron types (periglomerular cells, short-axon neurons and external tufted cells) were identified by comparing their positions and ultrastructural features with those described in unstained control samples. A C h E positivity was present in perikarya of external tufted cells, while the reaction product was absent from most of the periglomerular cells and from what appeared to be a subset of short-axon cells. A small n u m b e r of juxtaglomerular neurons displayed A C h E activity in the nuclear envelope and within the endoplasmic reticulum (Fig. 3B). Intercellular clefts along the

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surface membranes of juxtaglomerular cells were stained in restricted areas where one or several AChE-positive axons contacted it, regardless of whether the stained axon made a synapse on the soma or "simply" contacted the neuronal perikaryon (Fig. 3C). Heavy reaction end-product was present on the surface of stem dendrites Of some of the relay cells entering the glomerulus. When a cholinergic (ACHEpositive) axon contacted a short-axon cell (Figs 3A, 4) or made synaptic contact with a juxtaglomerular cell (Fig. 3C, D), the presence of the reaction product was evident between the contacting surfaces (Fig. 3C). Although most of the periglomerular cells exerted no AChE activity in perikarya, in rare cases an enzymepositive axon terminated there (Fig. 3C), and in some instances on the dendrite surfaces as well. Ultrastructurally, axon terminals with pleomorphic vesicles, contacting periglomerular cells with or without a synaptic junction (Fig. 3D), did not show A C h E reaction product.

The characteristic arrangement of acetylcholinesterasepositive structures within the glomeruli Within a given glomerulus, the arrangements of AChE-positive and AChE-negative structures displayed two characteristics: (i) the compartments and (ii) the triadic array of neuronal processes at cholinergic synapses. As already demonstrated at the light microscopy level (see Fig. IA, B), the inner glomerular neuropil was subdivided into two types of compartments. One compartment type forms shells around the other. In the shell compartment, AChE-positive structures are present, as well as processes of glial cells and blood capillaries, while chamber-like interior compartments are devoid of these structures. Such an arrangement of the two compartment types, present within a glomerulus, is depicted in Fig. 4. In the core compartment, no AChE-positive structures occur; it is filled with the terminals of sensory olfactory nerves and it contains terminal dendrites of relay cells and periglomerular cells (Fig. 4). In the shell compartment, the neuropil included many AChE-positive structures partly involved in specific arrangements of the neuronal processes, called triads. The triads 26 consisted of (i) a cholinergic axon profile identified by AChE reaction end-product completely surrounding its surface, (ii) a periglomerular cell dendrite and (iii) a relay

Fig. 3. (A) AChE distribution in the periglomerular area. AChE is present in the endoplasmic reticulum of an external tufted cell (ET), while it is absent from a short-axon neuron (S) and from the periglomerular cells (PG). In the upper part of the picture, the processes of the olfactory nerves (ON) are visible, while the EPL begins in the lower part of the picture. Scale bar = 3.2 #m. (B) Two juxtaglomerular cells; one has an AChE-positive label in the nuclear membrane and the endoplasmic reticulum (black star), while reaction product was absent from the other one (open star). Scale bar = 1.0 #m. (C) Along the surface membrane of an AChE-negative periglomerular cell, AChE is restricted to intercellular clefts surrounding an enzyme-positive axon and its synaptic contact (arrow). Scale bar = 1.0#m. (D) AChE staining surrounds a cholinergic axon (arrow) and axon terminal (black star), from where it diffuses into intercellular spaces between an axon terminal with pleomorphic vesicles (open star) and a periglomerular cell (PG). Scale bar = 1.0 #m.

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cell dendrite (Figs 4, 5A, B, D). In these triadic arrangements, relay cell dendrites often synaptically innervated a periglomerular cell dendrite, which was also innervated by an AChE-positive (cholinergic) axon terminal (Figs 4, 5D). In some cases, the AChE-positive axon varicosity was surrounded by several dendritic profiles, one of which was identified as a periglomerular cell dendrite, which received synaptic input from a relay cell dendrite (Fig. 5A). In other sections, the periglomerular cell dendrite was innervated by two relay cell dendrites, but the ACHEpositive axon profile was without a synaptic junction (Fig. 5B). Similar triadic arrangements were seen on the stem dendrites of the relay cells at their entrance into the glomerulus (Fig. 5D). At the periphery of the glomeruli, some of the periglomerular cells were in contact with ACHEpositive axon varicosities (Fig. 5C), and the stems of relay cell dendrites were surrounded by many ACHEpositive axons and/or axon varicosities (Fig. 5E). Distribution o f acetylcholinesterase-positive structures within other layers Triadic arrangements similar to those described in the GL were found in the EPL (Fig. 6A-C). In this case, the triad consisted of (i) an AChE-positive axon (with or without a synapse), (ii) a granule cell dendrite (or other interneuron cell dendrite) and (iii) a relay cell dendrite. The demonstration of synaptic junctions between a cholinergic axon and a granule cell dendrite, as well as between a relay cell dendrite and a granule cell dendrite, was very difficult in a given section (but see Fig. 5D for the glomerulus). However, by careful study of several sections, we were able to detect all the characteristic elements of the cholinergic triads (Fig. 6A-C) in the EPL. ACHEpositive axon varicosities were mostly apposed to dendrites of interneurons without forming synaptic junctions (but see Fig. 6C). Cholinergic axon varicosities were usually separated from relay cell dendrites of the interneurons (Fig. 6D), but they sometimes contacted the shaft of a secondary dendrite of a relay cell (Fig. 6E). Among the secondary dendrites of relay cells, an AChE-positive large dendrite profile was seen that possibly originated from an enzyme-positive short-axon cell (Fig. 6F). It was further noted that only a subset of the short-axon cells were AChE-positive (compare Fig. 6G and H). At the ultrastructural level, AChE-stained endoplasmic reticulum could be seen in several mitral cells and deep tufted cell perikarya (Fig. 7A). Although the AChE-positive fibres and axon varicosities were in close contact with mitral cell perikarya, asymmetric synaptic junctions have never been seen between them (Fig. 7B), although symmetric axosomatic synapses and somatodendritic synapses were found on AChE-positive mitral cell perikarya (Fig. 7C). Light microscopically, intense AChE staining was present in the IPL. Submicroscopically, AChE staining was confined to intercellular clefts, completely

surrounding unmyelinated nerve fibres and axon varicosities, as well as to surface parts of dendrites at sites of apposition to the former (Fig. 7D). In the GCL, a subset of short-axon cell perikarya was also stained. The surface membranes of the short-axon cells and the granule cells were stained only where an AChE-positive axon and/or axon varicosity contacted them (Fig. 7E). In the deepest part of the GCL, a large number of non-cholinergic asymmetric synapses was present, while the number of AChE-stained axon varicosities and synapses was greatly diminished (but see Fig. 7F). DISCUSSION The main findings of the present work are as follows: (i) histochemistry demonstrated the absence of AChE activity from the soma and dendrites of granule cells, while these cells receive cholinergic synaptic innervation; (ii) the presence of AChE within a subset of mitral cells and tufted cells, while cholinergic synapses could not be detected on them; (iii) the presence of AChE-positive and AChE-negative compartments within the glomerular neuropil; (iv) the triadic 26 arrangement of cholinergic axons (the term cholinergic axon is based on the observation 38 that in the human cortex there is a one-toone relationship between axons that are AChE-rich and those that are ChAT-positive, so that AChE-rich axons provide specific markers for cholinergic axons) and non-cholinergic dendritic structures; and (v) the release of ACHE, probably from the cholinergic axon varicosities into the intercellular clefts surrounding them. Our AChE histochemical results on the MOB of the rat are similar to those described in mice, 7 in that a few neurons of various types contained AChE product in all layers of the MOB, with the exception of granule cells. This result is in agreement with that of Le Jeune and Jourdan, 32 who similarly could not demonstrate enzyme activity in the granule cell perikarya. On the basis of the histochemical demonstration of AChE activity, Shute and Lewis 44'45introduced the concept that cholinoceptive non-cholinergic neurons which receive cholinergic innervation may contain AChE on the membranes of dendritic spines and in small or moderate amounts in the cell body, but not along their axons or on axon terminals.4~ Detailed ultrastructural analysis did not reveal AChE positivity in granule cells and in most of the periglomerular cells on which cholinergic synapses were found. 26 In contrast, a subset of external tufted cells lying near the glomerulus did exert AChE activity. Similarly, in the EPL, cells were observed that exerted "cholinoceptive features" (ACHE labelling) without receiving ChAT-positive synapses. 26 It is interesting to note that, in the cerebellum of the rat, some of the mossy fibres are cholinergic and innervate granule cell dendrites where no AChE activity can be demonstrated

Fig. 4. Ultrastructural details of the two types of neuropil compartments within a glomerulus. In those compartments (surrounded by black dashed lines) containing the olfactory sensory axons, no A C h E staining can be detected, while in the neuropil c o m p a r t m e n t devoid o f olfactory axons enzyme-positive structures are seen. In the latter compartment, a triad is present where an enzyme-positive unmyelinated axon (1) is apposed to a periglomerular dendrite (2), which in turn is synaptically innervated by a relay cell dendrite (3). At the bottom left corner, an enzyme-positive short-axon neuron is present. The surface of this neuron is stained only where unmyelinated AChE-positive axons are in close contact with it (arrow). Scale bar = 1.5 # m .

Cholinoceptive neurons without AChE activity in the MOB in the cholinoceptive neuronal perikarya. 23'25'2s Since A C h E is apparently absent from several different cholinoceptive neuron types and A C h E is widespread in the CNS, the suggestion based on A C h E histochemical work that A C h E is a marker for cholinoceptive neurons may need reinterpretation. A C h E is not present in cholinoceptive granule cells in the M O B and eholinoceptive granule cells in the cerebellar cortex, although these neurons receive cholinergie innervation on their perikarya. Thus, it is apparently necessary to suggest an alternative interpretation of the neuronal expression of A C h E and find new criteria for identification of cholinoceptive neurons.

Acetylcholinesterase-positive structures in the different layers" In the different layers of the MOB, cholinergic (AChE-positive) unmyelinated axon varicosities and/or terminals most likely made synapses with interneurons, e.g. some of the periglomerular cells, subpopulations of short-axon neurons and granule cells. 26 However, A C h E activity is absent from granule cells 3z and most, if not all, periglomerular neurons, but it is present in a subset of short-axon neurons, all of which receive cholinergic synapses. 26 In contrast, no direct cholinergic synapses were found on the surface of mitral cells and tufted cells, while A C h E staining was seen in the perikarya of relay cells. On the basis of light microscopic A C h E histochemistry, Nickell and Shipley 39 suggested that the enzyme-positive short-axon neurons in the juxtaglomerular area and in the inframitral region are the most likely candidates for cholinoceptivity. Our ultrastructural immunocytochemical demonstration of C h A T 26 and the ultrastructural localization of A C h E extends their suggestion, i.e. (i) ChAT-positive axons terminate not only on a subset of short-axon neurons, but also on the granule cells, which do not exert A C h E activity; (ii) A C h E activity was not demonstrated on those neurons (AChE-positive neurons in juxtaglomerular and inframitral positions), although the enzyme activity was present inside their perikarya; however, (iii) A C h E activity was always present where stained axons could be revealed, without exception, irrespective of which neuron type (or

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dendrite) was adjacent; (iv) AChE-positive neurons tended not to receive ChAT-positive synapses on their surfaces; and (v) AChE-positive neurons were unevenly distributed in the GL, EPL, IPL and G C L . Our ultrastructural C h A T immunocytochemica126 and A C h E histochemical findings therefore extend the suggestion that the inframitral and juxtaglomerular AChE-positive neurons are the primary sites of cholinergic synaptic transmission in the MOB. Our data suggest that a subset of periglomerular cells and the granule cells receive most of the cholinergic synaptic innervation, and short-axon neurons are not a dominant or even a primary site of cholinergic innervation in the M O B of the rat.

Origin of extracellular acetylcholinesterase Surprisingly, A C h E staining was present in the intercellular space in the vicinity of cholinergic unmyelinated axons, but in their absence did not appear adjacent to neurons with dendritic and perikaryal A C h E staining. This differential distribution raises the question of where the A C h E enters the extracellular space. It has been suggested that the extracellular A C h E may originate from AChE-positive neurotubules, and/or vesicular structures present inside the cholinergic axons 22'27 and cholinergic axon terminals. ~,7'~4This idea is supported by the present ultrastructural localization of the enzyme. In the MOB, cholinergic unmyelinated axons had enzyme-positive vesicles close to the outer membrane, contacted it and even opened towards the extracellular space, forming omega-shaped vesicle profiles. A C h E reaction endproduct within vesicles and in contact with the outer surface of the axons has also been described in the peripheral nervous system 2~'24'27for cholinergic axons in the sciatic nerve, in nerve terminals of the rat superior cervical ganglion t9 and in unmyelinated axons running between the chromaffin cells. 47 Similarly, the release and/or the secretion of A C h E from dendrites 3°'35 of non-cholinergic neurons and from the perikarya of non-cholinergic cells 47 has been documented by neurochemical and ultrastructural morphological means. A C h E release is Ca 2+ dependent and may involve an exocytotic process. 9 Thus, release of A C h E from

Fig. 5. (A) The AChE-covered axon terminal (1) is surrounded by several dendritic profiles. One of these dendrites (2) is innervated by a relay cell dendrite (arrow). Note the absence of AChE staining among olfactory nerves (ON). Scale bar = 1.0 ~m. (B) In a triad, note the AChE-covered unmyelinated axon (1) in contact with a periglomerular cell dendrite (2), which is synaptically innervated (arrow) by a relay cell dendrite (3). No staining is present in and around olfactory nerve fibres (ON). Scale bar = 1.0 #m. (C) AChE positivity is present in the nuclear membrane of a periglomerular cell. Note the presence of AChE staining around the surface membrane at a restricted part of the cell, where a stained axon terminal adheres (white asterisk). Scale bar = 1.0/~m. (D) This characteristic triadic arrangement was located at the entrance of a glomerulus. An unmyelinated cholinergic axon (1) innervates (small arrow) a periglomerular cell dendrite (2), which also receives synaptic input (large arrow) from a relay cell dendrite (3). Scale bar ~ 1.0 #m. (E) Intercellular clefts around primary dendrites of two relay cells (black stars) are filled with AChE reaction product, where AChE-positive unmyelinated axons adhere (arrows), while some of the stained intercellular clefts are separated from the dendritic surface by glial cell processes. Scale bar = 1.0 pm.

2

Cholinoceptive neurons without AChE activity in the MOB axons of cholinergic neurons and from dendrites of neurons which produce this enzyme without being cholinergic is apparently a widespread phenomenon in the central and peripheral nervous systems. It has been established 6 that Ca 2+ and Ca2+-transporting ionophores can stimulate protein secretion in many cellular systems. The increased intracellular Ca 2+ concentration can increase A C h E secretion. In the CNS, the release and/or secretion of A C h E from various central non-cholinergic neurons has been demonstrated by biochemical, electrophysiological and morphological methods. 3'5A1'16'2°'21'36'5°In peripheral cholinergic fibres, such release has been studied in Auerbach's plexus. 4 Morphological techniques do not allow differentiation between stimulated and/or spontaneous release, or between release from and/or uptake of A C h E into axons or dendrites. However, reduction or elimination of C h A T and A C h E activity from the M O B has been induced by electrolytic lesions in the basal forebrain of the hamster 37 and the rat (our unpublished observation). This firmly supports the interpretation that, in the olfactory bulb, A C h E may originate from the centrifugal cholinergic axon projections to the MOB. Demonstration of vesicles containing the A C h E reaction product in continuity with the extracellular staining may be due to secretion rather than an uptake of A C h E into the nerve fibres, because the incorporation of A C h E into synaptic vesicles has been shown to increase with the blockade of synaptic transmission. 4° Dickie et alJ ° presented

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experimental evidence that only the neurons in the substantia nigra were able to take up A C h E from the extracellular space. Nevertheless, reuptake of small amounts of released A C h E cannot be ruled out, since the bidirectional transport of A C h E in the cholinergic axons is well documented. Whether countercurrent transport of A C h E also occurs in cholinergic axons projecting to the M O B needs further investigation. Functional implications

Although A C h E plays an important role in cholinergic neuronal transmission, non-cholinergic functions of the enzyme ~'2'8'~5'~7'~8'46may exert noncholinergic effects on membrane channels and thus influence certain types of membrane conductance, enhance excitatory amino acid transmission and hydrolyse peptides. 8 Secretion of the enzyme is related to locomotor activity, and modulates the release of dopamine in the nigrostriatal pathway. TM Conversely, a significant decrease in the catalytic activity of the enzyme occurred in the presence of dopamine. 29 Since A C h E and dopamine are co-released from neurons undergoing degeneration in Parkinson's disease, the significance of this molecular interaction has been emphasized 29 in normal functioning and in pathological states. Vizi et al. 5~'52 provided evidence that axonal release of acetylcholine could occur. The function of A C h E may then involve an anticholinoceptive defence mechanism against the overexcitation caused by acetylcholine release and autoreceptors along cholinergic nerve fibres.

Fig. 6. (A~C) Triad-like array in the EPL. Note the characteristic sequence of structures: AChE-positive axon varicosity or terminal (1) in adhesion (A, B) or in a synaptic junction (C) with a granule cell dendrite (2), which is innervated by a relay cell dendrite (3). Scale bars = 1.0/am. (D) An AChE-positive axon varicosity (1) is separated from the relay cell dendrite (black star) by a presumptive process of an interneuron. Note weak AChE staining around the relay cell dendrite near to the positively stained axon. Scale bar = 1.0/am. (E) AChE staining around a relay cell dendrite (black star) is restricted to sites of apposition with unmyelinated cholinergic axons (arrow). No reaction end-product can be detected where the relay cell dendrite innervates (open arrow) what appears to be a granule cell dendritic process. Scale bar = 1.0/am. (F) AChE-positive short-axon cell dendrite (white star) is located between two relay cell dendrites (black stars). One of these dendrites innervates a granule cell dendritic process (straight arrow), while the other is in reciprocal synaptic contact (curved arrows) with the granule cell dendrites. Scale bar = 1.0/am. (G) AChE-negative short-axon neuronal perikaryon located in the EPL (black star) is in synaptic contact with a stained unmyelinated nerve fibre (arrow). Scale bar = 1.0/am. (H) AChE-positive short-axon neuronal perikaryon located in the EPL (black star), which is innervated (see enlargement at the left upper corner) by what might be a granule cell gemmule (arrow). Note the presence of enzyme staining in the rough surface endoplasmic reticulum in the perikaryon. Scale bar = 0.5/am; insert = 0.2/am. Fig. 7--overleaf. Fig. 7. (A) AChE staining within the perikarya of a deep tufted cell (black star) and a mitral cell (open star). Scale bar = 1.0 #m. (B) AChE-positive nerve fibre in close contact with an AChE-negative relay cell perikaryon. Although several axon varicosities (arrows) can be detected on the surface of the relay cell, none of them innervates the mitral cell. Scale bar = 1.0/am. (C) AChE-positive relay cell perikaryon (empty star) possibly innervated by a granule cell dendrite (arrow) and by a large axon varicosity (black triangle), where symmetric axosomatic synapses are present. Scale bar = 1.0/am. (D) Intense AChE staining (outlined by dashed lines) in the IPL is restricted to the surfaces of unmyelinated nerve fibres and axon varicosities. Scale bar = 1.0/am. (E) No AChE staining is visible within the granule cell perikaryon, but reaction product is present where axon varicosities occur either at a distance (white star) or at the surface (arrow) of the granule cell. Scale bar = 1.0/am. (F) Note the presence of an AChE-positive unmyelinated axon terminal which innervates a granule cell dendrite (open arrow) in the deep part of the GCL. There are two AChE-negative axon terminals, which possibly innervate (black arrows) granule cell dendrites. Scale bar = 1.0/am.

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Cholinoceptive neurons without AChE activity in the MOB T h e olfactory system is seriously affected in neurodegenerative disorders 12 a n d in Alzheimer's disease; 13 in particular, large a n d efferently projecting mitral cells degenerate. 48 It has even been suggested t h a t A l z h e i m e r ' s disease m a y begin in the " n o s e ''42 a n d lead to a n i m p a i r m e n t of olfactory f u n c t i o n w h e n elements of cholinergic n e u r o n a l t r a n s m i s s i o n are significantly reduced. Also, loss of A C h E from the M O B has been suggested to play a role in the initial stages of the disease. 12 In the rat M O B , cholinergic

843

synapses have n o t been f o u n d o n the soma a n d dendrites o f the relay cells (mitral a n d tufted cells), a l t h o u g h A C h E is present in the endoplasmic reticulum, suggesting t h a t A C h E is n o t related to cholinergic t r a n s m i s s i o n in these neurons. Acknowledgements--We are grateful to Mr R. Dungan and

Mrs Klara Zsidai Galgoczy for their expert photographic work. This work was supported by grants from the Hungarian Research Fund (OTKA; Grant 2723), the ETT, Hungary (Grant T-123 and T-04,602/93), the MKM 279 and the DFG, Germany (Wo279/8-4).

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