Synaptic and non-synaptic cholinergic innervation of the various types of neurons in the main olfactory bulb of adult rat: Immunocytochemistry of choline acetyltransferase

Neuroscience Vol. 67, No. 3, pp, 667-677, 1995

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SYNAPTIC A N D NON-SYNAPTIC CHOLINERGIC INNERVATION OF THE VARIOUS TYPES OF N E U R O N S IN THE MAIN OLFACTORY BULB OF A D U L T RAT: IMMUNOCYTOCHEMISTRY OF CHOLINE ACETYLTRANSFERASE P. K A S A , * t I. H L A V A T I , * E. DOBO,* A. W O L F F , * F. JOO~ and J. R. W O L F F § *Department of Neurology and Psychiatry, Alzheimer's Disease Research Unit, Albert Szent-Gy6rgyi Medical University, H-6720 Szeged, Hungary :~Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged0 Hungary §Department of Anatomy, University of G6ttingen, D-3400 Gfttingen, Germany Abstract--The cholinergic neuronal structures and their synaptic connections in the main olfactory bulb of adult rats were analysed by using choline acetyltransferase immunocytochemistry. Within the glomeruli, cholinergic nerve fibers were restricted to strands which subdivided the neuropil into small compartments, the interior of which contained sensory axons but was devoid of cholinergic axons. Small numbers of choline acetyltransferase neurons were detected in all layers. Ultrastructural analysis revealed selective triadic synaptic relationships with different neuron classes in the intraglomerular area and in the external plexiform layer. These triads were made up of (i) a cholinergic axon, (ii) one or several periglomerular or granule cell dendrites, and (iii) usually one relay cell dendrite. In these triads, asymmetric cholinergic synapses were selectively focused on dendrites (gemmules and spines) of periglomerular or granule cells. Within the glomerulus, mitral and tufted cell dendrites were closely apposed to some cholinergic axon varicosities, most abundantly near arborizations of the apical dendrites. However, cholinergic synapses were never seen on any relay cell dendrite. In the external plexiform layer, cholinergic synapses were present on all parts of the superficial short-axon cells. In the internal plexiform layer and the granule cell layer, cholinergic axon varicosities exhibited close apposition or asymmetric synapses with granule cell gemmules. The data suggest that cholinergic projections from the basal forebrain to the main olfactory bulb focus synaptic innervation on interneurons. On relay cells, direct acetylcholine effects may occur, but these must be based on non-synaptic acetylcholine release at the surface of their dendrites.

The cholinergic system (acetylcholine, ACh; choline acetyltransferase, E.C. 2.3.1,6, CHAT; acetylcholinesterase, E.C. 3.1.1.7, ACHE; nicotinic acetylcholine receptors, n A C h R ; and muscarinic acetylcholine receptors, m A C h R ) plays a significant role in modulating neuronal transmission, learning and memory functions 3'7 and neuronal plasticity 2'~9in various parts of the nervous system. In the main olfactory bulb (MOB), sensory information processing is controlled by various transmitters; 5'23'31'3z43'*~ the A C h system apparently plays a key role in this mechanism. 6'27'3~ It has also been shown that in Alzheimer's disease the olfactory sense is impaired, TM as may be the cholintTo whom correspondence should be addressed. Abbreviations: ACh, acetylcholine; ACHE, acetylcholin-

esterase; CHAT, choline acetyltransferase; DAB, 3,3'-diaminobenzidine; IR, immunoreactivity; mAChR, muscarinic ACh receptors; MOB, main olfactory bulb; nAChR, nicotinic A C h receptors; NBS, normal bovine serum; ON, olfactory nerve; PAP, peroxidase~ antiperoxidase; PBS, phosphate-buffered saline; TB, Tris-HCI buffer; TBS, TB containing 0.9% sodium chloride.

ergic forebrain system. Thus, the cholinergic system may serve various physiological and pathophysiological functions in the olfactory bulb. The presence of ACh, CHAT, ACHE, n A C h R and m A C h R in the M O B has been well documented by neurochemical and morphological means. 1'4's'17'34'35'39'46At the lightmicroscopic level, the ACh-synthetizing enzyme C h A T has been demonstrated in axons passing through all layers of the MOB. 14'17"29Since there has been no report on the ultrastructural localization of this enzyme, except in the atypical glomeruli, 16 a p r o o f of cholinergic synaptic transmission in the MOB, therefore, is still missing. This paper endeavors to provide ultrastructural immunocytochemical evidence for the structural organization of the cholinergic innervation in the MOB.

EXPERIMENTAPLROCEDURES Tissue preparation

Adult Wistar rats weighing 300-350 g were used in this study. The animals were transcardially perfused under ether anesthesia. Blood was first removed from the vascular 667

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system with 80-100 ml 0.9% sodium chloride buffered with 0.01 M sodium phosphate (PBS), at pH 7.4; the brain was then perfusion-fixed with 0.1 M PBS, pH 7.4, containing 4% freshly prepared formaldehyde. Thereafter, the fixative was removed by perfusion with 150ml PBS. The hydrostatic perfusion pressure was about one meter. The brains were dissected, and the hemispheres were immersed in 30% sucrose in 0.05 M Tris-HC1 buffer (TB), pH 7.6, overnight at 4°C. Frozen sections were cut coronally at a thickness of 30 p m and collected in 0.1 M TB containing 0.9% sodium chloride (TBS).

Light-microscopic immunocytochemistry Tissue specimens were processed according to the peroxidase-antiperoxidase (PAP) technique of Sternberger. 4° For light-microscopic investigations, sections were pretreated with 10% Triton X-100 in TBS for 1 h, and with a solution containing 50% normal bovine serum (NBS) and 50% TBS for 1 h at ambient temperature. Subsequently, the samples were incubated overnight in the following solutions: (l) rabbit antiserum raised against human placental ChAT (AB143; Chemicon, Temecula, CA) diluted 1/600-1/1,200 overnight at room temperature; (2) or goat anti-rabbit IgG (Nordic, Tilburg, The Netherlands) diluted 1/30; (3) or rabbit PAP (Nordic) diluted 1/400. The immunoreagents were diluted in the following solutions: (1) primary antibodies: 3% Triton X-100+ 20% N B S + 77% TBS, supplemented with 0.1% sodium azide; (2) linking antibodies: 20% normal rat serum + 20% NBS + 60% TBS; (3) PAP complex: 20% NBS + 80% TBS. The tissue-bound peroxidase was visualized in a solution containing 0.05% 3,Ydiaminobenzidine-4HC1 (DAB, Sigma), 0.15% nickel chloride and 0.005% H202 in TBS (pH 7.6) for 15 min. After each incubation step, sections were washed with three changes of TBS for 15 min each, then mounted on glass

slides, air-dried, dehydrated and finally enclosed in Entellan (Merck).

Electron-microscopic immunocytochemistry Without Triton pretreatment, samples were cryoprotected with 30% sucrose in 0.1 PBS (pH7.4), frozen in liquid nitrogen, sectioned on a freezing microtome at 30-40/zm thickness and processed for immunocytochemistry as described above. The sections were next treated with osmium tetroxide (1.0%) for 30 min, washed in two changes of PBS, dehydrated in alcohol, followed by isopropanol, and fiatembedded in Epon 812. After polymerization at 56°C for two days, single or serial ultrathin sections were cut (Reichert Ultracut S), placed on Formvar-coated single-hole copper grids and double-stained with uranyl acetate and lead citrate. Sections were investigated with a Zeiss EM 10 at 80 kV.

Control experiments Intermittent sections were processed as described above, but the primary ChAT antibodies were replaced by normal rabbit serum at the same concentrations. In these sections, no immunolabeling was observed. RESULTS

Light microscopy C h A T - i m m u n o r e a c t i v i t y ( C h A T - I R ) was f o u n d in varicose fibers a n d nerve cell bodies, which were located in all layers of the M O B except the olfactory nerve layer, where n e u r o n s were never observed, a n d very occasionally a terminal part Of a C h A T - I R nerve fiber was seen near a glomerulus. The highest density

Fig. 1. (A) Light-microscopic distribution of the ChAT-positive nerve fibers in the MOB. The highest density of stained axons is present in a profile of a 'modified' glomerulus (arrow). Note that differential ChAT-staining intensity can also be revealed inside all other glomeruli (stars) which show a higher density of cholinergic axons in periglomerular zones and irregular patterns in the glomerular neuropil. Scale bar = 50 #m. (B) Typical distribution of ChAT-positive fibers and varicosities within a glomerulns. Note the presence of small compartments completely devoid of any ChAT-IR structures (stars). Scale bar = 10 #m. (C) ChAT-positive axons and terminals accumulate at knot-like points (arrows) which may correspond to branching points of relay cell dendrites or overcrossing points of several dendrites (arrowhead). Scale bar = I0/~m. (D) Heavy ChAT staining is present in a "modified" glomerulus. At higher magnification than in Fig. 1A, empty compartments can be seen (star), as in other glomeruli. In the periglomerular zone a ChAT-positive neuron (white arrow) can be observed, which sends its dendrites into the periphery of the glomerulus and shows a stub of what appears to be the axon (black arrow). Scale bar = 25/~m. (E) This ChAT-IR neuron (arrow) is located in the periglomerular area. Its main dendrites run and branch in the periglomerular zone. A side-branch (arrowhead) of one dendrite enters the glomerular neuropil (star). Scale bar = 25/~m. (F) Laminar distribution of ChAT-positive fibers in the MOB. Note that the overall staining density is highest in the glomerular layer (gl), especially in septa between chamber-like compartments devoid of ChAT-IR fibers (e: external plexiform layer; m: mitral cell layer; i: internal plexiform layer; gr: granule cell layer). In the granular cell layer, a ChAT-positive neuron is present (arrow), which is enlarged in the insert. Scale bars = 100 #m; insert, 25 pm. Fig. 2. (A) Survey electron micrograph of a glomerular neuropil compartment that consists of a "core" rich in sensory axons (bottom right corner) and a "periphery" that contains many dendritic profiles and almost all ChAT-IR axon profiles (upper left corner). Note the triadic arrangement in the peripheral part of the intraglomerular compartment. The periglomerular cell dendrite (open star) is in close contact with a cholinergic axon terminal (arrow) and is innervated by a relay cell dendrite (black star). The overall distribution of cholinergic axon terminals and/or varicosities (arrows) is also revealed. Scale bar = 1.0 pm. (B) ChAT-positive terminals contacting one of the periglomerular cell dendrites (open star). One of the stained fibers makes an asymmetric synaptic junction (arrow) with this dendrite. In the lower half of the picture, a relay cell dendrite (black star) makes a dendrodendritic synapse (arrowhead) with a periglomerular cell gemmule (open star). Scale bar = 1.0 pm. (C) A ChAT-IR axon varicosity (arrow) makes close contact with a relay cell dendrite (star), but no synaptic junction can be seen. Scale bar = 1.0 #m. (D) ChAT-IR axon varicosities (arrows) are shown in the peripheral part of a glomerular neuropil compartment. One of these terminals contains synaptic vesicles and a synaptic junction (arrowhead) with a gemmule on a periglomerular cell dendrite. Scale bar = 1.0 p.m.

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Synaptic and non-synaptic cholinergic innervation in the MOB of stained fibers occurred in the "atypical" glomeruli ~>8 along the border of the MOB and the accessory olfactory bulb, in the ventrolateral bulbar area and ventrally close to the tip of the glomerular layer (Fig. 1A). In the MOB, the staining intensity varied in the different layers. In the glomerular layer, ChAT-IR fibers wove dense nets around the periglomerular cell bodies. From there, stained axons with varicosities entered the glomeruli. Within the glomerular neuropil, cholinergic axons were usually varicose and extremely thin. These axons appeared restricted to strands or sheath-like structures which surrounded neuropil compartments of variable size, where no ChAT-IR fibers could be demonstrated (Fig. 1B). At some points within these strands, small spherical structures appeared, richly surrounded by ChAT-IR varicosities forming knots, from where the stained fibers radiated in different directions. Such arrangements of the ChAT-IR fibers were suggestive of large dendritic profiles (possibly mitral cell and/or tufted cell) stem dendrites, which were richly innervated at their branching points (Fig. IC). Along thinner dendrites, the innervation was less pronounced. At overcrossing points of several dendritic branches, a similar accumulation of stained punctata was present. One of the most striking distribution patterns of cholinergic fibers in the MOB was their subdivision of the inner part of the glomerulus into compartments that could be revealed at a light microscopic level (Figs 1B, C). The actual size of these compartments varied not only from glomerulus to glomerulus, but also within a given glomerulus. The "core" of these compartments contained the terminal branches of the olfactory sensory axons, and the tufts of the relay (mitral and tufted) cell dendrites. These territories were devoid of cholinergic axon terminals. As a result of this cholinergic fiber distribution pattern, the compartmentalization of the glomeruli was very obvious in the rat MOB stained by means of ChAT-immunohistochemistry. ChAT-1R axons and punctate structures also accumulated near neuronal cell bodies within the periglomerular region, on some large dendrities and around some parts of the blood vessels. In other parts of the MOB, ChAT-1R fibers formed a relatively loose and diffuse network that did not show any preferential association with specific neuronal elements. Most of the ChAT-IR nerve fibers were thin, bore varicosities at variable distances and branched several times. Other cholinergic axons were thicker, did not show ramifications and ascended up to the glomerular layer. ChAT-IR fibers or punctatalike structures were relatively sparse in the external plexiform layer and granule cell layer as compared with the glomerular and/or internal plexiform layers. Some ChAT-IR neurons occurred in juxtaglomerular positions (Fig. 1D, E), and their somata were spherical or slightly ovoid, but usually larger than those of the periglomerular cells. The dendrites of these cells

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either ran between two glomeruli, or surrounded a glomerulus with a clamp-like formation of two dendrites, from which side-branches arose and penetrated the glomerular neuropil (Fig. 1E). Axons of these ChAT-positive cells could not be discerned from other stained fibers, but initial segments of axons were seen on such neurons, e.g. within an atypical or modified glomerulus (see Fig. 1D). A few ChAT-IR neurons were also present in the granule cell layer, where their dendrites could be followed for a long distance (Fig. I F and insert).

Electron microscopy Distribution of choline acetyltransferase-positive fibers in the glomerular layer. In the MOB, the periglomerular region receiving the highest density of cholinergic fibers also contained a large number of ChAT-IR varicosities and synapses. On the basis of ultrastructural criteria, two different types of neuronal perikarya were identified in this region: the periglomerular cells and short-axon cells, both of which received cholinergic innervation. The typical arrangement in glomeruli of ChAT-IR axon terminals, synapses and triadic arrangement of neuronal structures is depicted in Fig. 2A. The vast majority of stained fibers were intermingled with dendritic profiles in parts of the neuropil which also included glial processes and blood capillaries. Only rarely were ChAT-IR axons found between sensory axon terminals, and axoaxonic synapses never occurred between sensory and cholinergic axon terminals. In the ~'periphery" of the glomerular neuropil compartments, periglomerular cell dendrites received asymmetric synapses from cholinergic axon terminals or varicosities (Fig. 2B, D). Relay cell dendrites were also approached by immunopositive axon varicosities, but they never made synaptic contacts (either asymmetric or symmetric) with cholinergic axons (Fig. 2C).

Spec(fic arrangement of choline acetyltransJbrasepositive axon varicosities and synapses within the glomeruli. Most of the cholinergic axons, varicosities and synapses were seen in the periglomerular region. The number of immunostained structures gradually decreased towards those territories within glomeruli to which sensory fibers and their terminals were confined. In the periglomerular area, most of the synapses were situated on pale dendritic profiles containing large round vesicles (Fig. 3A, B). Two asymmetric synapses sometimes occurred on a dendritic spine, one of which may be ChAT-positive (Fig. 3C). Near the periglomerular zone, but inside the glomerulus, a ChAT-IR axon terminal was found to make an asymmetric synapse with a CHATpositive dendrite (Fig. 3D). This finding suggested that dendrites of cholinergic cells in the juxtaglomerular position may contribute to the glomerular neuropil and may be innervated by cholinergic axons. In the "peripheral" parts of the neuropil compartments, some dendritic profiles received several asymmetric synapses, one of which originated from a sensory

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afferent fiber, and another one from a centrifugal cholinergic axon; these junctions were at times adjacent to dendrodendritic synapses (Fig. 3E). In the periglomerular zone, many ChAT-positive axon terminals and/or varicosities, and synapses (Fig. 3F) were seen. Asymmetric axosomatic synapses could also be detected on the soma of short-axon cells, but very seldon (Fig. 3G). Some of the stained C h A T - I R varicosities were likewise found in close contact with the somata of the periglomerular cells (Fig. 3H). Cholinergic asymmetric synapses were never found on mitral and tufted cell dendrites.

Distribution of choline acetyltransferase-positive structures within other layers. In all layers, the most c o m m o n feature of the cholinergic fibers was that they were thin and most of the C h A T - I R varicosities apparently did not form synapses. In the external plexiform layer, a number of ChAT-positive varicosities were most c o m m o n l y found near gemmules of granule cells (Fig. 4A, E). These axons ran parallel to the secondary dendrites of mitral cells (Fig. 4A), and were sometimes seen even in close apposition to them (Fig. 4B). However, ChAT-positive synapses were present only on gemmules (Fig. 4C) and/or on dendritic spines (Fig. 4D) of the granule cells, but never on relay cell dendrites. A characteristic triadic arrangement among different neuronal structures ( C h A T - I R axons, granule cell gemmules and a mitral cell secondary dendrite) was revealed (Fig. 4E). Because of local variations, it was difficult to determine a general distribution pattern of cholinergic axon varicosities and cholinergic synapses within the external plexiform layer, but the impression emerged that there were more cholinergic structures in the deep part of this layer than in its superficial level. Many

C h A T - I R varicosities were also present in the internal plexiform layer and in the upper part of the granule cell layer. In the latter, asymmetric synapses were revealed on gemmules and spines of the granule cell dendrites (Fig. 4F, G). However, axosomatic cholinergic synapses were not observed, although C h A T - I R axon varicosities were sometimes in contact with granule cell bodies. DISCUSSION The overall synaptic organization of the M O B is well known. H'25'36 To date, however, only nerve fibers 14'17'29 and cells 17'29'38 immunoreactive for C h A T have been described in the M O B at the light microscopic level. There have been some reports on the distribution of C h A T - I R neurons, 29,3° but other authors did not confirm the presence of C h A T - I R neurons in the MOB. 15'2°'26 The present report is the first to demonstrate the ultrastructural distribution pattern of C h A T - I R nerve fibers, axonal varicosities, axon terminals and their synaptic and non-synaptic relationship with various cell types in different M O B layers of adult rats.

Cholinergic synaptic organization in the glomerular layer Histochemica137 and immunocytochemical morphological 24,47 studies have indicated both cholinergic projections from the basal forebrain and periglomerular cells. 17'29'3° Electrophysiological experiments have dmeonstrated that the periglomerular cells may play a key role in input processing as excitatory and inhibitory modulators in the first stage of synaptie integration in the olfactory pathwaysY

Fig. 3. (A, B) Two neighboring ultrathin sections show that the cholinergic synaptic contact zones (arrows) may be of considerable size. Comparable parts of the sections are labeled (white stars). Scale bar = 0.5 #m. (C) ChAT-positive fiber (arrow) and non-stained axon terminal (arrowhead) making a synaptic contact on the same dendritic profile. Scale bar = 0.5/~m. (D) A ChAT-positive terminal (white star) making an asymmetric synaptic junction with a dendrite that is also ChAT-immunoreactive (black star). Scale bar = 0.5 #m. (E) An asymmetric synapse between a cholinergic axon and what appears to be a periglomerular cell dendrite (white star). Synaptic junctions made by the cholinergic axon (arrow) and peripheral olfactory nerve fiber (arrowhead) are observed on the dendrite. Scale bar = 0.5 #m. (F) A cholinergic asymmetric synapse (arrow) on a small dendritic profile in the periglomerular area. Scale bar = 0.5 pm. (G) A cholinergic asymmetric synapse (arrow) on the soma of a short-axon cell in the juxtaglomerular position. Scale bar = 0.5/~ m. (H) A cholinergic axon varicosity (white star) in non-synaptic contact with a periglomerular cell body. Scale bar = 0.5 #m. Fig. 4. (A) ChAT-positive axon terminals (arrows) located near a relay cell dendrite (black star) in the external plexiform layer. One of them is surrounded by granule cell gemmules (white stars). Scale bar = 0.5 #m. (B) A ChAT-IR axon terminal (arrow) with spherical vesicles in close apposition to a relay cell dendrite (star) in the external plexiform layer. Scale bar = 0.5/~m. (C) A ChAT-positive axon terminal (arrow) making a synaptic contact with a granule cell gemmule in the external plexiform layer. Scale bar = 0.5 # m. (D) A ChAT-positive axon terminal in the external plexiform layer, making an asymmetric synaptic contact (arrow) on a spine of a granule cell dendrite. Scale bar = 0.5/~m. (E) Triadic arrangement in the external plexiform layer. Note the presence of a ChAT-positive terminal making an asymmetric synapse (arrow) on the dendrite gemmule of a granule cell. Two other gemmules (arrowheads) are innervated by a relay cell dendrite (star). Scale bar = 0.5/tm. (F) A ChAT-positive axon terminal (star) making an asymmetric synaptic contact on a spine of a granule cell dendrite in the granular cell layer. Scale bar = 0.5 12m. (G) A ChAT-positive axon terminal in contact with a cell body of a short-axon neuron (star) in the granule cell layer. Note the presence of a cholinergic asymmetric synapse on the GC dendrite spine (arrowhead). Scale bar = 0.5 # m

Synaptic and non-synaptic cholinergic innervation in the MOB

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Fig. 4

Synaptic and non-synaptic cholinergic innervation in the MOB The present results show that the periglomerular cells not only receive asymmetric (excitatory) synapses from the peripheral olfactory nerve (ON), and dendrodendritic synapses from relay cells, but are also synaptically innervated by central cholinergic projections from the basal forebrain. In this way, these glomerular interneurons may be involved in the modulation of sensory signal processing due to arousal. In contrast, relay cell dendrites within the glomerulus receive excitatory input from the ON, and inhibitory input 25 from the periglomerular cells, but cholinergic projections apparently do not make synaptic junctions with them. The ChAT-IR axon terminals and/or varicosities may aggregate around intraglomerular branching points of relay cell dendrites. This finding suggests that these points may be under firm synaptic inhibitory control by periglomerular cells, but may additionally receive nonsynaptic excitatory cholinergic centrifugal control. This may also hold true for cholinergic axon varicosities establishing a non-synaptic relationship with other parts of apical dendrites of relay cells. Cholinergic axon terminals having synapses on periglomerular dendrites, dendritic gemmules and spines, however, would facilitate the inhibitory effect on the relay cells. Neuropharmacological results suggest that ACh can be released along unmyelinated axons. 44'45 If this holds for cholinergic fibers in the MOB, excitatory effects of sensory neurons may be facilitated by ACh non-synaptically released from axon varicosities which are firmly attached to the relay cell dendrites. This non-synaptic ACh release within the glomerulus may also be responsible for the prolonged excitation, as has been demonstrated in the olfactory bulb after the blockade of synaptic inhibition. 2~ Thus, cholinergic forebrain projections may have a dual function in the glomeruli, depending on the actual conditions for information processing. Future electrophysiological experiments may prove or disprove the validity of this suggestion.

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Our immunocytochemical results show the presence of ChAT-IR asymmetric synapses on the perikarya, dendrites and dendritic spines of short-axon neurons, which are located near the glomerular region. Therefore, at least a subpopulation of these neurons may be under the effective control of ACh. Since the axons of short-axon cells form synapses with the granule cell dendrites, 25 which are both inhibitory, the final effect of cholinergic projections mediated by this cascade will be a drive of disinhibition of relay cells. Such a circuit has already been suggested on the basis of electrophysiological experiments) 6 Depending on the balance of cholinergic effects on short-axon neurons and granule cells, disinhibitory or inhibitory effects may dominate on the respective relay cells. In addition, inhibitory granule cells are surrounded by noradrenalinecontaining terminals, 5 and it is suggested that the effect of applied noradrenaline on granule cells is ," it

ON

"• %~

GL

EPL

MCL

Cholinergic.fibers in the other layers

IPL ] M,

Below the glomerular layer, cholinergic axon varicosities and terminals were predominantly located near gemmules and/or dendritic spines of granule cells, while few were seen in direct apposition to the surfaces of relay cell dendrites. The ratio between ChAT-IR profiles representing axon varicosities and those including synapses was about 50: 1. Because of tilting problems, we did not recognize all the cholinergic synapses present in the different layers, but the large number of varicosities suggest that in this part of the MOB too, both synaptic and non-synaptic relationships to dendrites may occur. Indeed, in electrophysiological experiments the microapplication of ACh revealed variable effects, 2tm which may be explained by differential distributions of nAChR and mAChR, 9A322'33but also by the different locations of cholinergic axon varicosities and cholinergic synapses.

Fig.' 5. Schematic representation of the proposed cholinergic synaptic and non-synaptic relations between cholinergic axons and interneurons, and non-synaptic interactions with relay cells in the MOB. The characteristic triadic relationship involving the different neuron processes within the glomerulus and in the EPL is depicted by the shadowed area. GL, glomerular layer; EPL, external plexiform layer; MCL, mitra/tufted cell layer; IPL, internal plexiform |ayer; GCL, granule cell layer; dG, deep granule cell; sG, superficial granule cell; M/T, mitral/tufted cell; ON, olfactory nerve; P, periglomerular cell; cP, cholinergic periglomerular cell; SA, short-axon cell; sT, superficial tufted cell. The cholinergic structures [cholinergic axon (--), cholinergic synaptic contact (--*), cholinergic non-synaptic contact (-@) and the cholinergic periglomerular cell] are represented in solid black.

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excitation 23 (but see Ref. 42). Both projection systems may then interact or cooperate in affecting relay cell activity. A schematic representation of the proposed cholinergic synaptic and non-synaptic relations between cholinergic axons and interneurons and nonsynaptic interactions with relay cells in the M O B is shown in Fig. 5. Functional implications f o r the cholinergic projection system

This immunocytochemical study has demonstrated that C h A T - I R synapses are mainly present on those neurons (periglomerular and granule cells) in which A C h E activity can rarely be revealed either in dendrites or in perikarya. Since most of the AChE-positive cells (short-axon cells in the external plexiform layer and granule cell layer, mitral cells and tufted cells) are not the targets for the ChAT-positive

synapses in the MOB, we consider that the classical idea that "cholinoceptive structures" are ACHEpositive can no longer be regarded as valid; at least, not in the MOB, where this has previously been suggested. 26 We have already suggested that A C h E may have other functions than the hydrolysis of ACh during development ~8 or in adult animals (for references, see Ref. 10). Physiological data are now urgently needed to improve our understanding of the presence of cholinergic synapses on those neurons where A C h E activity cannot be demonstrated in the perikaryon and dendrites, and in this manner the criteria of "cholinoceptive" neurons are not fulfilled. Acknowledgements--We are grateful to Mr R. Dungan for his expert photographic work. This work was supported by the DFG, Germany (Grant 279/8-4), and by the Hungarian Research Fund (OTKA: Grants 915 and 2723 and ETT, (Grants T-123 and T04,602/93) Hungary.

REFERENCES

1. Armstrong D. M., Saper C. B., Levey A. I., Wainer B. H. and Terry R. D. (1983) Distribution of cholinergic neurons in rat brain: demonstrated by the immunocytochemical localization of choline acetyltransferase. J. comp. Neurol. 216, 53-68. 2. Bear M. F. and Singer W. (1986) Modulation of visual cortical plasticity by acetylcholine and noradrenaline. Nature 320, 172-175. 3. Collerton D. (1986) Cholinergic function and intellectual decline in Alzheimer's disease. Neuroscience 19, 1-28. 4. Cuello A. C. and Sofroniew M. V. (1984) The anatomy of the CNS cholinergic neurons. Trends Neurosci. 7, 74 78. 5. Dahlstr6m A., Fuxe K., Olson and Ungerstedt U. (1965) On the distribution and possible function of monoamine nerve terminals in the olfactory bulb of the rabbit. Life Sci. 4, 2071 2074. 6. Elaagouby A., Ravel N. and Gervais R. (1991) Cholinergic modulation of excitability in the rat olfactory bulb: effect of local application of cholinergic agents on evoked field potentials. Neuroscience 45, 6534562. 7. Ferreyra-Moyano H. and Barragan E. (1989) The olfactory system and Alzheimer's disease. Int. J. Neurosci. 49, 157 197. 8. Fibiger H. C. (1982) The organization and some projections of cholinergic neurons of the mammalian forebrain. Brain Res. 4, 327 388. 9. Godfrey D. A., Ross C. D., Herrmann A. D. and Matschinsky F. M. (1980) Distribution and derivation of cholinergic elements in the rat olfactory bulb. Neuroscience 5, 273-292. 10. Greenfield S. (1984) Acetylcholinesterase may have novel functions in the brain. Trends Neurosci. 7, 364.368. 11. Greer C. A. (1991) Structural organization of the olfactory system. In Smell and Taste in Health and Disease (eds Getchell T. V., Doty R. L., Bartoshuk L. M. and Snow J. B. Jr), pp. 65-81. Raven Press, New York. 12. Greet C. A., Stewart W. B., Teicher M. H. and Shepherd G. M. (1982) Functional developmen( of the olfactory bulb and a unique glomerular complex in the neonatal rat. J. Neurosci. 2, 1744-1759. 13. Hunt S. P. and Schmidt J. (1978) Some observations on the binding patterns of ct-bungarotoxin in the central nervous system of the rat. Brain Res. 157, 213-232. 14. Ichikawa T. and Hirata Y. (1986) Organization of choline acetyltransferase-containing structures in the forebrain of the rat. J. Neurosci. 6, 281-292. 15. Le Jeune H. and Jourdan F. (1991) Postnatal development of cholinergic markers in the rat olfactory bulb: a histochemical and immunocytochemical study. J. comp. Neurol. 314, 383-395. 16. Le Jeune H. and Jourdan F. (1993) Cbolinergic innervation of olfactory glomeruli in the rat: an ultrastructural immunocytochemical study. J. comp. Neurol. 336, 279-292. 17. Kasa P. (1986) The cholinergic system in brain and spinal cord. Prog. Neurobiol. 26, 211-272. 18. Kasa P., Csillik B., Joo F. and Knyihar E. (1966) Histochemical and ultrastructural alterations in the isolated archicerebellum of the rat. J. Neurochem. 13, 173-178. 19. Kasamatsu T., Ohashi T. and Imamura K. (1989) Integration of adrenergic and cholinergic regulation in ocular dominance plasticity. Biomed. Res. 10, 43-53. 20. Kimura H., McGeer P. L. and Peng J. H. (1984) Choline acetyltransferase-containing neurons in the rat brain. In Handbook of Chemical Neuroanatomy (eds Bj6rklund A., H6kfelt T. and Kuhar M. J.), pp. 51457. Elsevier, Amsterdam. 21. Kunze W. A. A., Shafton A. D., Kemm R. E. and McKenzie J. S. (1991) Effect of stimulating the nucleus of the horizontal limb of the diagonal band on single unit activity in the olfactory bulb. Neuroscience 40, 21-27. 22. Large T. H., Lambert M. P., Gremillion M. A. and Klein W. L. (1986) Parallel postnatal development of choline acetyltransferase activity and muscarinic acetylcholine receptors in the rat olfactory bulb. J. Neurochem. 46, 6714580. 23. McLennan H. (1971) The pharmacology of inhibition of mitral cells in the olfactory bulb. Brain Res. 29, 177-184. 24. Mesulam M.-M., Mufson E. J., Wainer B. H. and Levey A. I. (1983) Central cholinergic pathways in the rat: an overview based on an alternative nomenclature (Chl-Ch6). Neuroscience 10, 1185-1201. 25. Mori K. (1987) Membrane and synaptic properties of identified neurons in the olfactory bulb. Prog. Neurobiol. 29, 275-320.

Synaptic and non-synaptic cholinergic innervation in the MOB

677

26. Nickell W. T. and Shipley M. T. (1988) Two anatomically specific classes of candidate cholinoceptive neurons in the rat olfactory bulb. J. Neurosci. 8, 4482-4491. 27. Nickell W. T. and Shipley M. T. (1988) Neurophysiology of the magnocellular forebrain inputs to the olfactory bulb in the rat: frequency potentiation of output neurons. J. Neurosci. 8, 4492-4502. 28. Nowicky M. C., Mori K. and Shepherd G. M. (1981) Blockade of synaptic inhibition reveals long-lasting synaptic excitation in isolated turtle olfactory bulb. J. Neurophysiol. 46, 649~58. 29. Ojima H., Yamasaki T., Kojima H. and Akashi A. (1988) Cholinergic innervation of the main and accessory olfactory bulbs of the rat as revealed by a monoclonal antibody against choline acetyltransferase. Anat. Embryol. 178, 481-488. 30. Phelps P. E., Houser C. R. and Vaughn J. E. (1992) Small cholinergic neurons within fields of cholinergic axons characterize olfactory-related regions of rat telencephalon. Neuroscience 48, 121-136. 31. Ravel N., Akaoka H., Gervais R. and Chouvet G. (1990) The effect of acetylcholine on rat olfactory bulb unit activity. Brain Res. Bull. 24, 1-5. 32. Ribak C. E., Vaughn J. E., Saito K., Barber R. and Roberts E. (1977) Glutamate decarboxylase localization in neurons of the olfactory bulb. Brain Res. 126, 1-18. 33. Rotter A. (1984) Cholinergic receptors. In Handbook of Chemical Neuroanatomy (eds Bj6klund A., H6kfelt T. and Kuhar M. J.), Vol. 3, pp. 273-303. Elsevier, Amsterdam. 34. Satoh K., Armstrong D. M. and Fibiger H. C. (1983) A comparison of the distribution of central cholinergic neurons as demonstrated by acetylcholinesterase pharmacohistochemistry and choline acetyltransferase immunohistochemistry. Brain Res. Bull. 11, 693-720. 35. Schambra U. B., Sulik K. K., Petrusz P. and Lauder J. M. (1989) Ontogeny of cholinergic neurons in the mouse forebrain. J. comp. NeuroL 288, 101-122. 36. Shepherd G. M. and Greer C. A. (1990) Olfactory bulb. In The Synaptic Organization of the Brain (ed. Shepherd G. M.), pp. 133-169. Oxford Univ. Press., New York--Oxford. 37. Shute C. C. D. and Lewis P. R. (1967) The ascending reticular cholinergic system: Neocortical, olfactory and subcortical projections. Brain 90, 497-520. 38. Sofroniew M. V., Campbell P. E., Cuello A. C. and Eckenstein F. (1985) Central cholinergic neurons visualized by immunohistochemical detection of choline acetyltransferase. In The Rat Nervous System (ed. Paxinos G.), Vol. 1, pp. 471-485. Academic Press, London. 39. Sofroniew M. V., Eckenstein F., Thoenen H. and Cuello A. C. (1982) Topography of choline acetyltransferase-containing neurons in the forebrain of the rat. Neurosci. Lett. 33, 7-12. 40. Sternberger L. A. (1979) Immunocytochemistry, 2nd edn. Wiley, New York. 41. Struble R. G. and Clark H. B. (1992) Olfactory bulb lesions in Alzheimer's disease. Neurobiol. Aging 13, 469-473. 42. Trombley P. Q. (1992) Norepinephrine inhibits calcium currents and EPSPs via a G-protein-coupled mechanism in olfactory bulb neurons. J. Neurosci. 12, 3992-3998. 43, Trombley P. Q. and Westbrook G. L. (1990) Excitatory synaptic transmission in primary cultures of rat olfactory bulb. J. Neurophysiol. 64, 598~606. 44, Vizi E. S., Gyires K., Somogyi G. T. and Ungvfiri G. (1983) Evidence that transmitter can be released from regions of the nerve cell other than presynatic axon terminal: axonal release of acetylcholine without modulation. Neuroscience 10, 967-972. 45. Vizi E. S. and L~bas E. (1991) Non-synaptic interactions at presynaptic level. Prog. Neurobiol. 37, 145 163. 46. Wainer B. H., Levey A. I., Mufson E. J. and Mesulam M.-M. (1984) Cholinergic systems in mammalian brain identified with antibodies against choline acetyltransferase. Neurochem. Int. 6, 163 182. 47. Zaborszky L., Carlsen J., Brashear H. R. and Heimer L. (1986) Cholinergic and GABAergic afferents to the olfactory bulb in the rat with special emphasis on the projection neurons in the nucleus of the horizontal limb of the diagonal band. J. comp. Neurol. 243, 488-509. 48. Zheng L. M., Ravel N. and Jourdan F. (t987) Topography of centrifugal acetylcholinesterase-positive fibers in the olfactory bulb of the rat: evidence for original projections in atypical glomeruli. Neuroscience 23, 1083-1093. (Accepted 4 January 1995)