Atypical olfactory glomeruli contain original olfactory axon terminals: An ultrastructural horseradish peroxidase study in the rat

Neuroscience Vol. 26, No. 2, pp. 367-378, 1988 Printed in Great Britain

0306-4522/88$3.00+ 0.00 Pergamon Press plc IBRO

ATYPICAL OLFACTORY GLOMERULI CONTAIN ORIGINAL OLFACTORY AXON TERMINALS: AN ULTRASTRUCTURAL HORSERADISH PEROXIDASE STUDY IN THE RAT L. M. ZHENG and F. JOURDAN Physiologie Neurosensorielle, CNRS-Universite Claude Bernard/Lyon, France

F-69622 Villeurbanne Cidex,

Abstract-The labelling of olfactory bulb glomeruli following horseradish peroxidase lavage of the nasal cavity has been studied in the rat. In such conditions, atypical glomeruh, previously described according to their high acetylcholinesterase content, display a strong tracer accumulation. The course of afferent olfactory fibres could be followed along the lateral and dorsal surface of the olfactory bulbs. The primary olfactory axons ending in atypical glomeruli have been identified with horseradish peroxidase in electron microscopy. They differ significantly from classical olfactory terminals owing to the presence of large dense-cored vesicles accompanying small clear ones. Moreover, the olfactory terminals do not gather in dark nodules as they do classically in olfactory glomeruli. The study demonstrates that a subset of olfactory neuroreceptors displaying original ultrastructural characteristics projects selectively into atypical olfactory glomeruli. Ultrastructural features indicate that olfactory information processing taking place in the neuropil might be similar to that which occurs in typical glomeruli. Considered together, the atypical olfactory neuroreceptors, glomeruli and acetylcholinesterase-containing centrifugal fibres could constitute a new olfactory subsystem. This hypothesis is discussed by taking into account previous demonstration of other olfactory subsystems devoted to the processing of olfactory cues of fundamental biological importance.

In a previous work devoted to the study of acetylcholinesterase (AChE)-positive fibres innervating the olfactory bulbs,27 we described a set of atypical glomeruli receiving a particularly high concentration of AChE-containing bulbopetal fibres. Some of these atypical glomeruli might correspond to the modified glomerular complex (MGC) previously described in the rat with the aid of 2-deoxyglucose (2-DG) metabolic mapping,6,26 but others differed from MGC by their position in the ventrolateral, dorsolateral and dorsal bulbar areas.27 The fact that these glomeruli displayed original characteristics according to structural, topological and neurochemical criteria led us to propose that they could form part of a new olfactory subsystem located inside the main olfactory system. However, maintaining this hypothesis required the demonstration of true primary olfactory projections reaching the atypical glomeruli. The purpose of the present study was to investigate whether olfactory neuroreceptors do project into atypical glomeruli, using horseradish peroxidase (HRP) anterograde axonal transport following HRP lavage of the nasal cavity.25 Moreover, we have studied the atypical glomeruli at the ultrastructural

AChE, acetylcholinesterase; AOB, accessory olfactory bulb; 2-DG, 2-deoxyglucose; HRP, horseradish peroxidase; LHRH, luteinizing hormonereleasing- hormone; MGC, modified- glomerular complex; MOB, main olfactory bulb; PBS, phosphatebuffered saline; TMB, tetramethyl benzidine.

Abbreviations:

367

level, particular attention being paid to the identification of primary olfactory terminals with HRP labelling. All ultrastructural features were compared with those of typical olfactory glomeruli in order to estimate the originality of atypical glomeruli and the possible functional implications of such anatomical specificities. EXPERIMENTAL PROCEDURES Intranasal

irrigation

with horseradish peroxidase

Fifteen Wistar (Iffe-Credo, France) adult rats of both sexes, weighing 300-400 g received an i.p. injection of 0.5 ml atropine sulphate. Ten min later, they were anaesthetized with Equithesin (chloral hydrate 4%, pentobarbital, 16%) and put on their backs. A polyethylene catheter, with o.d. 0.7mm, was fixed on the needle of a 100 ~1 Hamilton syringe. The syringe and catheter were filled with a 30% solution of HRP type VI (Sigma) in sterile saline and fixed vertically on a syringe holder. The tip of the catheter was gently pushed into one nostril until 1.5 cm had penetrated inside the nasal cavity. Then 30-50 ~1 of the HRP solution were delivered dropwise over a 15min period. Afterwards, the animals were left on their backs for a 30-min period. Horseradish microscopy

peroxidase

labelling

in light

and

electron

Following a 24-h post-injection survival time, the animals were perfused intracardiallv using a rapid-flow perfusion methdd.5 Bach animal received 1i. of fixative made up of elutaraldehvde (Taab) (1%) and oaraformaldehvde (1%) in phosphate-buffered saline (PBS), ‘0.4 M at pH 7.4. Then the brains were dissected out and dropped into a washing solution containing PBS (0.1 M) and sucrose (15%). For the light microscopic localization of HRP, the olfactory bulbs were frozen in isopentane cooled to -40°C with liquid nitrogen, and serial frontal sections, 20pm thick,

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L. M. ZHENG

were made with a motorized cryostat. The sections were collected on gelatin-coated slides and processed for HRP localization, using tetramethyl benzidine (TMB) as a chromogen” and hydrogen peroxide as a substrate. The sections were counterstained with Neutral Red or Cresyl Violet. Four rats were processed for the alternate labelling of AChE and HRP on successive sections. AChE labelling was performed on free-floating sections according to the method reported previously for the description of atypical glomeruli.27 For the electron microscopic localization of HRP, we used the TMB method proposed by Mesulam.” Following the post-fixation washing, the bulbs were cut into serial frontal sections, 4Opm thick, using a Vibratome (Oxford Instruments). The free-floating sections were placed in the incubation medium containing TMB and hydrogen peroxide. Then they were post-fixed in 2% buffered osmic acid, at pH 6 and at a temperature of 45°C for 20 min, dehydrated in graded alcohols and flat-embedded in Epon. For flat-embedding, each section was gently placed in a drop of epoxy resin on the surface of a cubic block of polymerized Epon. Pieces of a solid Teflon film had been previously glued on glass coverslips which were placed on the epoxy drop containing the sections, the Teflon-coated side being in contact with the section to be embedded. The flat-mounted sections were baked at 37°C for 24 h and then at 60°C for 48 h. Following the polymerization of the epoxy resin, the coverslips could be easily separated from the block by pulling the Teflon film sharply. Owing to the limited section thickness, it was easy to select areas of interest, according for instance to HRP or AChE labelling, under a stereo-

and F. JOURVAN scopic microscope. could be observed

Then blocks were cut so that such areas on semithin and ultrathin sections.

Standard ultrastructural studies For standard ultrastructural studies of selected areas of the glomerular layer, the procedure was similar to that described above, without the histoenzymologic reactions. The semithin sections (1 pm thick) were stained with Richardson’s stain (Methyl Blue-AzurII). Ultrathin sections were contrasted with uranyl acetate and lead nitrate. Morphometric study of synaptic vesicles Measurements of the synaptic vesicle diameter were performed on electron micrographs of constant magnification with the aid of a digitizer table (Houston Inst.). The vesicle areas and diameters were systematically computed and submitted to a modified Student-Fisher test (6 test) to compare the mean values obtained from large samples.

RESULTS Horseradish Light

peroxidase

microscopic

labelling observations

in light microscopy reveal

a

dense

labelling confined to the olfactory nerve and glomerular layers of the main olfactory bulb (MOB), without any indication of transneuronal transport in deeper layers (Fig. 1). In one case, a weak but significant labelling of the vomeronasal nerve and accessory olfactory bulb (AOB) was observed, probHRP

Fig. 1. HRP labelling of olfactory nerve and glomeruli. Frontal section of the posterior part of the olfactory bulbs (OB). Atypical glomeruli lying in the lateral and dorsal areas have accumulated HRP (large arrows). Labelled afferent fibres innervating the atypical glomeruli can be followed on the lateral and dorsal surfaces of the OB (small arrows). GLL, Glomerular layer; MCL, mitral cell layer; GRL, granule cell layer.

HRP labelling of atypical olfactory glomeruli

ably due to a certain rate of HRP diffusion to the vomeronasal epithelium during intranasal irrigation. This animal was discarded from further analyses. Most olfactory bulbs displayed a full labelling of the glomerular layer (Fig. 1) and gave rise to the following observations. The analyses of either HRP-labelled serial sections, or alternate AChE-HRP labelling, demonstrate unambiguously that all the atypical glomeruli are innervated by primary olfactory axon terminals. Examples of labelled dorsolateral and dorsal atypical glomeruli are shown in Figs 1 and 2. Atypical glomeruli lying in most anterior and ventral (Fig. 1) bulbar levels also display a strong tracer accumulation. No significant difference in labelling intensity could be noted between atypical and typical glomeruli. The labelled fibres afferent to atypical glomeruli have been frequently observed running at the surface of the olfactory bulb (Figs 2, 3a). The complete lack of any HRP-labelling in the accessory olfactory system (Figs 1, 2) demonstrates that olfactory neuroreceptors sending their axons into atypical glomeruli are unambiguously located in the main olfactory mucosa. Particular attention has been paid to the spatial distribution of olfactory fibres afferent to the dorsal atypical glomeruli. As shown in Fig. 2, bundles of olfactory axons come from the dorsolateral glomerular area (Fig. 2a), leave the glomerular layer by turning medially and terminate in atypical glomeruli lying on the dorsal face of the anterior AOB (Fig. 2b, c, d). Whereas some lateromedial labelled fibers make a loop to reach the largest atypical glomerulus in this area (Fig. 2c), others continue their route medially by running over the dorsal AOB (Fig. 2d), turn round the unlabelled afferent fibres of the vomeronasal nerve (Fig. 2d, e) and terminate in atypical glomeruli located along the medial border of the vomeronasal nerve, close to the AOB (Fig. 2g, h). On the contrary, other glomeruli lying farther from the AOB, but outside the typical glomerular layer are innervated by afferent olfactory fibres coming from the medial bulbar aspect (Fig. 2f, g). Finally, we can conclude that most dorsal atypical glomeruli are innervated by lateral fibres although some displaced glomeruli in this area receive afferent fibres from the medial bulbar surface. The possible heterogeneity of the mediodorsal glomerular region will be further discussed by taking into account the description of a modified glomerular complex (MGC) in the same area.26 Horseradish

peroxidase

Iabelling in electron

micro -

scoPY

Atypical glomeruh could be easily identified on unstained semithin sections owing to their unambiguous position and their strong reactivity on AChE-processed sections.27 Thus, we could select areas containing only one atypical glomerulus and its afferent fibres (Fig. 3a) on HRP-labelled sections embedded in Epon. Electron microscope obser-

369

vations of such unstained sections reveal the HRP reaction product typical of tetramethyl benzidine (TMB) (Fig. 3b-d). Numerous crystalline aggregates are observed in the atypical periglomerular and glomerular areas, most often superimposed on small or medium-sized profiles. The large, electron-lucent, dendritic profiles remain unstained (Fig. 3b), confirming the lack of transneuronal transport of HRP. On the other hand, numerous fibres and axon terminals contain significant amounts of reaction product. In spite of the imperfect cytological preservation due to the conditions of fixation required for TMB use, the main cytological characteristics of labelled profiles can be depicted. Most HRP labelling is associated with axon terminals containing numerous densely packed clear vesicles whose diameter has been estimated at 44.0 f 5.5 nm (N = 150). Moreover, large dense-cored vesicles are often present in these axonal endings (Fig. 3~). Such terminals make numerous asymmetrical synaptic contacts (Figs 3d, 5a) on small dendrites, the small clear vesicles being in contact with the presynaptic membrane. Although the frequency of labelled terminals is not homogeneous in the whole atypical glomerular neuropil, they do not form dense clusters and remain relatively scattered.

Synaptic organization of atypical glomeruli in standard electron microscopy: a new type of olfactory axon terminal

As previously underlined,27 atypical and typical glomeruli display both structural similarities and differences. Such features are well visible on stained semithin sections (Fig. 4a, c). The two glomerular types share common characteristics such as their shape and relative position with respect to the deeper bulbar layers. However, they obviously differ according to the density of AChE-positive centrifugal fibres” and, as shown in Fig. 4, to the structural organization of their neuropil. Whereas the classical glomerular neuropil contains dark nodules of densely packed axon terminals (Fig. 4a), that of atypical glomeruli looks much more homogeneous (Fig. 4c). This observation has been fully confirmed by electron microscope investigations. The clusters of olfactory axon terminals found in typical glomeruli, and containing densely packed vesicles in a dark cytoplasmic matrix (Fig. 4b), are not found in the neuropil of atypical glomeruli (Fig. 4d). However, several types of neuronal profiles can be depicted. Large roundshaped profiles with a clear cytoplasm and numerous neurotubules look very similar to the mitral/tufted cell dendrites (Figs 4d, 5). Their identification can be considered as quite certain since such dendrites con: tribute to the atypical glomeruli, as shown on semithin sections (Fig. 4c). Much more original is the occurrence of numerous profiles with a darker cytoplasmic matrix, containing two types of vesicles and making numerous asymmetrical synaptic contacts

Fig. 2. HRP labelling of dorsal atypical glorneruli. Successive OB frontal sections made along the rostrocaudal axis (a-h) at the level of the AOB. Sections were separated by the following intervals: a b, bc, c-d, d-e, e-f, f-g: 100 pm; g-h: 300 pm. The atypical glomeruli (large arrows), identified according to their position and strong AChE labelling on adjacent sections, have accumulated HRP. The course of the labelled afferent fibres (small arrows) can be followed on the dorsal AOB (b, c, d, e) and round the vomeronasal nerve (d, e, f, g). Large displaced glomeruli (broken arrows) receive libres from the medial OB and could not be definitely identified as atypical glomeruli. L, Lateral olfactory bulb; M, medial olfactory bulb: VNN, vomeronasal nerve. 370

Fig. 3. HRP labelling of a lateral atypical glomerulus. (a) The HRP accumulation is restricted to the glomerular neuropil, without any indication of transneuronal labelhng (large arrow). Afferent labelled fibres can be individually identified as they enter the glomerulus (small arrows). (b)-(d) HRP labelling at the ultrastructural level: crystalline aggregates (large arrows) are visible in neuronal profiles containing synaptic vesicles while large dendritic profiles lack reaction product (b). HRP is often associated with terminal containing large dense-cored vesicles (c, small arrows) accompanying small clear ones. Labelled terminals make numerous synaptic contacts with dendritic profiles (d, broken arrows). d, Dendrite; EPL, external plexiform layer; GRL, granule cell layer; MCL, mitral cell layer. 371

Fig -1 C’~~npar~son ofcytolog~cal organizations in normal (a. b) and atyptcal (c. d) glomeruli. A classical olf~tory glomerulus obscrwd on sennthm sectlon (a) displays the dense nodules corresponding to clusters of cllfactory axon terminals. as confirmed at the electron microscope level (h) Olfactory terminals arc easily identifiable owing to then dark cyroplasrn and numerous small clear vesicles. They make synaphc contacta (broken arrow) with dendrites. The neuropil of the atypical glomerulus shown in (c) proves much mom h~~ln~~gcncous. A large dendrite belonging to a second-order olfactory neuron enters and branches into the neuropil (c. arrow), At the ullrastructuml level (d). the atypical neuropil contains numerous clear dendruic profiles and large scattered axonal endings (asterisks) containing large dense-cored vesicles (arrows) accompanying small clear ones. Such terminals make numerous synaptic contacts with clear dcndrittc protiles (broken arrow). d. Dendrite: GL, glomerulus; EPL. external plewifarm layer: MCL. mitral cell layer 372

313

HRP labelling of atypical olfactory glomeruli with clear dendritic profiles (Figs 4d, 5a). Small clear

vesicles are found close to tbe synaptic contact, while the large dense-cored ones are scattered in the whole terminal (Fig. 5a). The axonal nature of such terminals is illustrated by Fig. 5a, where the distal part of the axon is clearly visible. Whereas such axons and their terminals are found scattered in the atypical glomerular core, more densely packed aggregates can be observed at the glomerular periphery, most often close to the outer layer of the olfactory bulb (Fig. Sb): large mitral/tufted cell dendrites are surrounded by bundles of parallel axons of various diameters ranging from 0.25 to 0.5 pm. Regularly arranged neurotubules are well visible in both transversely and longitudinally sectioned axons. Some contain the same two types of vesicles as those encountered in axon terminals, often ,concentrated at the periphery, near the cell membrane. Finally, some of these axons are seen making asymmetrical synapses with large dendritic profiles in this area as in the glomerular core. Owing to its frequency, spatial distribution and cytological characteristics, this type of axon is the best candidate for identification as the one labelled with HRP, following intranasal HRP irrigation. Systematic measurements of the clear vesicle diameters in such axon terminals (Fig. 5a) were performed. We have found a mean diameter of 45.3 f 5.4nm (N = 150) which does not differ significantly (t-test, 6 = 1.95) from diameter of clear vesicles in HRP-labelled profiles (44.0 + 5.5 nm). In fact, such axonal profiles are the only ones to contain a rather numerous population of dense-cored vesicles and do not correspond to any type of synaptic terminals encountered in typical glomeruli.i6 As a consequence, we can assume that atypical glomeruli contain a new type of primary axon terminals issued from olfactory neuroreceptors located in the nasal olfactory mucosa. These terminals are characterized by large dense-cored vesicles found in addition to the more classical small clear ones. They do not gather in densely packed nodules as the classical olfactory terminals do in typical glomeruli. Finally, these primary axon terminals make asymmetrical synaptic contacts with dendritic profiles, some undoubtedly belonging to mitral or tufted cells. Synaptic neurons

organization

of atypical

glomeruli:

inter-

Atypical glomeruli are surrouned by a row of neuronal perikarya which look similar to periglomerular interneurons, as observed on semithin sections (Fig. 4a, c). This similarity has been confirmed at the ultrastructural level. Two types of neurons commonly found peripheral to atypical glomeruli are shown in Fig. 6. Their ultrastructural characteristics are identical with those of periglomerular and short-axon cells.” On the basis of observations of the atypical periglomerular and glomerular areas, we can postulate that the inter-

neuronal synaptic organization does not differ strikingly from that of typical glomeruli. Symmetrical synaptic contacts on mitral/tufted cell dendrites (Fig. 7a) are probably made by periglomerular cell dendrites. Typical reciprocal synaptic complexes have been occasionally observed between periglomerular and mitral cell dendrites (Fig. 7b). Other complexes involving axonal afferents on mitral or tufted cell dendrites and mitral-periglomerular dendrodendritic contacts could also be depicted in the neuropil (Fig. 7~). Finally, symmetrical synapses on mitral cell dendrites have been found in the periglomerular area (Fig. 7d) and might belong to periglomerular axons. In the same region, asymmetrical synapses containing small clear vesicles cannot be definitely identified with primary terminals and might correspond to centrifugal fibres, which are numerous in the area. DISCUSSION

The observations reported in this study demonstrate that the atypical glomeruli, first identified according to their high content in AChE-containing centrifugal fibres, *’ do receive olfactory axon terminals making synaptic contacts with second-order olfactory neurons. This observation, as well as the presence of typical olfactory interneurons, supports the idea that processing of neuronal information taking place in atypical glomeruli might be similar to that occurring in classical olfactory glomeruli.24 However, several structural characteristics depicted in our study differentiate strictly atypical glomeruli from normal ones. They mainly concern the occurrence of a new type of olfactory axon terminals and their spatial organization inside the atypical glomerular neuropil. A new subset of olfactory neuroreceptors?

a

The ultrastructural characteristics of olfactory terminals in atypical glomeruli are original enough to give rise to the hypothesis that they belong to a well-specified subset of olfactory receptors. Although the olfactory neuroreceptor population has often been considered as homogeneous, the existence of minority subsets of olfactory neurons has already been postulated. In 1975, Jourdan9 described in the rat olfactory mucosa a population of bipolar receptor cells sharing numerous characteristics with olfactory receptors but bearing microvilli instead of cilia at the apical pole of their dendrite. These “B” receptor cells have been systematically observed since this date in the rat mucosa, where their frequency could be estimated to 2-3% of the total receptor population (Jourdan, unpublished observations). Several authors reported further observations of microvillar receptor cells in other species such as the dog,” and some primates “x2’ including man. I4 More recently, olfactory receptor subsets were identified in the olfactory system of mammals according to different criteria such as the presence of carbonic anhydrase4 and

Fig. 5. Ultrastructure of atypical glomerular neuropil. (a) A small axon containing neurotubules (large arrow) makes a large synaptic terminal which contacts a small dendrite (broken arrow). The axon terminal contains numerous small clear vesicles and a few large dense-cored ones (small arrows). Other axonal profiles display the same cytological characteristics (asterisks) and are seen contacting large dendritic profiles (d) typical of second-order olfactory neurons. (b) At the outer periphery of the atypical glomerulus, numerous axons (ax) containing regularly arranged neurotubules gather to form large bundles surrounding large clear dendritic profiles (d). Some axons are locally enlarged (asterisks) and then contain large dense-cored vesicles (arrows) with small clear ones and neurotubules. It is most likely that this type of axon gives rise to synaptic contacts similar to that shown in (a). ax, Axon; d, dendrite. 374

HRP labelling of atypical olfactory glomeruli

315

Fig. 6. Periglomeridar area of an atypical glomerulus. Two periglomerular cells display different cytological characteristics; the smaller one, with a large nucleus in a sparse dark cytoplasm, looks like the typical periglomerular cells (PGC). The other, with large nuclear indentations and a clear abundant cytoplasm is very similar to the second interneuron type, namely the short axon cells (SAC). Roth are wrapped in thin processes of glial cells (small arrows). Many myelinated processes (large arrows) are present in this periglomerular area and can be interpreted as centrifugal afferent fibres. PGC, Periglomerular cell; SAC, short-axon cell.

immunoreactivity to anti-neurofilaments23 or antineuronal’ monoclonal antibodies. In every case, the proportion of atypical receptor cells was consistent with that of “B” receptor cells.9 Whether olfactory neurons ending in atypical glomeruli correspond to one of the subsets already described is not known at this time, since ultrastructural data concerning axon terminals cannot be related either to structural specificities of soma and dendrites, or to antigenic characteristics. However, experiments of retrograde HRP labelling of the olfactory mucosa following focal HRP injections in the olfactory bulb’.‘5,22have given rise to results consistent with this hypothesis. In particular, HRP injections in the modified glomerular complex (MGC)” must have involved some atypical glomeruli located in this area. They resulted in the labelling of olfactory receptor cells whose distribution is consistent with that of “B” cells’ or immunoreactive olfactory subsets.7*23Studies at the ultrastructural level are now needed to look for

possible ultrastructural specificities of neuroreceptors projecting to the atypical glomeruli. In any case, the cytological peculiarities of olfactory terminals in atypical glomeruli give some indications about the possible characteristics of this contingent olfactory receptor subclass. First, the occurrence of numerous dense-cored vesicles might represent a neurochemical specificity related to neurotransmission or neuromodulation. Second, the absence of densely packed clusters of axon terminals in atypical glomeruli suggests that some factors of aggregation are probably lacking in those axons when compared with normal olfactory terminals. Important functional implications could be inferred from such a hypothesis, but the lack of information about the role of olfactory axon nodules in normal olfactory glomeruli keeps us from further speculation. Finally, the well-focused projection of atypical receptor terminals onto topographically defined areas in the glomerular layer implies mechanisms of

Fig. 7. Synaptic contacts mvolving interncur~)~s in the ncuropil ~4’alypical ~f~)r~lerl~l~Arrow indicate the presynaptic to postsynaptic orientations. (a) Asymmetrical sytxcptlc wntact ot‘ 3 pe~~glr~merular dendrite an a large round-shaped mitral cell dendrite. (b) Reciprocal synaptic complex between mitral ccl1 (MC) and p~rigfomeru~r ccl! (PGC) dendriter. fc) Synaptic compku. A clear mitral wll dendrite (dMC’) receives 3 synaptic contract of the asymmetricat type from a primary ax** trrminat c~~~taioj~~ 17003ctear and targe dense-cored vzskles. This dendrite is presynaptrc to another dcndritc prob&y b&wginp kt a periglomerular celf. fd) Symmetrieai axodendritic synapse located in the peri~l~)rnerui~lr region. The axon cunnot be definitely idcntdied but might belong to a periglomrrular cell according to the six and shape of vesicles and the symmetrical membrane thrkcning. Two myelinated fihres. prtlh;ihly of centrifugal origin, are also identifiable. ax. Axon; d. dendrite; MC. mttral cell; PGC. pcriglomerular cell.

HRP labelling of atypical olfactory glomeruli spatially organized neuronal connection in the olfactory system. Such an organization has been shown for the whole system I,**but we can postulate that specific events, perhaps involving trophic factors, could occur during the growing of an axonal subset towards chemically defined glomerular areas. At this moment, the neurochemical specificity of atypical glomeruli is restricted to the presence of numerous AChE-positive*’ and a few luteinizing hormonereleasing hormone (LHRH)-containing”,** centrifugal fibres. Further experiments are now needed to estimate the possible function of those centrifugal fibres with regard to the peripheral connectivity, and to look for other neurochemical specificities of atypical glomeruli. Atypical

gIomeruli

and

the

modljied

glomerular

complex

Since the first demonstration by Teicher et a1.26 that suckling pheromone stimulation resulted in 2-deoxyglucose (2-DG) labelling of a group of dorsomedial glomeruli in the developing olfactory bulbs, several lines of evidence have confirmed the original properties of this “modified glomerular complex” (MGC). It has been proposed that the MGC may mature earlier than other regions of the olfactory bulbs6 so that it could be well adapted to the processing of odour cues relevant to suckling behaviour in neonatal rats. Finally, HRP retrograde tracing studies I5 demonstrated that the MGC received olfactory afferents from the main olfactory system. Neuroreceptors projecting onto the MGC were present in large but topographically defined areas of the olfactory mucosa. Some atypical glomeruli of the dorsomedial bulbs, as shown by AChE histochemistry,*’ lie in a position similar to that of the MGC, as demonstrated by 2-DG studies.6.26 Whether these anatomical units correspond to a unique morphofunctional complex cannot be asserted in the absence of double labelhng studies. However, our present observations are consistent with retrograde HRP tracing studies’.** since we confirm that atypical glomeruli of this area are actually innervated by neuroreceptors of the main olfactory system. Furthermore, we have demonstrated that this glomerular subset is mainly innervated by laterodorsal olfactory fibres. This finding is consistent with previous retrograde HRP tracing studies’,** since dorsolateral olfactory fibres have been shown to originate in the anterior dorsal recess and some turbinates which were also labelled following an HRP injection in the MGC.15 Our study cannot give rise to definitive functional interpretations. However, it confirms that all the

311

atypical glomeruli previously described with the aid of AChE histochemistry*’ do receive olfactory afferents and, as a consequence, might be involved in processing of some olfactory information. In this respect, the whole subset of atypical glomeruli looks homogeneous and could be involved in common functional processes, possibly including the processing of olfactory cues related to suckling behaviour, as demonstrated for the MGC area.26 This hypothesis might explain why lesions of the MGC, not altering the ventral and lateral atypical glomeruli, did not result in significant alterations of suckling pheromone detection in neonate ratsIs and rabbits.8 However, we cannot state definitely whether atypical glomeruh and the MGC share common properties or not. Since the MGC was described according to morpho-functional criteria (2-DG studies), double labelling experiments (AChE-2-DG) will be necessary to clear up this question. At any rate, our observations of a new type of olfactory terminal which ends in atypical glomeruli receiving simultaneously a specific centrifugal innervation, strongly supports the hypothesis that a new selective subsystem is present in the main olfactory system of the rat. Furthermore, we suggest that particular attention should be drawn to this olfactory subset in the framework of the recent hypothesis involving the olfactory system in the aetiology of Alzheimer’s disease.*~” Tracing studies using peroxidaseconjugated wheat germ agglutinin have demonstrated that substances applied to the olfactory epithelium could reach the basal forebrain by retrograde transneuronal transport along centrifugal fibres innervating the glomerular layer.3 Atypical glomeruli receive olfactory afferents (this study) and are simultaneously innervated by an exceptional number of AChE-positive centrifugal fibres originating in the nuclei of the basal forebrain.27 Since neuronal degeneration affects this area in Alzheimer’s disease, the olfactory subsystem depicted in the present study, if present in human species, could constitute a preferential route for those possible pathological processes using the olfactory entry.”

Acknowledgements-The authors wish to thank M. Vigouroux and A. M. Brandon for technical assistance, and Prof. A. Holley for comments on the manuscript. Electron microscopy was carried out at the Centre de Microscopic Slectronique appliqute a la Biologie et la Geologic de Wniversiti Cl. Bernard. One of the authors (L.M.Z.) is the recipient of pre-doctoral fellowships from the E. Roudnitska Foundation and the Association francochinoise pour la Recherche en Biologie et en Medecine.

REFERENCES

1. Astic L. and Saucier D. (1986) Anatomical mapping of the neuroepithelial projection to the olfactory bulb in the rat. Brain Res. Bull. 16, 445451. 2. Baker H. and Margolis F. L. (1986) Deafferentation-induced alterations in olfactory bulb as a model for the etiology of Alzheimer’s disease. Neurobiol. Aging 7, 568-569.

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3. Baker

4. 5. 6.

13 14 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

H. and Spencer R. F. (1986) Transneuronal transport of peroxidase-conjugated wheat germ agglutinin (WGA-HRP) from the olfactory epithelium to the brain of the adult rat. Expl Brain Res. 63, 461-473. Brown D. L., Garcia-Segura M. and Orci L. (1984) Carbonic anhydrase is present in olfactory receptor cells. Histochemistry SO, 307-309. Descarries L. and Schroeder J. M. (1968) Fixation du tissu nerveux par perfusion a grand debit. J. Microsc. 7,281-286. Greer C. A.. Stewart W. B.. Teicher W. H. and Sheoherd G. M. (1982) Functional develooment of the olfactorv bulb and a unique glomerular complex in the neonatal rat. J. Neurosci. 2(12), 17441759. L Hempstead J. L. and Morgan J. I. (1985) Monoclonal antibodies reveal novel aspects of the biochemistry and organization of olfactory neurons following unilateral olfactory bulbectomy. J. Neurosci. S(9), 23822387. Hudson R. and Distel H. (1987) Regional autonomy in the peripheral processing of odor signals in newborn rabbits. Bruin Res. 421, 85-94. Jourdan F. (1975) Ultrastructure de l’tpithelium olfactif du rat: polymorphisme des rtcepteurs. C.r. hebd. S&K. Acad. Sci., Paris 280, 443446. Loo S. K. (1977) Fine structure of the olfactory epithelium in some primates, J. Ati&. 123, 1355145. Menco B. Ph.M. (1980) Qualitative and quantitative freeze-fracture studies on olfactory and nasal respiratory structures of frog, ox, rat and dog. I-A general survey. CeN. Tiss. Res. 207, 183~.209. Mesulam M. M. (1978) Tetramethylbenzidine for horseradish peroxidase neurohistochemistry: a non-carcinogenic blue reaction-product with superior sensitivity for visualizing neural afferents and efferents. J. Histochem. Cytochem. 26, 106-l 17. Mesulam M. M. (ed.) (1982) Tracing Neural Connections with Horseradish Peroxidase. John Wiley, New York. Moran D. T., Carter-Rowley J., Jafek B. W. and Love11 M. A. (1980) The fine structure of the olfactory mucosa in man. J. Neurocyfol. 11(5), 731-746. Pedersen P. E., Jastreboff, P. J., Stewart W. B. and Shepherd G. M. (1986) Mapping of an olfactory receptor population that projects to a specific region in the rat olfactory bulb. J. camp. Neural. 250, 93--108. Pinching A. J. and Powell T. P. S. (1971) The neuropil of the glomeruli of the olfactory bulb. J. Cell Sci. 9, 3477377. Pinching A. J. and Powell T. P. S. (1971) The neuron types of the glomerular layer of the olfactory bulb. J. Cell Sci. 9, 305-345. Risser J. M. and Slotnick B. M. (1987) Suckling behavior in rat pups with lesions which destroy the modified glomerular complex. Brain Res. Bull. 19, 275528 1. Roberts E. (1986)Alzheimer’s disease may begin in the nose and may be caused by aluminosilicates. Neurobiol. Aging 7, 561-567. Rosser A. E., Hokfelt T. and Goldstein M. (1986) LHRH and catecholamine neuronal systems in the olfactory bulb of the mouse. J. camp. Neural. 250, 352-363. Saini K. D. and Breipohl W. (1976) Surface morphology in the olfactory epithelium of normal and female rhesus monkeys. J. Anat. 107, 433446. Saucier D. and Astic L. (1986) Analysis of the topographical organization of olfactory epithelium projections in the rat. Bruin Res. Bull. 16, 455462. Schwab J. E.. Farber N. B. and Gottlieb D. I. (1986) Neurons of the olfactory . epithelium in adult rats contain vimentin. _ J. Neurosci. 6(l), 208-217. Shepherd G. M. (1972) Synaptic organization of the mammalian olfactory bulb. Physiol. Rer. S2(4), 864917. Stewart W. (1985) Labelling of olfactory bulb glomeruli following horseradish peroxidase lavage of the nasal cavity. Bruin Res. 347, 2OG203. Teicher M. H., Stewart W. B., Kauer J. S. and Shepherd G. M. (1980) Suckling pheromone stimulation of a modified glomerular region in the developing rat olfactory bulb revealed by the 2-deoxyglucose method. Bruin Res. 194,530-535. Zheng L. M., Ravel N. and Jourdan F. (1987) Topography of centrifugal acetylcholinesterase-postive fibres in the olfactory bulb of the rat: evidence for original projections in atypical glomeruli. Neuroscience 23, 1083-1093. Zheng L. M., Caldani M. and Jourdan F. (1988) Immunocytochemical identification of luteinizing hormone-releasing hormone positive fibres and terminals in the olfactory system of the rat. Neuroscience 24, 5677578. (Accepted 19 January 1988)