Expression of netrin-1 and netrin-1 receptor, DCC, in the rat olfactory nerve pathway during development and axonal regeneration.

Netrin-1 is a bifunctional secreted protein that directs axon extension in various groups of developing axonal tracts. The transmembrane DCC (deleted in colorectal cancer) receptor is described as netrin-1 receptor and is involved in the attractive effects of netrin-1. In this study, we examined the spatio-temporal expression patterns of both netrin-1 and DCC in the rat olfactory system at different stages of development and during axonal regeneration following unilateral bulbectomy. High DCC expression was detected on the pioneer olfactory axons as they are extending toward the telencephalon. This expression was transient since from embryonic day 16 onwards, DCC was no longer detected along the olfactory nerve path. From embryonic day 14 until birth, DCC was also expressed within the mesenchyme surrounding the olfactory epithelium. During the same period, netrin-1 protein was detected along the trajectory of olfactory axons up to the olfactory bulb and its expression pattern in the nasal mesenchyme largely overlapped that of DCC. Moreover, netrin-1 continued to be present during the two first post-natal weeks, and a weak protein expression still persisted in the dorso-medial region of the olfactory epithelium in adult rats. While unilateral bulbectomy induced a transient up-regulation of netrin-1 in the lamina propria, particularly in the dorso-medial region of the neuroepithelium, no DCC expression was detected on the regenerating olfactory axons. In the developing olfactory bulb, the extension of mitral cell axons was associated with DCC presence while netrin-1 was absent along this axonal path. DCC was also highly expressed in the newly formed glomeruli after birth, and a weak DCC expression was still detected in the glomerular layer in adult rats. Taken together, these data support the notion that netrin-1, via DCC expressed on axons, may play a role in promoting outgrowth and/or guidance of pioneering olfactory axons toward the olfactory bulb primordium. Moreover, association of netrin-1 with mesenchymal DCC may provide a permissive environment to the growth of both pioneer and later-growing axons. The maintenance of netrin-1 expression in the nasal mesenchyme of adult rats as well as its regional up-regulation following unilateral bulbectomy infer that netrin-1, even in the absence of DCC, may be involved in the process of axonal growth of newly differentiated olfactory receptor neurons probably through the use of other receptors.

Neuronal plasticity and regeneration in the olfactory system of mammals: morphological and functional recovery following olfactory bulb deafferentation.

The mammalian olfactory system has the unique property in the permanent turnover of the olfactory sensory neurons under normal conditions and following injury. This implies that the topographical map of the epithelium-to-bulb connections generated during ontogenesis has to be maintained despite neuron renewal in order to insure olfactory information processing. One way to investigate this issue has been to disrupt the peripheral connections and analyze how neural connections may be reestablished as well as how animals may perform in olfactory-mediated tasks. This review surveys the main data pertaining to both morphological and functional recoveries taking place in the peripheral olfactory system following olfactory bulb deafferentation. Conclusions from these studies are enlightened by recent data from molecular biology.

Expression of SCG10 and stathmin proteins in the rat olfactory system during development and axonal regeneration.

The membrane-associated protein SCG10 is expressed specifically by neuronal cells. Recent experiments have suggested that it promotes neurite outgrowth by increasing microtubule dynamics in growth cones. SCG10 is related to the ubiquitous but neuron-enriched cytosolic protein stathmin. To better understand the role played by SCG10 and stathmin in vivo, we have analyzed the expression and localization of these proteins in both the olfactory epithelium and the olfactory bulb in developing and adult rats, as well as in adult bulbectomized rats. The olfactory epithelium is exceptional in that olfactory receptor neurons constantly regenerate and reinnervate the olfactory bulb throughout animal life-span. SCG10 and stathmin expression in the olfactory receptor neurons was found to be regulated during embryonic and postnatal development and to correlate with neuronal maturation. Whereas SCG10 expression was restricted to immature olfactory receptor neurons (GAP-43-positive, olfactory marker protein-negative), stathmin was also expressed by the basal cells. In the olfactory bulb of postnatal and adult rats, a moderate to strong SCG10 immunoreactivity was present in the olfactory nerve layer, whereas no labeling was detected in the glomerular layer. Olfactory glomeruli also showed no apparent immunoreactivity for several cytoskeletal proteins such as tubulin and microtubule-associated proteins. In unilaterally bulbectomized rats, SCG10 and stathmin were seen to be up-regulated in the regenerating olfactory epithelium at postsurgery stages corresponding to olfactory axon regeneration. Our data strongly suggest that, in vivo, both SCG10 and stathmin may play a role in axonal outgrowth during ontogenesis as well as during axonal regeneration.

Decline and recovery of olfactory receptor expression following unilateral bulbectomy.

The effects of unilateral olfactory bulb ablation upon the odorant receptor expression were studied during the degeneration/regeneration process in the olfactory epithelium of adult rats. Using the in situ hybridization approach, we compared the time course of decay and recovery of expression for three different receptor subtypes (OR14, OR5, OR124). The number of neurons expressing receptor subtypes dramatically decreased in the olfactory epithelium on the lesioned side and reached a minimum at day 5 postsurgery. A progressive recovery was then observed from day 5 to day 15 postlesion, when a plateau was reached. Noticeable differences in the recovery level of receptor expression were observed according to the zonal patterning: the recovery level for neurons located in the lateral zone reached 70% of the control side value while the recovery levels in the dorsal and medial zones represented 35% and 53% of this value, respectively. Axotomy experiments suggest that zone-specific differences in receptor reexpression reported after bulbectomy might be related to the trophic influence of the olfactory bulb.

Spatio-temporal patterns of ensheathing cell differentiation in the rat olfactory system during development.

An immunocytochemical approach with specific glial markers was used to investigate the temporal and spatial patterns of differentiation of ensheathing glia wrapping axon fascicles along the primary olfactory pathway of the rat during development. The two glial markers tested, the proteins S-100 and glial fibrillary acidic protein, are known to be expressed at different stages of maturation in glial cells. The S-100 protein was first weakly expressed in cells accompanying the olfactory axons at embryonic day 14 (E14), while a first faint glial fibrillary acidic protein staining was detected along the olfactory axons at E15 and along the vomeronasal nerves at E16. A strong S-100 immunoreactivity was already present from E16 onwards along the axon fascicles through their course in both the nasal mesenchyme and the subarachnoid space before entering the olfactory nerve layer of the olfactory bulb. A gradual increase in glial fibrillary acidic protein expression was observed along this part of the developing olfactory pathway from E16 up to E20, when an adult-like pattern of staining intensity was seen. By contrast, most of the ensheathing cells residing in the olfactory nerve layer exhibited some delay in their differentiation timing and also a noticeable delayed maturation. It was only from E20 onwards that a weak to moderate S-100 expression was detected in an increasing number of cells throughout this layer, and only few of them appeared weakly glial fibrillary acidic protein positive at postnatal days 1 and 5. The immunocytochemical data indicate that there is a proximodistal gradient of differentiation of ensheathing cells along the developing olfactory pathway. The prolonged immaturity of ensheathing cells in the olfactory nerve layer, which coincides with the formation of the first glomeruli, might facilitate the sorting out of olfactory axons leading to a radical reorganization of afferents before they end in specific glomeruli.

Ultrastructural and cytochemical identification of apoptotic cell death accompanying development of the fetal rat olfactory nerve layer.

It has been previously shown that the embryonic olfactory nerve contains, in addition to glial ensheathing cells, a large population of differentiated neurons that migrate from the developing olfactory epithelium, in close association with the olfactory axon fascicles. The purpose of our study was to verify the hypothesis according to which a process of physiological cell death might be involved in the progressive disappearance of these migrating neurons that has been reported during late embryonic stages in several immunocytochemical studies. To do so, we have investigated the development of the olfactory nerve layer in rat embryos by using light and electron microscopy, with special reference to the presence of cell death processes within this structure. We have also applied the histochemical TUNEL method allowing in situ visualization of cells degenerating by apoptosis. In order to determine if neurons were present among dying cells, a procedure of double-labeling was performed by combining the DNA-specific bisbenzimide with two neuronal markers, the protein B-50/GAP-43 and the lectin Ulex europaeus I. Results brought out the precise temporal and spatial patterns of programmed cell death accompanying the morphogenesis of the olfactory nerve layer. A cell death process was observed within the olfactory nerve layer from its onset at embryonic day 13 (E13). While only few pycnotic cells were observed in E13 and E14 embryos, their number increased from E15 to reach a maximum at E16 and then diminished. Few dying cells were also observed along the olfactory axon fascicles when they penetrated the olfactory nerve layer. Degenerating cells appeared strongly TUNEL-labeled and exhibited morphological features of cell death by apoptosis. Double-labeling experiments revealed that some of the apoptotic cells were neurons. These observations indicate that apoptosis may account for the progressive decrease in the number of migrating neurons present within the embryonic olfactory nerve layer. Otherwise, a zone of massive cell death by apoptosis was observed at E14 within the nasal mesenchyme located ventrally and caudally to the olfactory nerve layer. Double-labeling experiments showed that apoptotic cells present within this zone were not neurons. Our findings strongly suggest that apoptotic cell death of migrating neurons may allow the elimination of non-functional cells whereas that of mesenchymal cells may facilitate outgrowth of the newly formed olfactory axon fascicles by pathway formation.

Time-course of apoptosis in the olfactory epithelium of rainbow trout exposed to a low copper level.

Copper at low doses is known to specifically induce olfactory neuron death in fish olfactory epithelium. Using light and electron transmission microscopy, we have investigated the features and the time-course of receptor cell death in rainbow trout exposed for 15 days to 20 mug Cu(2+)/l. Ultrastructural observations demonstrate that degenerating cells, which included both mature and immature neurons, exhibited morphological changes characteristic of a cell death by apoptosis. Quantitative analysis shows that the number of apoptotic cells increased significantly already after 1 day of exposure, reaching a peak at day 5. From this timepoint of exposure, no more mature neuron was noted in the olfactory epithelium. Following a significant decrease in the number of apoptotic cells at day 10, a second wave of neuron death was noted at day 15. These findings argue for the occurrence of a neurogenesis process to balance the receptor cell death, despite continued copper exposure, and for a higher vulnerability to the metal of olfactory neurons presenting more advanced stages of cell differentiation. The molecular mechanisms by which copper may induce olfactory neuron apoptosis are discussed.

Analysis of the possible altering function of the septal organ in rats: a lesional and behavioral study.

The septal olfactory organ is a small patch of sensory epithelium located on the septal wall at the entry of the nasopharynx. There is a general consensus that by sampling olfactory stimuli during periods of rest, this organ may have an alerting function. To verify this hypothesis, we have lesioned by electrocoagulation the septal organ of male rats and recorded by polygraphy their awakening reaction in response to biologically meaningful (trimethyl-thiazoline, dimethyl sulfite, food) and meaningless (geraniol, eucalyptol) odorants. The awakening reactions of both lesioned and intact rats in response to these odorants were studied according to 3 parameters, frequency, latency and duration of awakening, and were analyzed using three-way analyses of variance. Data show that no significant difference in the awakening reactions was observed between control and lesioned animals. In all cases, the biologically meaningful odors presented the highest awakening influence. In addition, two of these odors (trimethyl-thiazoline and dimethyl sulfite) elicited a later habituation in comparison to biologically meaningless odors. From our results, it could be inferred that the hypothesis regarding an alerting function that would be specific to the septal organ, appears no longer current.

[Modifications of the olfactory bulb electrocorticogram and trans-glomerular evoked potentials in staggerer mutant mice]. / Modifications de l'électrocorticogramme du bulbe olfactif et des potentiels évoqués transglomérulaires chez la souris mutante cérébelleuse staggerer.

Electrophysiological observations have been made on normal C57-BL/6J and staggerer mutant mice. Morphological observations have given evidence it existed various neurones modifications which affected the olfactory bulb in the mutant mice. Olfactory bulb electrocorticograms (ECoG) of mutant have shown rare bursts of potentials of longer time duration than in normal mice. These bursts were less affected by odor stimulations (ammonia and urine of opposite sexes) than in the normal mice and never varied under urine odor influence in female mutant. Evoked potential induced by the odors had long latency and long duration (up to 50 ms vs 30 ms). In a large amount of them, the late phases and the late oscillatory potentials, which generally followed the evoked potential, were absent. All these results improved the idea that staggerer mutation, which mainly affected the N-CAM gene, not only induced cerebellar diseases, but also functionally affects the olfactory bulb.

Immunocytochemical study of the differentiation process of the septal organ of Masera in developing rats.

The septal organ of Masera is a small patch of olfactory epithelium located near the base of the nasal septum. Using the growth-associated protein B-50/GAP-43 as neuronal marker, we have studied the differentiation process of this organ from the olfactory sheet in embryonic and newborn rats. Results show that the septal organ first appeared at embryonic day 16. Even though it was included in the olfactory sheet, the presumptive septal organ could be distinguished by a higher density of B-50/GAP-43-positive neurons. Concomitantly to its morphological development, the septal organ progressively isolated from the main olfactory epithelium. This isolation resulted from the extension of a transitional area which progressively lost its typical features of olfactory epithelium to become a putative respiratory epithelium in late embryonic stages. Results strongly suggest that the septal organ should be a proper chemosensory system with its own time-course of development.

Detection of apoptosis by electron microscopy and in situ labelling in the rat olfactory pit.

The purpose of this study was to provide further information upon the cell death process by apoptosis occurring in the olfactory pit during the primary palate formation and the vomeronasal organ detachment. Apoptotic cells were detected by coupling ultrastructural observations and in situ end-labelling of DNA breaks (TUNEL labelling) in E12-E15 rat embryos. During the primary palate formation and the vomeronasal organ closure, a strong apoptotic cell death process was observed along the midline epithelial seam after the epithelial fusion. The topographical distribution of labelled nuclei was in agreement with the morphological distribution of dying cells. One day before the nasal swellings fused, numerous degenerating cells were also detected in the regions of prospective contact which thus appeared as regions of programmed cell death.

B-50/GAP-43 expression by the olfactory receptor cells and the neurons migrating from the olfactory placode in embryonic rats.

B-50/GAP-43 is a growth-associated phosphoprotein that is commonly expressed in all developing neuronal systems. Using an immunocytochemistry approach, we have investigated the expression of this protein in the rat olfactory system during embryogenesis and neonatal development with a particular emphasis on the early developmental stages of the olfactory placode. Data show that already at embryonic day 12 (E12), a strong B-50/GAP-43 immunoreactivity was detected in few olfactory receptor cells well-recognizable by their positive short neuritic processes. The B-50/GAP-43 expression in the placodal epithelium thus appeared to coincide with the onset of neurite outgrowth. From E13 onwards, there was a rapid increase in the number of B-50/GAP-43-positive olfactory neurons and from E18, the protein was strongly expressed by nearly all neurons. In addition, results clearly demonstrate that as early as E13, B-50/GAP-43 was strongly expressed by many migrating cells which were seen leaving the pit epithelium in association with the first olfactory axons that penetrated the nasal mesenchyme. Many immunoreactive cells were also observed in the presumptive olfactory nerve layer. Experiments of double-labeling showed that B-50/GAP-43-immunostained migrating cells were also stained with anti-neuron-specific enolase (NSE). This confirms the neuronal nature of these early labeled migrating cells. The progressive disappearance of migrating neurons noted during the late stages of embryonic development is discussed in relation with their possible function in the early stages of development of the peripheral olfactory system.

Cell death in the developing olfactory epithelium of rat embryos.

Cell death process in the developing olfactory epithelium was studied by light and electron microscopy in rat embryos from embryonic days 12-18. A massive wave of cell death was observed at embryonic days 12 and 13 and two types of dying cells were seen coexisting. The first type of dying cells exhibited morphological features of apoptosis while the second showed characteristics of a cell death by non-lysosomal disintegration with cytoplasmic swelling and absence of phagocytosis. From embryonic day 14 onward, only apoptotic figures could be still occasionally observed. The significance of such a massive wave of cell death occurring during the earliest developmental stages of the rat olfactory epithelium is discussed in relation with the morphogenesis of the olfactory system.

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode.

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.

Biotransformation enzyme activities in the olfactory organ of rainbow trout (Oncorhynchus mykiss). Immunocytochemical localization of cytochrome P4501A1 and its induction by ß-naphthoflavone.

Olfaction is a crucial function in most fish species, but little is known about biotransformation enzymes in the olfactory organ. This study demonstrates that biotransformation enzymes usually found in the rainbow trout liver, are present in the olfactory organ as well. While microsomal cytochrome P450 reductase, p-nitrophenol hydroxylase and cytosolic glutathioneS-transferase presented similar levels in both the olfactory organ and the liver, microsomal 7-ethoxyresorufinO-deethylase (EROD), 7-ethoxycoumarinO-deethylase, and 7-pentoxyresorufinO-deethylase were much lower in the olfactory organ (77-, 35-, 200-times respectively). Furthermore, microsomes from the olfactory organ were able to perform testosterone hydroxylation only in the 16α-position while testosterone was hydroxylated in the 16ß-position by liver microsomes. Using polyclonal antibodies raised against perch cytochrome P4501A1, the immunoreactive protein was shown to be strongly expressed in various cellular types forming the nonsensory epithelium. Some immunostaining was also reported in the nonsensory cellular elements constituting the sensory epithelium, while olfactory receptor cells failed to show cytochrome P4501A1-immunoreactivity. Finally, the exposure of rainbow trout to waterborne ß-naphthoflavone (0.1 µg ml(-1)) for 2 or 4 days resulted in a higher induction of EROD activity in the olfactory organ compared to the liver. The presence of biotransformation enzymes in the olfactory organ of rainbow trout addresses the question of their involvement in the detoxication/toxication of pollutants as well as in the olfactory function.

Effects of chronic low-level copper exposure on ultrastructure of the olfactory system in rainbow trout (Oncorhynchus mykiss).

This study investigated the effects of a chronic exposure to a low level of copper on cell populations of the olfactory system in yearling rainbow trout. Fish were sacrificed after 15, 30 and 60 days of copper exposure. Transmission electron microscopy was used to describe the sequence of subcellular changes occurring in three tissues, the sensory epithelium, the olfactory nerve and the olfactory bulb. Data show that a 15-day exposure to 20 micrograms/l of copper causes specific degeneration of all mature receptor cells as well as numerous immature neurons. Moreover, degenerating receptor cells exhibited morphological features of a cell death by apoptosis. After 30 days, and more specifically after 60 days of exposure, numerous clusters of cells were observed in the basal region of the epithelium, suggesting a great mitotic activity in this area. In parallel, an increased number of maturing receptor cells and goblet cells were observed, but no fully mature neurons were noted even after 60 days of exposure. In both the olfactory nerve and the olfactory bulb, the number of degenerating axons and terminals, which was high at 15 days, decreased with time and some process of glomerular reinnervation was detected after 60 days. A reactive hypertrophy of supporting, ensheathing and astrocytic cells was also observed in exposed fish, which demonstrates that these cell types are actively involved in the process of tissue scarring. Even though some signs of neuronal regeneration were reported during the time-course of exposure, indicating some fish acclimation, results raise the question of the olfactory function during such environmental stress.

The CVS strain of rabies virus as transneuronal tracer in the olfactory system of mice.

The sequential distribution of transneuronally infected neurons was studied in the olfactory pathway of mice after unilateral inoculation of the challenge virus standard (CVS) strain in the nasal cavity. A first cycle of viral multiplication was observed in a subpopulation of receptor cells scattered in the main olfactory epithelium and in the septal organ. No viral spread from cell body to cell body was reported even in later stages of infection. The second round of viral replication which took place in the ipsilateral main olfactory bulb at 2 and 2.5 days post-inoculation (p.i.), involved second order neurons and periglomerular cells, known to be directly connected with the axon terminals of receptor cells. Also reported as a result of a second cycle of viral replication, was surprisingly the spread of CVS at 2 and 2.5 days p.i. in bulbar interneurons located in the internal plexiform layer and in the superficial granule cell layer, as well as that of 2 ipsilateral cerebral nuclei, the anterior olfactory nucleus and the horizontal limb of the diagonal band. From day 3, a rapid spread of CVS was suggested by detection of virus in all ipsilateral direct terminal regions of the second order neurons and in most tertiary olfactory projections. The locus coeruleus, a noradrenergic nucleus which sends direct afferents to the olfactory bulb, never appeared immunoreactive. In spite of a certain inability of CVS to infect some neuron types, the virus appears relevant to provide new information regarding the complex network of olfactory-related neurons into the CNS.

Organization of the septal organ projection to the main olfactory bulb in adult and newborn rats.

The septal organ, which is regarded as an olfactory subsystem, is a small patch of sensory epithelium located ventral to the main olfactory sheet on the septal wall of the nasal cavity. The only consensus to date regarding some proper area of projection of this subsystem is that the septal organ projects to the medial aspect of the main olfactory bulb. The purpose of our study was to analyze precisely the topographical organization of the bulbar projection of the septal organ in adult rats and in 3- to 15-day-old rats following WGA-HRP placements at the level of the septal epithelium. Results show that the septal organ projects exclusively to the posterior half of the main olfactory bulb and its projection area is mainly restricted to the ventromedial bulbar aspect. When the septal organ was fully injected, the pattern of bulbar projection was characterized by two types of glomerular labeling: 1) presence of single heavily labeled glomeruli identified as "septal" glomeruli, since they were mainly built up by afferents coming from the septal organ and (2) presence of a thin network of labeled septal fibers distributed in glomeruli which were mainly formed by afferents coming from the main olfactory epithelium. Although the pattern of mucosobulbar projection of the septal organ is already established in newborns, a significant increase in the number of "septal" glomeruli occurs during the first 15 postnatal days. Anatomical data indicate that even if the projection of the septal organ does not appear completely segregated in the olfactory bulb, this projection is not either exactly similar to that of the main olfactory epithelium.

Spread of the CVS strain of rabies virus and of the avirulent mutant AvO1 along the olfactory pathways of the mouse after intranasal inoculation.

After intranasal instillation in the mouse, rabies virus (CVS strain) selectively infected olfactory receptor cells. In the main olfactory bulb (MOB), infection was observed in periglomerular, tufted, and mitral cells and in interneurons located in the internal plexiform layer. Beyond the MOB, CVS spread into the brain along the olfactory pathways. This infection is specific to chains of functionally related neurons but at the death of the animal some nuclei remain uninfected. CVS also penetrated the trigeminal system. The avirulent mutant AvO1, carrying a mutation in position 333 of the glycoprotein, infected the olfactory epithelium and the trigeminal nerve as efficiently as CVS. During the second cycle of infection, the mutant was able to infect efficiently periglomerular cells in the MOB and neurons of the horizontal limb of the diagonal band, which indicates that maturation of infective particles is not affected in primarily infected neuronal cells. On the other hand, other neuronal cells permissive for CVS, such as mitral cells or the anterior olfactory nucleus, are completely free of infection with the mutant, indicating that restriction is related to the ability of AvO1 to penetrate several categories of neurons. From these observations, we concluded that CVS should be able to bind several different receptors to penetrate neurons, while the mutant would be unable to recognize some of them.

Cellular expression of H and B antigens in the rat olfactory system during development.

Developmental expression of H and B antigens in the rat olfactory system was studied from the embryonic day 14 up to the postnatal day 30. The H antigen was detected in the olfactory and vomeronasal epithelia as early as fetal day 14, whereas the B antigen first appeared 2 days later. The anti-H reagent reacted strongly with sensory receptors and weakly with supporting cells in both epithelia, whereas the anti-B reagent was specific for olfactory receptors. In the main olfactory epithelium, the H antigen was expressed from fetal day 19 by most of the receptor cells, whereas the B determinant was expressed from fetal day 16 to postnatal day 3 by only a few neuroreceptors mostly located near the epithelial surface. After the postnatal day 3, B positive neurons increased in number from the periphery toward the deeper mucosal layers and they were distributed over 3/4 of the epithelial thickness in 15- and 30-day-old rats. In the main olfactory bulb, a widespread glomerular B staining with variable binding intensity between adjacent glomeruli was already observed at birth. The vomeronasal receptor cells and their axon terminals in the accessory olfactory bulb exhibited a comparable developmental expression of the B antigen. Results suggest that the B antigen could be regarded as a marker of neuronal maturation of both the olfactory and vomeronasal receptor cells; moreover, its first appearance in the receptor cells might be temporally related to the formation of synapses between receptor axons and deutoneurons in the bulb.