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.

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.

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.

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.

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.

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.

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.

Topographical projection of the septal organ to the main olfactory bulb in rats: ontogenetic study.

Using the horseradish peroxidase (HRP) retrograde tracing method, we have studied the topographical organization of the projection of the septal organ to the main olfactory bulb in rats varying in age from 22 fetal days up to 15 postnatal days. For all developmental stages studied, receptor cells of the septal organ had their axons ending in a relatively circumscribed region of the olfactory bulb, which was the posterior ventromedial bulbar aspect. In younger animals, olfactory receptor cells were observed within the epithelial area isolating the septal organ from the olfactory epithelium, whereas this area was reported to be an exclusive respiratory region in adult rats. The complete disappearance of these receptor cells noted in 12-day-old rats was related to some ontogenetic process.

Ontogenesis of the functional activity of guinea-pig olfactory bulb: autoradiographic study with the 2-deoxyglucose method.

An ontogenetic study of [14C]2-deoxyglucose (2-DG) uptake within the olfactory bulbs was performed in 1-, 4-, 7- and 21 day-old guinea-pigs exposed to a pure chemical odor, ethyl acetoacetate (EAA). Whatever the age studied, the patterns of glomerular activity are characterized by restricted spots which extend along the bulb, and by sheets of increased density overlapping 10-30 contiguous glomeruli, found only on the lateral aspect of the bulb. Even if the main features of the 2-DG patterns related to EAA are already present at birth, a perceptible evolution of these patterns is observed during the first 3 postnatal weeks. The changes include on the one hand, an increase of the spot-like glomerular labeling which appears particularly prominently on the medial and dorsal aspects. On the other hand, whereas the restricted spots overlaying 1-3 glomeruli become more numerous with age, a decrease in size and in extension of the sheet-like glomerular activity is noted. The results confirm the high maturation level of the olfactory bulbs in newborn guinea-pigs. Some implications related to the mainly prenatal maturation of the olfactory system are discussed.

Metabolic mapping of functional activity in the rat olfactory system after a bilateral transection of the lateral olfactory tract.

The effects of the bilateral transection of the LOT on the patterns of 2-deoxyglucose uptake within the olfactory bulb and the olfactory projections were studied in adult rats. Animals were exposed to a pure neutral odor, ethyl acetoacetate (EAA) or to a biological alarming odor, fox odor. In intact animals, the patterns of glomerular activity elicited by EAA and fox odor appeared complex. An average number of 30-40 foci of 2-DG uptake was noted in each bulb and the optical density of the foci spread out according to a continuous gradient. These patterns were largely overlapping on the lateral aspect and the third-to-posterior part of the medial aspect of the bulb. Nevertheless, they were somewhat different and the spatial distribution of the darkest foci seemed particularly relevant for the pattern recognition. In lesioned animals, the same spatial distribution of the foci as in intact rats, was observed in the lateral and the medial aspects of the bulb. Nevertheless, lesioned animals presented some quantitative changes in their patterns of glomerular labeling. Moreover, these patterns appeared different according to the biological meaning of the odor tested. The bilateral transection of the LOT brought about a very strong decrease of the optical density in the direct olfactory projections. No evident change of 2-DG uptake was noted in the different tertiary olfactory projections. These 2-DG results confirm the anatomical data relating to the LOT projections.

Metabolic mapping of functional activity in the olfactory projections of the rat: ontogenetic study.

An ontogenetic study of the uptake of [14C]2-deoxy-D-glucose (2-DG) within the direct olfactory bulb projections and the tertiary olfactory projections was performed on rats of 1, 9 and 21 days old. Animals were exposed either to ethyl acetoacetate or to nest odor. In newborns, most of the direct olfactory bulb projections - anterior olfactory nucleus, anterior part of the olfactory tubercle, piriform cortex and nucleus of the lateral olfactory tract - appear labelled on films and therefore seem functional. No evidence of 2-DG uptake can be brought out in the cortical amygdala nucleus. AS regards the tertiary olfactory projections, there is no apparent functional activity, neither in the medio-dorsal and medio-ventral thalamic nuclei nor in the hypothalamic nuclei, e.g. the lateral preoptic area and the lateral hypothalamus. In 9-day-old pups, the direct olfactory bulb projections and the tertiary olfactory projections appear well-contrasted. Moreover, the patterns of labelling within the direct olfactory bulb projections are comparable to those observed in 21-day-old rats and in adult. These data are correlated with the postnatal development of the discriminating ability of the rat.

Ontogenesis of the functional activity of rat olfactory bulb: autoradiographic study with the 2-deoxyglucose method.

An ontogenetic study of the uptake of [14C]2-deoxyglucose (2-DG) within the olfactory bulb was performed on rats of 3 age groups, 1, 9 and 21 days. Animals were exposed either to ethyl acetoacetate (EAA) or to a nest odor. Already in newborns, a glomerular activity characterized by small spots of increased 2-DG uptake, appeared in response to either odor stimulation. An ontogenetic development of this glomerular activity was observed with age: whereas the labeled glomerular foci were scanty in newborns, their number constantly increased with age until weaning; moreover, an increased proportion of foci overlaying 2 or 3 single glomeruli was noted in parallel with the postnatal maturation of the olfactory system. For the 3 age groups, whatever the odor stimulus, the glomerular activity seemed located in 2 main regions of the olfactory bulb, the lateral aspect and the posterior part of the medial aspect of the bulb. In newborns, no clear difference could be brought out between the patterns of glomerular activation related to EAA and to nest odor, respectively. In 9-day-old rats, the spatial patterns of distribution associated with the 2 odors were overlapping, but nevertheless different. These data are correlated with the postnatal maturation of the olfactory system.

Topographical relationships between olfactory receptor cells and glomerular foci in the rat olfactory bulb.

We have studied the distribution patterns of olfactory receptor cells that project to glomerular foci after HRP injections in the different bulbar surfaces of 15-day-old rats. HRP label overlapped only 2-4 contiguous glomeruli and had a maximal extent of 260 micron along the antero-posterior (A-P) axis. HRP injections were confined enough to allow labeled individual receptor cells to be discerned in the epithelial sheet. Results have shown that neurons ending in a few contiguous glomeruli of a given bulbar surface were distributed in most of the regions projecting to this bulbar surface. Moreover, within these epithelial areas, labeled cells were largely dispersed in both the frontal and the A-P axes of the nose. When few HRP-filled neurons were observed on a turbinate or a recess, their distribution seemed organized according to a certain alignment along the A-P nasal axis. Our findings demonstrate for the first time that the principal features of the regional organization of epithelium projections to a given bulbar surface are reproduced in any glomerular focus of this bulbar surface, and suggest that it could be the same for any single glomerulus. Such a principle of projection supports the concept of a large redundance in the anatomical relationship between periphery and bulb.

Spatial distribution of [14C]2-deoxyglucose uptake in the olfactory bulbs of rats stimulated with two different odours.

The uptake of [14C]2-deoxy-D-glucose (2-DG) has been studied by autoradiography in the olfactory bulbs of control and odour-stimulated rats. The sites of highest 2-DG-uptake coincide very accurately with individual glomeruli. The other bulbar histological layers appear to be far less metabolically affected by the olfactory stimulation. The mapping of the glomerular activation has been compared in two groups of animals stimulated with two different odours. The patterns of selective glomerular 2-DG-uptake are rather similar within each group. They differ from one group to the other by the number and localization of the highly labelled glomeruli. It can be inferred from our observations that a few glomeruli are metabolically highly activated by a strong and pure odour stimulation. A correlation between the quality of the odour and the pattern of glomerular activation may be supposed but has to be confirmed with other compounds.

Metabolic mapping of 2-deoxyglucose uptake in the rat piriform cortex using computerized image processing.

Using a computerized image processing method, the metabolic activity of the unfolded molecular and pyramidal layers of the piriform cortex was studied in rats during odorous stimulation. Animals were either intact or had sustained a bilateral transection of the lateral olfactory tract. 2-DG labelling was observed in corresponding areas across these two layers. No spatial pattern of 2-DG uptake could be seen in correlation with the odorant quality. These results are discussed with respect to the known anatomical and functional properties of the piriform cortex.

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.

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.

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.

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.

Anatomical mapping of the neuroepithelial projection to the olfactory bulb in the rat.

The topographical organization of the epithelium-to-bulb projections was studied in 15-day-old rats by the retrograde tracing method using horseradish peroxidase (HRP). Injections of HRP were placed in the glomerular layer of the 4 bulbar surfaces; the location and the maximal extent of labeled epithelial areas were assessed. Results have shown an important but variable degree of overlapping of peripheral projections to the bulb. The dorsal aspect received inputs from only the anterior dorsal recess and endoturbinate II. Epithelial projections to the lateral side included the anterior dorsal recess and turbinates 1, 2-2' and II-II'. Peripheral projections converging to the medial surface were the most various; they comprised the septal wall, the posterior dorsal recess, ectoturbinate 3 and all endoturbinates. The ventral pole received afferents from the lateral recess, lower parts of the septal wall and turbinates 2-2', 3, III, IV and also recesses between these turbinates. Otherwise, three epithelial areas showed divergent projections to the bulb. Information processing via convergence and divergence of epithelium-to-bulb connections may represent differential functions in olfactory information coding.