Olfactory ensheathing cells: the primary innate immunocytes in the olfactory pathway to engulf apoptotic olfactory nerve debris.

The olfactory system is an unusual tissue in which olfactory receptor neurons (ORNs) are continuously replaced throughout the life of mammals. Clearance of the apoptotic ORNs corpses is a fundamental process serving important functions in the regulation of olfactory nerve turnover and regeneration. However, little is known about the underlying mechanisms. Olfactory ensheathing cells (OECs) are a unique type of glial cells that wrap olfactory axons and support their continual regeneration from the olfactory epithelium to the bulb. In the present study, OECs were identified to exist in two different states, resting and reactive, in which resting OECs could be activated by LPS stimulation and functioned as phagocytes for cleaning apoptotic ORNs corpses. Confocal analysis revealed that dead ORNs debris were engulfed by OECs and co-localized with lysosome associated membrane protein 1. Moreover, phosphatidylserine (PS) receptor was identified to express on OECs, which allowed OECs to recognize apoptotic ORNs by binding to PS. Importantly, engulfment of olfactory nerve debris by OECs was found in olfactory mucosa under normal turnover and was significantly increased in the animal model of olfactory bulbectomy, while little phagocytosis by Iba-1-positive microglia/macrophages was observed. Together, these results implicate OEC as a primary innate immunocyte in the olfactory pathway, and suggest a cellular and molecular mechanism by which ORNs corpses are removed during olfactory nerve turnover and regeneration.

Cytokines and olfactory bulb microglia in response to bacterial challenge in the compromised primary olfactory pathway.

BACKGROUND: The primary olfactory pathway is a potential route through which microorganisms from the periphery could potentially access the central nervous system. Our previous studies demonstrated that if the olfactory epithelium was damaged, bacteria administered into the nasal cavity induced nitric oxide production in olfactory ensheathing cells. This study investigates the cytokine profile of olfactory tissues as a consequence of bacterial challenge and establishes whether or not the bacteria are able to reach the olfactory bulb in the central nervous system. METHODS: The olfactory epithelium of C57BL/6 mice was damaged by unilateral Triton X-100 nasal washing, and Staphylococcus aureus was administered ipsilaterally 4 days later. Olfactory mucosa and bulb were harvested 6 h, 24 h and 5 days after inoculation and their cytokine profile compared to control tissues. The fate of S. aureus and the response of bulbar microglia were examined using fluorescence microscopy and transmission electron microscopy. RESULTS: In the olfactory mucosa, administered S. aureus was present in supporting cells of the olfactory epithelium, and macrophages and olfactory nerve bundles in the lamina propria. Fluorescein isothiocyanate-conjugated S. aureus was observed within the olfactory mucosa and bulb 6 h after inoculation, but remained restricted to the peripheral layers up to 5 days later. At the 24-h time point, the level of interleukin-6 (IL-6) and tumour necrosis factor-α in the compromised olfactory tissues challenged with bacteria (12,466 ± 956 pg/ml and 552 ± 193 pg/ml, respectively) was significantly higher than that in compromised olfactory tissues alone (6,092 ± 1,403 pg/ml and 80 ± 2 pg/ml, respectively). Immunohistochemistry confirmed that IL-6 was present in several cell types including olfactory ensheathing cells and mitral cells of the olfactory bulb. Concurrently, there was a 4.4-, 4.5- and 2.8-fold increase in the density of iNOS-expressing cells in the olfactory mucosa, olfactory nerve and glomerular layers combined, and granule layer of the olfactory bulb, respectively. CONCLUSIONS: Bacteria are able to penetrate the immunological defence of the compromised olfactory mucosa and infiltrate the olfactory bulb within 6 h even though a proinflammatory profile is mounted. Activated microglia may have a role in restricting bacteria to the outer layers of the olfactory bulb.

Transient ischemia-induced changes of neurofilament 200 kDa immunoreactivity and protein content in the main olfactory bulb in gerbils.

This study was carried out to investigate alterations of neurofilament 200 kDa (NF-200) and its polyphosphorylation form (RT97) immunoreactivity and protein content in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. In the sham-operated group, weak NF-200 immunoreactivity was detectable in a few somata of mitral cells, which projected weak NF-200-immunoreactive processes to the external plexiform layer (EPL). At 1-5 days after ischemia, strong NF-200 and RT97 immunoreactivity was shown by the mitral cell processes; however, somata of mitral cells did not show NF-200 immunoreactivity. At this time point, strong NF-200-immunoreactive mitral cell processes ran to the EPL and glomerular layer (GL). Thereafter, NF-200 and RT97 immunoreactivity was decreased up to 30 days after ischemia. In the 15 days post-ischemic group, the distribution pattern of NF-200 and RT97 immunoreactivity was slightly lower than that in the 1-5 days post-ischemic groups. In the 30 days post-ischemic group, moderate NF-200 and RT97 immunoreactivity was found in the mitral cells processes, but the immunoreactivity in the EPL and GL nearly disappeared. A Western blot study showed a pattern of NF-200 and RT97 expression at all post-ischemic time points similar to that of immunohistochemistry after ischemia. This result indicates that NF-200 and RT97 accumulates in injured mitral cell processes a few days after transient ischemia, which suggests that the axonal transport in the MOB may be disturbed during this period after transient ischemia.

Systematic screening and identification of antigens recognized by monoclonal antibodies raised against the developing lateral olfactory tract.

During development, olfactory bulb axons navigate a complex microenvironment composed of myriad molecules to construct a bundle called the lateral olfactory tract. The axons themselves also express thousands of different molecules. In the present study, we produced and characterized six monoclonal antibodies that label the lateral olfactory tract and its surroundings in a unique pattern. The labeling profiles suggested that the antigen molecules recognized by each antibody are heterogeneously distributed around the developing lateral olfactory tract. We developed an efficient screening method to identify the antigen molecules by combining expression of a cDNA library in COS-7 cells and the subsequent immunohistochemical staining of the cells. The systematic screening successfully identified specific cDNA clones for all of the monoclonal antibodies, which highly probably coded for the antigen molecules, and therefore unveiled the molecular nature of local components that embrace the developing lateral olfactory tract in mice.

c-Fos expression in the rat cerebral cortex during systemic GvH reaction.

OBJECTIVE: It is becoming clear that the CNS receives signals from the peripheral immune system. In order to identify the areas of the brain that receive information about a specific immune response to allogeneic antigens, we studied the expression of c-Fos, a neural activation marker, in the cerebral cortex following the induction of a graft-vs.-host reaction (GvHR) in rats. METHODS: C-Fos expression in the brain was studied by immunohistochemistry. GvHR was induced in (WKY x PVG)F(1) rats by injecting 5 x 10(8) spleen cells from PVG rats. Control rats received syngeneic cells. RESULTS: No c-Fos immunoreactivity (IR) was observed in animals undergoing GvHR in the nucleus tractus solitarii (NTS), the locus coeruleus (LC), the organum vasculosum of lamina terminalis (OVLT), the paraventricular nucleus (PVN) or the central amygdaloid nucleus (Ce). In contrast, 3 days after GvH induction c-Fos IR was observed in the piriform cortex and several other olfactory-related regions indicating the stimulation of the olfactory pathway during GvHR. Strong c-Fos IR was also observed in the occipital visual cortex of animals undergoing a GvHR, suggesting that GvHR can affect visual functions. In addition, GvHR induced c-Fos IR in the prefrontal cortex (Cg3, orbital cortex), a region that has interconnections with most sensory modalities. Double-staining studies indicate that the cells that express the c-Fos signal are neurons. CONCLUSION: We have defined the distribution of brain neurons that are affected during the induction phase of GvHR. Our results also indicate that the integration and processing of information from the immune system at CNS levels involve different areas during different types of immune responses.

Immunolocalization of synaptotagmin for the study of synapses in the developing antennal lobe of Manduca sexta.

In the mature olfactory systems of most organisms that possess a sense of smell, synapses between olfactory receptor neurons and central neurons occur in specialized neuropil structures called glomeruli. The development of olfactory glomeruli has been studied particularly heavily in the antennal lobe of the moth Manduca sexta. In the current study, we address the development of synapses within the antennal lobe of M. sexta by reporting on the localization of synaptotagmin, a ubiquitous synaptic vesicle protein, throughout development. A cDNA clone coding for M. sexta synaptotagmin was characterized and found to encode a protein that shares 67% amino acid identity with Drosophila synaptotagmin and 56% amino acid identity with human synaptotagmin I. Conservation was especially high in the C2 domains near the C-terminus and very low near the N-terminus. A polyclonal antiserum (MSYT) was raised against the unique N-terminus of M. sexta synaptotagmin, and a monoclonal antibody (DSYT) was raised against the highly conserved C-terminus of D. melanogaster synaptotagmin. In Western blot analyses, both antibodies labeled a 60 kD protein, which very likely corresponds to synaptotagmin. On sections, both antibodies labeled known synaptic neuropils in M. sexta and yielded similar labeling patterns in the developing antennal lobe. In addition, DSYT detected synaptotagmin-like protein in three other insect species examined. Analysis of synaptotagmin labeling at the light microscopic level during development of the antennal lobe of M. sexta confirmed and extended previous electron microscopic studies. Additional synapses in the coarse neuropil and a refinement of synaptic densities in the glomeruli during the last one-third of metamorphic development were revealed.

Localization of FMRFamide-like immunoreactivity in the brain of the viviparous skink (Chalcides chalcides).

Neuroanatomical distribution of FMRFamide-like immunoreactivity was investigated in the brain and olfactory system of the viviparous skink, Chalcides chalcides. In the adult brain FMRFamide immunoreactive (ir) perikarya were observed in the diagonal band of Broca, medial septal nucleus, accumbens nucleus, bed nucleus of the anterior commissure, periventricular hypothalamic nucleus, lateral forebrain bundle, and lateral preoptic, subcommissural, suprachiasmatic and lateral hypothalamic areas. This pattern was seen in both male and female brains. Though all major brain areas showed FMRFamide-ir innervation, the densest ir fiber network was observed in the hypothalamus. During development, ir elements were observed for the first time in embryos at mid-pregnancy. FMRFamide perikarya were located along the ventral surface of the vomeronasal nerve, in the olfactory peduncle mediobasally, as well as in the anterior olfactory nucleus and olfactory tubercle. Furthermore, some ir neurons were observed in the rhombencephalic reticular substance; however, the ir fiber network was poorly developed. Later in development FMRFamide-ir neurons appeared also in the bed nucleus of the anterior commissure as well as the rhombencephalic nucleus of solitary tract and the dorsal motor nucleus of vagus nerve. In juveniles, the distribution profile of FMRFamide immunoreactivity was substantially similar to that of the adults, with a less widespread neuronal distribution and a more developed fiber network. Ontogenetic presence of FMRFamide immunoreactivity in the nasal area has been linked to the presence of a nervus terminalis in this reptile.

Bovine herpesvirus 5 glycoprotein E is important for neuroinvasiveness and neurovirulence in the olfactory pathway of the rabbit.

Glycoprotein E (gE) is important for full virulence potential of the alphaherpesviruses in both natural and laboratory hosts. The gE sequence of the neurovirulent bovine herpesvirus 5 (BHV-5) was determined and compared with that of the nonneurovirulent BHV-1. Alignment of the predicted amino acid sequences of BHV-1 and BHV-5 gE open reading frames showed that they had 72% identity and 77% similarity. To determine the role of gE in the differential neuropathogenesis of BHV-1 and BHV-5, we have constructed BHV-1 and BHV-5 recombinants: gE-deleted BHV-5 (BHV-5gEDelta), BHV-5 expressing BHV-1 gE (BHV-5gE1), and BHV-1 expressing BHV-5 gE (BHV-1gE5). Neurovirulence properties of these recombinant viruses were analyzed using a rabbit seizure model (S. I. Chowdhury et al., J. Comp. Pathol. 117:295-310, 1997) that distinguished wild-type BHV-1 and -5 based on their differential neuropathogenesis. Intranasal inoculation of BHV-5 gEDelta and BHV-5gE1 produced significantly reduced neurological signs that affected only 10% of the infected rabbits. The recombinant BHV-1gE5 did not invade the central nervous system (CNS). Virus isolation and immunohistochemistry data suggest that these recombinants replicate and spread significantly less efficiently in the brain than BHV-5 gE revertant or wild-type BHV-5, which produced severe neurological signs in 70 to 80% rabbits. Taken together, the results of neurological signs, brain lesions, virus isolation, and immunohistochemistry indicate that BHV-5 gE is important for efficient neural spread and neurovirulence within the CNS and could not be replaced by BHV-1 gE. However, BHV-5 gE is not required for initial viral entry into olfactory pathway.

Neural invasion of two virulent suid herpesvirus 1 strains in neonatal pigs with or without maternal immunity.

The neural invasion of two virulent Suid Herpesvirus 1 (SHV1) strains was examined in neonatal pigs with or without maternal immunity. One-week-old pigs with comparable levels of maternal immunity (SN-titer = 12-48) were intranasally inoculated with 10(7.0) TCID50 of either of the Ka or E21 strains. The invasion of the strains was examined in the nasal mucosa and in three neuronal levels of the trigeminal nervous pathway as well as in three levels of the olfactory nervous pathway by virus titration and immunohistochemistry (IHC). In control pigs without specific antibodies, both strains invaded up to the end level of each neural pathway. In pigs with maternal immunity, the Ka strain invaded only up to the 2nd level of each pathway with titers being significantly lower (p<0.05) than in the negative controls. However, the E21 strain invaded up to the end levels in both neural pathways of immune pigs with virus titers being similar to those observed in non-immune pigs (p>0.05). IHC revealed that maternal antibodies can protect against a fibroblast-mediated spread of the Ka strain in the lamina propria of the nasal mucosa, as well as against a local spread of the Ka and E21 strains from neurons to their satellite cells in the trigeminal ganglion. In conclusion, the nature of virus strain determines the invasion of SHV1 within the nervous system of maternally-immune neonatal pigs.

Differential antigen expression during metamorphosis in the tripartite olfactory system of the African clawed frog, Xenopus laevis.

In the adult African clawed frog, Xenopus laevis, olfactory epithelium is housed in three separate nasal cavities: the principal cavity, the middle cavity, and the vomeronasal organ. The sensory epithelium in each of these cavities has distinct cellular features, and presumed physiological and behavioral functions, which arise during metamorphosis. Most notably, the middle cavity is formed de novo, and the principal cavity is transformed from a larval sensory epithelium with water exposure to an adult olfactory epithelium with air exposure. To understand the cellular nature of this plasticity more clearly, we characterized the staining patterns generated in the olfactory system of X. laevis with a new monoclonal antibody, anti-E7. The olfactory epithelium is first stained with anti-E7 during late embryonic development. Transection of the olfactory nerves during metamorphosis eliminates all staining and indicates that the staining is associated with mature or nearly mature olfactory receptor neurons. The antibody diffusely stains the vomeronasal organ throughout development and in adults. In the larval principal cavity, the olfactory receptor neurons are brightly stained, but this cellular staining is lost after metamorphosis. The mucus from Bowman's glands in the principal cavity, however, is intensely stained in adults. The middle cavity, throughout development and in adulthood, has the same staining characteristics as the larval principal cavity. Thus, the E7 antibody can distinguish the three areas of the olfactory epithelium, allowing measurement of sensory epithelium volume, and serves as an excellent marker for the changes in the sensory epithelium that occur during metamorphosis.

Tripartite mushroom body architecture revealed by antigenic markers.

We have explored the organization of the axonal lobes in Drosophila mushroom bodies by using a panel of immunohistochemical markers. These markers consist of antibodies to eight proteins expressed preferentially in the mushroom bodies: DAMB, DCO, DRK, FASII, LEO, OAMB, PKA RII, and RUT. Previous to this work, four axonal lobes, two projecting dorsally (alpha and alpha') and two medially (beta and gamma), had been described in Drosophila mushroom bodies. However, our analysis of immunohistochemically stained frontal and sagittal sections of the brain revealed three medially projecting lobes. The newly distinguished lobe, which we term beta', lies along the dorsal surface of beta, just posterior to gamma. In addition to resolving a fifth lobe, our studies revealed that there are specific lobe sets defined by equivalent marker expression levels. These sets are (1) the alpha and beta lobes, (2) the alpha' and beta' lobes, and (3) the gamma lobe and heel (a lateral projection formed by a hairpin turn of some of the peduncle fibers). All of the markers we have examined are consistent with these three sets. Previous Golgi studies demonstrate that each mushroom body cell projects one axon that branches into a dorsal lobe and a medial lobe, or one unbranched axon that projects medially. Taken together with the lobe sets listed above, we propose that there are three major projection configurations of mushroom body cell axons: (1) one branch in the alpha and one in the beta lobe, (2) one branch in the alpha' and one in the beta' lobe, and (3) one unbranched axon projecting to the heel and the gamma lobe. The fact that these neuron types exhibit differential expression levels of a number of mushroom body genes suggests that they may have corresponding functional differences. These functions may be conserved in the larvae, as several of these genes were expressed in larval and embryonic mushroom bodies as well. The basic mushroom body structure, including the denritic calyx, peduncle, and lobes, was already visible by the late stages of embryogenesis. With new insights into mushroom body organization, and the characterization of markers for developing mushroom bodies, we are beginning to understand how these structures form and function.

Spread of measles virus through axonal pathways into limbic structures in the brain of TAP1 -/- mice.

The spread of measles virus into the brain was studied exploiting the olfactory pathway, which represents an important route of neuroinvasion by viruses. The virus was injected into the main olfactory bulb of wild-type mice and mice with disrupted TAP1 gene (TAP refers to the Transporter associated with Antigen Presentation), which codes for products essential for the cell-mediated immune response. Virus invasion was monitored for 4 weeks by immunohistochemistry. The distribution of measles virus was found to be restricted to brain areas connected with the olfactory bulbs. However, in the wild-type mice there was a marked infiltration of lymphocytes in the infected brain structures, and the virus did not pass beyond the piriform cortex. In the TAP1 -/- mice the virus spread more extensively along olfactory projections into the limbic system and monoaminergic brainstem neurons. Infected mice of both types developed seizures, which may have been focally evoked from the piriform cortex. This study provides evidence that measles virus can spread through axonal pathways in the brain. The findings obtained in the gene-manipulated mice point out that a compromised immune state of the host may potentiate targeting of virus to the limbic system through olfactory projections.

Differential expression of vomeromodulin and odorant-binding protein, putative pheromone and odorant transporters, in the developing rat nasal chemosensory mucosae.

Expression of the putative pheromone and odorant transporter, vomeromodulin, was characterized in developing rat nasal mucosae using in situ hybridization and immunocytochemistry. Initial expression of vomeromodulin mRNA and protein was detected at embryonic day (E)16 in the maxillary sinus component of the lateral nasal glands. The abundance of mRNA and protein in the lateral nasal glands increased with age and reached a peak at postnatal day (P)27. Also at P27, vomeromodulin mRNA and protein expression was initiated in vomeronasal glands and posterior glands of the nasal septum. Comparison of the developmental expression of odorant-binding protein, another carrier protein synthesized in the lateral nasal glands, with that of vomeromodulin demonstrated major differences. In contrast to vomeromodulin, odorant-binding protein was not detected until postnatal day 2 in the ventral component of the lateral nasal glands and anterior glands of the nasal septum. These results suggest that the expression of vomeromodulin and odorant-binding protein is developmentally and differentially regulated and confirms the suggestion that vomeromodulin may function in olfactory and vomeronasal perireceptor processes as a transporter for pheromones and odorants. In addition, the embryonic expression of vomeromodulin suggests its involvement in olfactory perireceptor processes in utero.

Analysis of immunocytochemical staining patterns in the antennal system of Drosophila melanogaster.

By immunizing mice with homogenized brains, heads, or a mixture of heads and antennae of D. melanogaster, we obtained six monoclonal antibodies (mabs) that bind to the olfactory system of Drosophila with various degrees of specificity. They can be divided into three groups with respect to their staining pattern: (1) The antibodies ca51/2, na21/2, and nb230 label both in the third (olfactory) antennal segment and in the visual ganglia. All of them bind to antennal structures that can be correlated with basiconic sensilla. The antibody ca51/2 labels sensory neurons of these sensilla. In the antenna of the lozenge mutant, which lacks basiconic sensilla, no labeling is present. In Western blots ca51/2 recognizes in the antenna an antigen of 43.5 kDa, which is expressed in the antenna only in the presence of basiconic sensilla. The antibody na21/2 binds to basiconic and coeloconic sensilla, most likely to the apical part of sheath cells. In immunoblots it recognizes in the antenna two antigens of 42.2 kDa and 46.7 kDa. The latter appears to be correlated in the antenna with the presence of basiconic sensilla. (2) The staining pattern of antibody nc10 is associated with the sheath cells of basiconic and coeloconic sensilla. Moreover, nc10 binds to a subset of glomeruli in the antennal lobe. (3) The staining pattern of the antibodies VG2 and I24B5 is restricted to the antenna. I24B5 recognizes coeloconic sensilla and VG2 recognizes both coeloconic and basiconic sensilla. Staining patterns in both cases include sheath cells.

Luteinizing hormone-releasing hormone (LHRH) and neural cell adhesion molecule (NCAM)-immunoreactivity in development of the forebrain and reproductive system.

The origin and migration of LHRH neurons (detected by immunocytochemical procedures) is preceded by a migration of NCAM-immunoreactive cells from the olfactory epithelium, and the formation of an NCAM-immunoreactive cellular aggregate between the olfactory epithelium and the developing forebrain. The central processes of the olfactory nerves grow into the lateral parts of this aggregate and the terminal and vomeronasal nerves grow into the medial parts. No nerve fibers of the main or accessory olfactory systems grow directly into the forebrain. The LHRH neurons, following the course of the terminal and vomeronasal nerves, traverse the medial edge of the NCAM-immunoreactive cellular aggregate before they enter the medial forebrain caudal to the developing olfactory bulbs. The LHRH neurons do not migrate through the olfactory bulbs. After formation of the olfactory bulbs, the cellular aggregate disappears and is replaced by the olfactory nerve layer of the olfactory bulb. The NCAM and LHRH-immunoreactive cells on the medial side appear to the retained in the ganglion terminale of the terminal nerve. The fate of the NCAM-immunoreactive cells that formed the aggregate could not be determined by the methods used in these studies. The early-appearing NCAM-immunoreactive cells may function to separate and direct axons of the olfactory, vomeronasal and terminal nerves (and the LHRH neurons) to their respective targets in the forebrain. The development and migration of neurons from both the lateral and medial parts of the olfactory placode appears to be essential for the normal development of the forebrain and reproductive system.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunoelectron microscopic analysis of a novel carbohydrate differentiation antigen (CDA-3C2) in the developing rat olfactory and otic systems.

A carbohydrate differentiation antigen (CDA-3C2) exhibits a highly specific and restricted pattern of expression during rat embryogenesis. In the periphery of the embryo, this antigen is associated transiently with the lateral ectoderm but is retained only in the olfactory and otic epithelium throughout morphogenesis. At the light microscopic level, CDA-3C2 immunoreactivity appears mostly along cell periphery and in the extracellular matrix. The aim of the present study was to determine the specific cellular and subcellular distribution of CDA-3C2 in vivo in order to identify potential sites of cellular and tissue function of the antigen during embryogenesis. There was a strikingly similar subcellular distribution of CDA-3C2 in the developing otic and olfactory systems, found mostly along cell membranes, microvillar projections and acellular secretions of the epithelium. Mature sensory components of the epithelia were not immunoreactive, whereas supportive cells and their secreted structures were densely stained. The highly coincident nature of CDA-3C2 in both sensory epithelia suggests that this carbohydrate epitope, and possibly its carrier macromolecule, participate in a morphogenetic function common to these two sensory epithelia.

Immunocytochemical analysis of a novel carbohydrate differentiation antigen (CDA-3C2) associated with olfactory and otic systems during embryogenesis in the rat.

Carbohydrate differentiation antigens are known to display specific patterns of expression during mammalian development and are thought to participate in significant morphogenetic events. In the present study, two monoclonal antibodies that react with a novel carbohydrate differentiation antigen (CDA-3C2) were used to analyze, by light microscopy, the spatiotemporal distribution of this unique high molecular weight antigen during embryogenesis in the rat. Correlative analysis of the development of peripheral neural structures, in which CDA-3C2 was expressed, was carried out with an anti-neurofilament antibody. Enzymatic digestion, combined with Western blots, reveal that the CDA-3C2 epitope is a carbohydrate which is carried on a high molecular weight glycoprotein with a mass of greater than 1 million Daltons. Characteristic of carbohydrate antigens, immunoreactivity was found in several distinct cellular patterns: only along the apical border of cells, along lateral and basal membranes of cells, and extracellular-like staining in the mesenchyme. During neurulation, CDA-3C2 showed differential staining in the ectoderm, distinguishing lateral from neural regions. Following closure of the neural tube, there was a striking specificity of expression of CDA-3C2 in the periphery, found almost exclusively in olfactory and otic epithelial structures. While CDA-3C2 is found in placode-derived tissues that subserve sensory transduction, it appears to be primarily associated with the supportive cells (and their secretions) in both otic and olfactory regions and less so with the sensory cells. The data suggest that a unique carbohydrate antigen on a large macromolecule may play a role in neurulation and/or morphogenesis of the placode-derived otic and olfactory structures.

Insect olfactory neurons in vitro: morphological and immunocytochemical characterization of male-specific antennal receptor cells from developing antennae of male Manduca sexta.

Sex-pheromone components released by Manduca sexta females are detected solely by male-specific olfactory receptor neurons that innervate long sensilla trichodea on the male antennae. To facilitate studies of the development and physiology of these receptor cells, we have produced primary in vitro cultures of cells dissociated from pupal male antennae. These cultures comprise several morphological types of cells, 2 of which have been characterized immunocytochemically with a pair of monoclonal antibodies that were shown previously to recognize certain antigens in olfactory receptor neurons at defined stages of development. The good correlation between in vivo and in vitro expression of these antigens suggests that the immunocytochemically recognized cells are olfactory receptor neurons that follow at least partially their normal course of differentiation in vitro.