Olfactory neurons express a unique glycosylated form of the neural cell adhesion molecule (N-CAM).

mAb-based approaches were used to identify cell surface components involved in the development and function of the frog olfactory system. We describe here a 205-kD cell surface glycoprotein on olfactory receptor neurons that was detected with three mAbs: 9-OE, 5-OE, and 13-OE. mAb 9-OE immunoreactivity, unlike mAbs 5-OE and 13-OE, was restricted to only the axons and terminations of the primary sensory olfactory neurons in the frog nervous system. The 9-OE polypeptide(s) were immunoprecipitated and tested for cross-reactivity with known neural cell surface components including HNK-1, the cell adhesion molecule L1, and the neural cell adhesion molecule (N-CAM). These experiments revealed that 9-OE-reactive molecules were not L1 related but were a subset of the 200-kD isoforms of N-CAM. mAb 9-OE recognized epitopes associated with N-linked carbohydrate residues that were distinct from the polysialic acid chains present on the embryonic form of N-CAM. Moreover, 9-OE N-CAM was a heterogeneous population consisting of subsets both with and without the HNK-1 epitope. Thus, combined immunohistochemical and immunoprecipitation experiments have revealed a new glycosylated form of N-CAM unique to the olfactory system. The restricted spatial expression pattern of this N-CAM glycoform suggests a possible role in the unusual regenerative properties of this sensory system.

[Immunohistochemical study on the primary olfactory neuron of rat and suncus].

Carnosine (beta-alanyl-L-histidine), a putative neurotransmitter, was identified immunohistochemically in the primary olfactory neuron, using newly developed anti-carnosine antiserum. Similar results were obtained in the rat and suncus. Carnosine-like immunoreactivity was observed in the olfactory cells and its apical dendrites of the olfactory epithelium. The olfactory nerve and glomerular layer of the olfactory bulb showed positive reaction. At electron microscopic level, carnosine-immunoreactive end products widely spread out in the cytoplasm of olfactory nerve cells and also on microtubules of olfactory cilia. In the glomerular layer, reaction products were found diffusely in many axon terminals. These terminals had small spherical vesicles and often made asymmetric synaptic contacts to the second neuron. Unilateral closure of the olfactory naris resulted lower immunoreactivity of tyrosine hydroxylase in periglomerular cells, which are dopaminergic interneurons and are thought to regulate neurotransmission of olfactory input. In contrast no remarkable changes were seen on carnosine immunoreactivity in olfactory bulb. The present results suggest that carnosine may play some important roles in the olfactory mucosa. The functional role of carnosine remarks to be further examined.

Darkfield illumination improves microscopic detection of metals in Timm's stained tissue.

Deposits of trace or toxic metals can be quickly identified by light microscopical surveys of tissue sections stained for metals by variants of Timm's silver enhancement method. The present work shows that the small, isolated silver grains that label isolated deposits of metal in tissue are undetectable in brightfield light microscopy but are easily detected in darkfield microscopy. Darkfield illumination is therefore recommended for improving the detection of trace or toxic metals in tissue.

Carnosine-like immunoreactivity in the olfactory bulb of the rat: an electron microscopic study.

Carnosine-immunoreactive primary olfactory nerve terminals are demonstrated in the glomerular layer of the rat olfactory bulb by immunoelectron microscopy. Asymmetrical synapses between dendrites of mitral/tufted cells and that of periglomerular cells could be observed. In the accessory olfactory system, carnosine-like immunoreactivity is also detected in the vomeronasal neurons.

High substance P-like immunoreactivity (SPLI) in the olfactory and electrosensory cells of gymnotid fish.

Substance P has been proposed as a candidate neurotransmitter or neuromodulator in the nociceptive system. Using a light microscopial immunohistochemical peroxidase-anti-peroxidase technique we have detected high substance P-like immunoreactivity (SPLI) in several types of sensory organs of 4 species of gymnotiform teleost fish: olfactory epithelium, vestibular, lateral line and electrosensory organs. The olfactory nerve and its endings within the olfactory bulb and the telencephalon were also strongly labelled. At these sites no SPLI was revealed in other teleosts (Carassius auratus, Gnathonemus petersii). The findings suggest that substance P may be involved in neurotransmission or neuromodulation in these specific sensory systems of these species.

Axon pathway boundaries in the developing brain. I. Cellular and molecular determinants that separate the optic and olfactory projections.

When optic fibers first approach the chiasmatic region of the diencephalon in the chick embryo on days 3 and 4 (E3-4), they rarely grow rostrally into the olfactory region of the telencephalon. Conversely, olfactory tract axons grow as far as, but never cross the diencephalic/telencephalic (D/T) boundary to enter the optic chiasm. In this study, a region of specialized neuroepithelium, originally named the "knot" in mouse by Silver (1984), has been identified at the D/T border of chick embryos. At pre-axonal stages, the presumptive knot region undergoes a cataclysmic cell death, with concomitant phagocytosis of necrotic debris by the remaining cells. When fibers subsequently appear in the chiasm and olfactory tracts, the knot consists of a very dense, interwoven cluster of non-neuronal cells that lack marginal radial processes, and whose cell bodies directly abut the glial limiting membrane. Thus, the morphology of the knot is in sharp contrast to the cell body-free marginal zone and endfoot regions along which axons tend to grow. In addition, we found that the neural cell adhesion molecule (N-CAM), which is expressed on neuroepithelial cell processes within the central optic and olfactory pathways, is not present on cells in the knot region during periods of axon growth. These results suggest that the knot, through its elimination of the marginal zone processes, absence of large extracellular spaces, and relative absence of adhesion molecules, functions as an axon-refractory barrier that effectively separates the optic and olfactory projections.

Nervus terminalis, olfactory nerve, and optic nerve representation of luteinizing hormone-releasing hormone in primates.

The luteinizing hormone-releasing hormone (LHRH) system was examined immunocytochemically in olfactory bulbs of adult monkeys, including two New World species (squirrel monkey, Saimiri sciureus and owl monkey, Aotus trivirgatus) and one Old World species (cynomolgus macaque, Macaca fasciculata), and in the brain and nasal region of a fetal rhesus macaque Macaca mulatta. LHRH neurons and fibers were found sparsely distributed in the olfactory bulbs in all adult monkeys. There was more LHRH in the accessory olfactory bulb (which is absent in Old World monkeys). In the fetal macaque there was a rich distribution of LHRH neurons and fibers along the pathway of the nervus terminalis, anterior and ventral to the olfactory bulb, and in the nasal septum, with fibers branching into the olfactory epithelium. In addition, there were LHRH neurons and fibers in the optic nerve.

Soybean agglutinin binding to the olfactory systems of the rat and mouse.

The binding of the lectin soybean agglutinin (SBA) to the olfactory system of both the rat and mouse was investigated histochemically. SBA bound to fibers in the accessory olfactory nerve and to glomeruli in the accessory olfactory bulb. In addition, SBA binding sites were present in some, but not all, glomeruli in the ventrolateral and ventromedial portions of the main olfactory bulb of only the rat. Under standard experimental conditions, SBA did not bind to neurons in other regions of the olfactory system nor to any other neurons in the brain. This selective binding of SBA to only some glomeruli in the olfactory bulb provides additional support for the presence of, at least, two subclasses of olfactory receptor cells in the nasal cavity. Whether these neuronal subclasses are the same as those previously characterized by monoclonal antibodies in rabbit remains to be determined.

Immunohistochemical study of subclasses of olfactory nerve fibers and their projections to the olfactory bulb in the rabbit.

The organization of the olfactory nerve projection to the olfactory bulb was studied immunohistochemically in the rabbit by using monoclonal antibodies (MAbs). Out of 42 MAbs raised against the homogenate of the olfactory bulb, two types of MAbs that strongly stained the olfactory nerve fibers (axons of olfactory receptor cells) were selected and their staining patterns were analysed in detail. MAbs of one type (represented by MAb R2D5) specifically labeled all olfactory receptor cells in the nasal epithelium and all olfactory nerve fibers and their terminal portions in the bulb. The other type of MAbs (represented by MAb R4B12) recognized only a subgroup of olfactory nerve fibers. The R4B12-positive fibers were distributed over the ventrolateral areas but not in the dorsomedial areas of the epithelium. Similarly in the bulb, the R4B12-positive fibers terminated in the glomeruli in the ventrolateral and the caudal regions but not in the dorsomedial region. These results demonstrate for the first time the cellular heterogeneity among olfactory receptor neurons at the molecular level. The segregated distribution of the subtypes of olfactory receptor cell axons both in the epithelium and the bulb indicates a defined topographical organization of the olfactory nerve projection. These results also suggest a functional division between dorsomedial and ventrolateral areas both in the epithelium and the bulb.

Olfactory marker protein in the human olfactory pathway.

The presence of olfactory marker protein (OMP) in the olfactory tissue of rats, gerbils, and humans was demonstrated with goat antiserum to rat OMP. In control studies on rat neural tissue, OMP was found to be located in the olfactory receptor cells, nerves, and bulbs. Likewise, staining of OMP was found in the olfactory tissues of gerbils. Olfactory marker protein reactivity was also demonstrated in human olfactory receptor cells, nerves, and bulbs. The reactivity of OMP in human olfactory neurons to goat antiserum has allowed determination of the exact junction of olfactory and respiratory epithelia immunohistochemically.

Immunohistopathology of human olfactory epithelium, nerve and bulb.

The immunohistochemical characteristics of the human olfactory system were (OMP). OMP was detected in the olfactory receptor neurons and processes extending from the olfactory neuroepithelium to the olfactory bulb. The olfactory receptor cells located close to the epithelial surface also contained OMP. In severely degenerate regions, only a few OMP-containing cells were observed. Differences in OMP-staining intensity were noted among the olfactory receptor cells. The thick neuroepithelium. Proliferating olfactory neuroepithelium contained OMP reactive and nonreactive olfactory receptor cells. The presence of OMP reactive and nonreactive olfactory neurons indicates the coexistence of two functionally different phases of olfactory neurons. These findings suggest that continuous cell turnover is occurring in human olfactory neuroepithelium.

Failure of chronic haloperidol treatment to alter levels of cholecystokinin in the rat brain striatum and olfactory tuberclenucleus accumbens area.

We have studied the effect of chronic haloperidol (HAL) treatment on CCK-8 levels in two rat brain regions. HAL administration using two different protocols, daily injections and infusion with subcutaneously implanted minipumps, did not produce any significant changes in CCK-8 levels in the striatum or olfactory tubercle-nucleus accumbens area.

Localization of herpes simplex virus in the trigeminal and olfactory systems of the mouse central nervous system during acute and latent infections by in situ hybridization.

The precise anatomical location of latent herpes simplex virus (HSV) infection of the mouse central nervous system (CNS) has been identified by application of a 3H-labeled HSV-specific probe to deparaffinized sections of mouse brain tissue in situ. At times after corneal inoculation with HSV type 1 (HSV-1), strain F, representing the acute and latent phases of infection, BALB/c mice were perfused with a fixative containing sodium m-periodate, lysine, and paraformaldehyde and their CNS tissues and trigeminal ganglia embedded in paraffin, sectioned, and and subjected to hybridization. During the acute phase, HSV-1 was localized to neurons and some small supporting cells in the sensory portion of the 5th cranial nerve including the trigeminal ganglia and nerve root, principal sensory nucleus, mesencephalic nucleus, descending tract and nuclei, and cerebral cortex. During the latent phase, HSV-1 was found only in neurons located primarily in the descending nuclei and mesencephalic nucleus. Evidence was also obtained that implicated the olfactory tract as an additional route of entry into the CNS, in that positive hybridization was found in the olfactory bulb, the entorhinal cortex, and adjacent cerebral cortex. Additionally, HSV-1 established latent infections in neurons of the olfactory system. HSV-1-specific RNA was detected in ganglionic and CNS neurons throughout the acute and latent phases of infection, whereas HSV-1-specific DNA was detected only during the acute phase, indicating that the relationship between HSV and latently infected CNS and ganglionic neurons involves limited transcription of the viral genome.

Mitogenic effect of myelinated vs unmyelinated garfish nerve extracts.

Mitogenic activities in crude extracts of unmyelinated olfactory nerves and myelinated trigeminal nerves of the garfish Lepisosteus osseus were compared. Extracts of each nerve type were added in a range of protein concentrations to serum-starved, subconfluent cultures of BALB/c 3T3 cells. At low protein concentrations (50-250 micrograms/ml) myelinated nerve extracts produced more [3H]thymidine incorporation in the cultured cells than unmyelinated nerve extracts, while at higher concentrations (500-1000 micrograms/ml), the latter caused as much DNA synthesis as the myelinated nerve extracts, surpassing them at the highest concentrations tested. The results suggest that both axonal and myelin components contribute to the growth-promoting activity in nerve tissue.

Composition and subcellular distribution of glycoproteins and glycosaminoglycans undergoing axonal transport in garfish olfactory nerves.

The study examined the subcellular distribution of [3H]glucosamine-labeled glycoconjugates undergoing axonal transport in 100,000 x g soluble and two membranous subfractions of the garfish olfactory nerve. Analysis was made of intact glycoconjugates and of glycopeptides and glycosaminoglycans derived from these molecules by limit protease digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed labeling of a variety of high-molecular-weight molecules with a lower molecular weight distribution in the soluble fraction than in the membranous fractions. Following protease digestion, nearly two-thirds of transported radioactivity in glycopeptides was recovered in the plasma membrane-enriched subfraction, with the remainder equally divided between soluble and higher density membrane fraction. Comparison of the distribution of glycopeptide radioactivity and chemically assayed hexosamine revealed transport labeling of a large variety of different-sized neutral and acidic glycopeptides in all subfractions. Transport labeling of most glycoprotein carbohydrate chains was in proportion of their hexosamine content. Transported glycosaminoglycan label was most heavily concentrated in the plasma membrane fraction, whereas hexosamine was most concentrated in the higher density membrane fraction. The labeling pattern suggested both transported and nontransported pools of these molecules. The specific glycosaminoglycans chondroitin sulfate and heparan sulfate were recovered in all subfractions, whereas hyaluronic acid was confined to the soluble fraction.

Chemical and morphological studies on garfish peripheral nerves.

Gangliosides were extracted, separated by thin layer chromatography, and quantitated in three cranial nerves of the garfish (Lepisosteus osseus): the completely unmyelinated olfactory nerve (OLF), and two nerves composed of both myelinated and unmyelinated fibers, viz., the main trunk of the maxillary nerve (MAX) and a branch of the maxillary nerve (BR-MAX). Morphological studies on each of these nerves were done to verify that the OLF had been excised free of any contamination from the accompanying myelinated BR-MAX, to aid in the interpretation of the biochemical findings, and to clarify the nature of the OLF supporting cell. The chief chemical findings were (1) documentation of the presence of gangliosides in nerves previously thought not to contain them, (2) demonstration that gangliosides can be associated with unmyelinated nerves, (3) demonstration of a greater proportion of one simple ganglioside (G-6) in the OLF but greater proportions of two complex gangliosides (G-2 and G-0) in the MAX and BR-MAX, and (4) that either GM4 or a variant of the GM3 is present in OLF. The morphological findings with respect to the difficulty of ascribing only peripheral nervous system characteristics to the OLF supporting cell are discussed in relation to the ganglioside band chromatographing slightly ahead of GM4 in the OLF.

Differences in the composition of the polypeptides deposited in the axon and the nerve terminals by fast axonal transport in the garfish olfactory nerve.

Proteins transported by the fast wave of axonal transport have been shown to be deposited both in the axon and in the nerve terminals. Differences in the nature of the molecules deposited in these two areas were studied in the garfish olfactory system. In order to avoid analysis of transported molecules in two different types of tissue like the olfactory nerve and the olfactory bulb, the study was conducted (1) by comparing the composition of the moving crest of radioactivity at two different points along the nerve: when the crest enters the axon and when it reaches a distance of approximatively 5 cm from the nerve endings, (2) by determining the composition of the molecules remaining in the axon behind the moving crest. Three subcellular fractions (two membranous fractions and a mitochondrial pellet) were investigated. In both membranous fractions the majority of the polypeptides deposited in the axon ranged from 50 to 150,000 daltons. No outstanding peak of radioactivity was found in either fraction. Radioactivity was relatively evenly distributed among the various polypeptides. In the lightest membranous fraction, however, a peak (mol. wt., 54-58,000) was more particularly deposited in the axon. The opposite situation was found for the molecules moving toward the synapses: transported radioactivity was concentrated in a few distinct polypeptides, while the others were significantly less labeled. Three peaks were found in the lightest membranous fraction (mol. wt., 35,000, 54-58,000 and 126,000). Only two peaks were determined in the heaviest fraction (mol. wt., 58,000 and 126,000). The 126,000 mol. wt. peak increases with distance in both membranous fractions from 9 to 12% of the total radioactivity and moves mainly toward the synapses. The 35,000 mol. wt. polypeptide presented some interesting properties: it was found in larger quantities in the lightest membranous fraction; labeling was very poor in the heaviest membranous fraction, and finally this polypeptide appeared to be largely transported to the synapses. Results concerning the polypeptide composition and the composition of the transported molecules indicated that the lightest fraction may contain more synaptosomal material. From this study it appears that most transported polypeptides are distributed in both the axon and the nerve terminals, but that the percentage delivered to each area varies. A few distinct polypeptides on the contrary are more selectively transported to the synapses and are even differently localized in subcellular fractions.