Atypical olfactory glomeruli subset of the rat: quantitative study and organization of the peripheral afferents

The atypical glomeruli constitute a particular subset of olfactory glomeruli in the rat olfactory bulb which is mainly characterized by a strong centrifugal cholinergic innervation. In the present study, the topographical organization of the mucoso-bulbar projection of these glomeruli was analysed using small injections of WGA-HRP into the anterior nasal cavity of adult rats. The atypical olfactory glomeruli were visualized on adjacent bulbar sections using acetylcholinesterase histochemistry. A mean of 29 atypical glomeruli per bulb was observed in several areas of the posterior half of the olfactory bulb. Following the rostro-caudal axis of the olfactory bulb, the first atypical glomeruli were located in lateral positions, then in dorsal and ventral ones. The most posterior atypical glomeruli were located in the bulbar medial side. Concerning the projections from the periphery to the atypical glomeruli, various WGA-HRP patterns of labelling were observed. When the surface area of injection sites in the anterior part of the olfactory sheet was between 30 and 40 mm2, half of the atypical population was labelled with the atypical glomeruli being heavily labelled. All sites of distribution previously described were represented. When the surface area of injection sites was inferior to 20 mm2, only some positions distributed along the bulbar antero-posterior axis were represented. These atypical glomeruli were generally partially labelled. Taken together, these results suggest that, although atypical glomeruli are restricted in the posterior olfactory bulb, they receive peripheral projections diffusely organized along the antero-posterior axis of the olfactory mucosa. This profile was compared with that of other classical olfactory glomeruli.     

Distribution of astroglia in glomeruli of the rat main olfactory bulb: exclusion from the sensory subcompartment of neuropil

During an entire lifetime, sensory axons of regenerating olfactory receptor neurons can enter glomeruli in the olfactory bulb and establish synaptic junctions with central neurons. The role played by astrocytes in this unique permissiveness is still unclear. Glomerular astrocytes have been identified by immunocytochemistry for glial fibrillary acidic protein and S100 proteins at the light and electron microscopic levels. The latter labeling included submicroscopic lamellar and filopodial extensions of astroglial processes. Cell bodies and processes accumulate along the border between juxtaglomerular walls and glomerular neuropil. Within glomeruli, a network of astroglial processes encloses mesh-like neuropil zones devoid of astroglia. Electron microscopy confirmed the division into subcompartments of glomerular neuropil: 1) The "sensory-synaptic subcompartment" includes all sensory axon terminals and terminal dendritic branches receiving sensory input, whereas astroglia are excluded; 2) in the "central-synaptic subcompartment," astroglial processes are intermingled with other neuropil components: dendrites of relay cells and interneurons, dendrodendritic synapses, centrifugal (cholinergic and serotonergic) axons, their axodendritic synapses, and blood vessels. Unevenly distributed astroglial processes in this subcompartment are attached to vascular basal laminae, stem dendrites, and subpopulations of dendrodendritic synapses, especially those colocalized with centrifugal projections ("triadic synapses"). Astroglia-free parts of the "central" subcompartment contain segments of dendrites and subpopulations of dendrodendritic synapses. Because of the subdivision of the glomerular neuropil into portions with and without glial components, glia do not completely demarcate the border between the "sensory" and the "central" subcompartments. Interdigitation between the subcompartments varies among glomeruli and even within a single glomerulus. The mesh width of astroglial networks covaries with numerical relations between sensory and dendrodendritic synapses. This distribution pattern of astrocytes suggests that these glial cells monitor brain-derived effects on olfactory glomerular neuropil rather than olfactory input and that astroglial processes are (re-)arranged accordingly.     

Oncoprotein v-Myb and retinoic acid receptor alpha are mutual antagonists

The expression of nitric oxide synthase (NOS) in the olfactory bulb was compared between two mouse strains, CD-1 and BALB/c, that differ in the connectivity within their olfactory glomeruli, their content of tyrosine hydroxylase, and their response to olfactory deafferentation. Labelled cells were qualitatively and quantitatively analyzed by both immunohistochemistry for NOS and histochemistry for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND). Both periglomerular cells and short-axon cells were observed with both techniques employed, and their colocalization in the same neurons demonstrated that ND is a reliable marker for NOS-expressing cells in the mouse olfactory bulb (OB). The histochemical technique differentiates two types of glomeruli: ND-positive and ND-negative. Olfactory glomeruli in the CD-1 strain were about 7% larger than those in the BALB/c animals. While the density of NOS/ND-containing periglomerular cells was similar between both strains studied, more NOS/ND-labelled cells were observed in the ND-positive glomeruli (P = 0.002). Since periglomerular cells in the BALB/c strain do not receive direct olfactory receptors synapses, the present results indicate that such inputs do not regulate the expression of NOS and ND activity in the periglomerular cells. The different densities of NOS/ND-expressing periglomerular cells may indicate that nitric oxide is implicated in a differential modulation of the odor response within both types of chemically distinct glomeruli in the mouse 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.     

CSF levels of HVA and 5-HIAA in drug-free schizophrenic patients and healthy controls: a prospective study focused on their predictive value for outcome in schizophrenia

Recent progress in the studies of the olfactory system, especially in the molecular biological studies, makes it one of the useful sensory model systems for understanding neural mechanisms for the information processing. In the olfactory bulb, the primary center of the olfactory system, glomeruli are regarded as important functional units in the transmission of odorant signals and in processing the olfactory information, but have been believed to be composed by only a small number of neuronal types and thus to be simple in their neuronal and synaptic organization. However, accumulating morphological data reveal that each type of neurons might further consist of several different subpopulations, indicating that the organization of glomeruli might not be so simple as it was believed. Here we describe an aspect of the structural organization of glomeruli, focusing on the heterogeneities of periglomerular neurons in mammalian main olfactory bulb.     

Non-conventional role of lysosomal acid phosphatase in olfactory receptor axons: co-localization with growth-associated phosphoprotein-43

Olfactory receptor neurons undergo a continuous turnover in adult mammals. It is largely unknown how their axons invade the olfactory bulb and induce synaptic re-organization in glomeruli. Here, the cytochemical localization of lysosomal acid phosphatase has been studied in olfactory bulbs of adult rats and mice. The enzyme has been identified by specific substrate, inhibitors and absence in lysosomal acid phosphatase-knockout mice. Lysosomal acid phosphatase is located in primary and secondary lysosomes, which are unevenly distributed in the olfactory nerve layer and among olfactory glomeruli. In consecutive sections of glomeruli, the intensity of lysosomal acid phosphatase immunoreactivity co-varied with that of growth-associated phosphoprotein. Electron microscopically, differential lysosomal acid phosphatase staining in glomeruli corresponded to different proportions of labelled and unlabelled axons. Quantification revealed that lysosomal acid phosphatase labelling was strongest in non-synaptic profiles of terminal axons, while it was weak in or even missing from most synaptic profiles. Hence, growing olfactory axons apparently carry more lysosomal acid phosphatase than those which have established synaptic contacts. Following olfactory deafferentation both lysosomal acid phosphatase activity and growth-associated phosphoprotein-43 are lost from glomeruli, suggesting that both proteins are expressed in olfactory sensory axons during growth, while lysosomal acid phosphatase is apparently not a marker of anterograde terminal degeneration.     

A subset of olfactory neurons that selectively express cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D define a unique olfactory signal transduction pathway

Odorant information is encoded by a series of intracellular signal transduction events thought to be mediated primarily by the second messenger cAMP. We have found a subset of olfactory neurons that express the cGMP-stimulated phosphodiesterase (PDE2) and guanylyl cyclase-D (GC-D), suggesting that cGMP in these neurons also can have an important regulatory function in olfactory signaling. PDE2 and GC-D are both expressed in olfactory cilia where odorant signaling is initiated; however, only PDE2 is expressed in axons. In contrast to most other olfactory neurons, these neurons appear to project to a distinct group of glomeruli in the olfactory bulb that are similar to the subset that have been termed "necklace glomeruli." Furthermore, this subset of neurons are unique in that they do not contain several of the previously identified components of olfactory signal transduction cascades involving cAMP and calcium, including a calcium/calmodulin-dependent PDE (PDE1C2), adenylyl cyclase III, and cAMP-specific PDE (PDE4A). Interestingly, these latter three proteins are expressed in the same neurons; however, their subcellular distribution is distinct. PDE1C2 and adenylyl cyclase III are expressed almost exclusively in the olfactory cilia whereas PDE4A is present only in the cell bodies and axons. These data strongly suggest that selective compartmentalization of different PDEs and cyclases is an important feature for the regulation of signal transduction in olfactory neurons and likely in other neurons as well. In addition, the data implies that an olfactory signal transduction pathway specifically modulated by cGMP is present in some neurons of the olfactory neuroepithelium.     

Neurocalcin immunoreactivity in the rat main olfactory bulb

The morphological characteristics and distribution of neurocalcin (NC)-immunoreactive elements were studied in the rat main olfactory bulb (OB) using a polyclonal antibody and the avidin-biotin immunoperoxidase method. NC-positive elements were abundant in the glomerular layer (GL), where numerous immunostained external tufted cells and periglomerular cells were detected. Other less abundant NC-immunolabeled populations included middle and internal tufted cells, Van Gehuchten cells, horizontal cells, vertical cells of Cajal, deep short-axon cells and granule cells. This study demonstrates the presence of NC immunoreactivity in subsets of different neuronal types in the rat main OB. This calcium-binding protein has been found in interneurons, and no evidence of immunoreactivity to NC is detected in projecting neurons. Despite the large population of labeled external tufted cells, most of them belong according to morphological criteria to the local circuit group and some others to those with interbulbar and/or intrabulbar connections. The identification of neuronal subpopulations expressing NC provides a further characterization and shows the existence of biochemical differences within morphologically identical neurons. Thus, this marker may be a useful tool in unravelling the circuitries of the rodent OB in both normal and experimental conditions. The exact physiological function of NC in the olfactory system remains unknown. On the basis of similarities to recoverin, it could be involved in mechanisms responsible for sensory adaptation. Additionally, its calcium-binding abilities may contribute to improve the temporal precision of stimuli transmission, or be concerned with general calcium-related events occurring in specific interneuronal groups.     

Comparative laminar distribution of various autoradiographic cholinergic markers in adult rat main olfactory bulb

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (alpha 4 beta 2-like) and bungarotoxin-sensitive (alpha 7-like) receptors were visualized using [3H]pirenzepine, [3H]AF-DX 384, [3H]cytisine and [125I] alpha-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [3H]vesamicol and [3H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [125I] alpha-BTX binding sites were mostly concentrated in the superficial bulbar layers, [3H]cytisine binding was found in the glomerular layers, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [3H]vesamicol and [3H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [3H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors have been hypothesized to be mostly involved in cholinergic olfactory processing and short-term memory in the OB, nicotinic receptors, especially of the cytisine/ alpha 4 beta 2 sub-type, may have important roles in mediating olfactory transmission of efferent neurons as well as in a subset of olfactory glomeruli.     

Beta-galactosidase-labelled relay neurons of homotopic olfactory bulb transplants establish proper afferent and efferent synaptic connections with host neurons

The vertebrate olfactory system has long been an attractive model for studying neuronal regeneration and adaptive plasticity due to the continuous neurogenesis and synaptic remodelling throughout adult life in primary and secondary olfactory centres, its precisely ordered synaptic network and accessibility for manipulation. After homotopic transplantation of fetal olfactory bulbs in bulbectomized neonatal rodents, newly regenerated olfactory neurons form glomeruli within the graft, and the efferent mitral/tufted cells of the transplant innervate the host brain, terminating in higher olfactory centres. However, the synaptic connections of the transplanted relay neurons within the graft and/or host's olfactory centres could not be characterized mainly because of lack of suitable cell-specific markers for these neurons. In this study, we have used olfactory bulbs from transgenic fetuses, in which the majority of the mitral/tufted cells express the bacterial enzyme beta-galactosidase, for homotopic olfactory bulb transplantation following complete unilateral bulbectomy. In the transplants, the cell bodies and terminals of the donor mitral/tufted cells were identified by beta-galactosidase histochemistry and immunocytochemistry at both light and electron microscope levels. We demonstrate that transplanted relay neurons re-establish specific synaptic connections with host neurons of the periphery, source of the primary signal and central nervous system, thereby providing the basis for a functional recovery in the lesioned olfactory system.     

A histological and immunohistochemical study of medical-grade fluid silicone

BACKGROUND: Growth cone-associated protein (GAP43) is found in growing axons and we hypothesized that systemic treatment with antineoplastic agents should disrupt regeneration of olfactory receptor cells. Disruption of regeneration should be evidenced by decreased presence of growing axons in the olfactory bulb. OBJECTIVE: To evaluate GAP43 in human olfactory bulb in normal controls and in individuals receiving treatment for neoplasms. DESIGN: Immunocytochemical studies were performed on autopsied human olfactory bulbs to identify both GAP43 and olfactory marker protein immunoreactivity. The former recognizes growing axons and the latter is a definitive marker of adult olfactory nerve. SUBJECTS: Twenty-seven subjects were evaluated. Seven had received either antineoplastic agents and/or x-irradiation of the whole head. Four subjects were young, untreated controls, 10 were age matched to the treated group, and 2 had neoplasms but did not receive antineoplastic agents or irradiation of the head. In addition, 3 subjects with end-stage renal disease were immunostained. RESULTS: Subjects treated with antineoplastic agents or x-irradiation of the whole head displayed no statistically significant loss of olfactory bulb glomeruli, but GAP43 immunoreactivity was markedly reduced in all but 1 subject (P<.32). The subjects with end-stage kidney disease showed frank loss of both GAP43 immunoreactivity and olfactory glomeruli. CONCLUSIONS: Treatment with antineoplastic agents apparently does not damage olfactory epithelium directly but inhibits growth of new axons into the olfactory bulb. This observation suggests that the quality of olfactory experience may change during the course of treatment with antineoplastic agents because the olfactory nerve is not replaced.     

Development and further characterization of a small subclass of rat olfactory receptor neurons that shows immunoreactivity for the HSP70 heat shock protein

We previously described a rat olfactory receptor neuron (ORN) subpopulation [the 2A4(+) ORNs] that shows uniquely strong reactivity with antibodies to the 70-kD heat shock protein (HSP70) family of molecular chaperones (Carr et al. [1994] J. Comp. Neurol. 348:150-160). The 2A4(+)ORNs are dispersed through zones II-IV of the olfactory epithelium (OE), and their axons project to only two or three glomeruli that are located consistently in each olfactory bulb (OB). To date, the 2A4(+)ORN subpopulation is the only cell population to show such distinct HSP70 immunoreactivity as well as the most discrete ORN subpopulation to be so labeled. The present report shows that 2A4(+)ORN neurons first appear between postnatal days 7 (P7) and P10. Initially, low cell numbers rise to a density of 0.1 2A4(+)ORNs/mm OE length by P14, plateau at 0.9 2A4(+)ORNs/mm by P49, then fall to adult values of 0.4 cells/mm. Autoradiographic birthdating indicates that almost all of these early appearing 2A4(+)ORNs are generated postnatally, in contrast to the prenatal generation of all ORN subpopulations characterized to date by their expression of olfactory receptor protein mRNAs. A developmentally related increase in the mean depth of 2A4(+)ORNs within the OE also occurs. In the OB, initial 2A4(+)axonal projections are to only two or three glomeruli, as in adults. Slight but significant rostral shifts in (+)glomerular location occur with development. The 2A4(+)ORN immunoreactivity was found to be due to expression of HSP70, the dominant stress-inducible member of the HSP70 family, rather than constitutively expressed HSC70. In addition, despite their presence in rat OE, no 2A4(+)ORNs were found in mice, gerbils, guinea pigs, or hamsters.     

Odorant response properties of convergent olfactory receptor neurons

Information about odorant stimuli is thought to be represented in spatial and temporal patterns of activity across neurons in the olfactory epithelium and the olfactory bulb (OB). Previous studies suggest that olfactory receptor neurons (ORNs) distributed in the nasal cavity project to localized regions in the glomerular layer of the OB. However, the functional significance of this convergence is not yet known, and in no studies have the odorant response properties of individual ORNs projecting to defined OB regions been measured directly. We have retrogradely labeled mouse ORNs connecting to different glomeruli in the dorsal OB and tested single cells for responses to odorants using fura-2 calcium imaging. ORNs that project to clusters of dorsomedial (DM) glomeruli exhibit different odorant response profiles from those that project to dorsolateral (DL) glomeruli. DL-projecting ORNs showed responses to compounds with widely different structures, including carvone, eugenol, cinnamaldehyde, and acetophenone. In contrast, DM-projecting neurons exhibited responses to a more structurally restricted set of compounds and responded preferentially to organic acids. These data demonstrate that ORN afferents segregate by odorant responsiveness and that the homogeneity of ORN and glomerular input varies with different OB regions. The data also demonstrate that a subpopulation of ORNs projecting to DM glomeruli is functionally similar.     

Effects of fenvalerate on the development of olfactory perception in a beetle

Low doses of fenvalerate (a Type II pyrethroid) were applied to the beetle Tenebrio molitor at pupation, to ascertain its effects on the developing olfactory system. Doses of fenvalerate that prevent the formation of glomeruli in the primary olfactory neuropil (antennal lobes) also inhibit olfactory orientation behavior for different odors, despite the fact that sensory neurons developed responses to these odors. Even when lower amounts of fenvalerate that allowed glomeruli to develop were applied to pupae, the olfactory behavior was affected. Therefore, the formation of glomerular structures within the antennal lobe is not sufficient to establish olfactory behavior. A possible reason for this developmental effect of fenvalerate is a change in the odotopic arrangement of sensory axons within the glomeruli.     

Early formation of sexually dimorphic glomeruli in the developing olfactory lobe of the brain of the moth Manduca sexta

The antennal lobes (ALs), the primary olfactory centers, of the moth Manduca sexta are sexually dimorphic. Only ALs of males possess the macroglomerular complex (MGC), the site of primary processing of information about the female's sex pheromone. To understand the development of identified, odor-specific olfactory glomeruli, we investigated the cellular events involved in the morphogenesis of the MGC by means of various fluorescence staining techniques and laser-scanning confocal microscopy. The MGC lies near the entrance of the antennal nerve into the AL of the adult male and comprises three glomeruli, the globular cumulus and two toroidal structures. The MGC forms during early stages of metamorphic adult development through a stereotyped sequence of coordinated changes in MGC-specific receptor axons, glial cells, and early-ingrowing projection neurons of the medial group of AL neurons. The MGC divisions are the earliest glomeruli to form in the male AL, and their basic organization is established within about 3 days after ingrowth of the first sensory axons. Despite their special anatomical features, the MGC glomeruli develop in a manner similar to that of the ordinary glomeruli. Comparison of the ALs of males and females reveals that two relatively large and early-developing glomeruli that are situated dorsolaterally in the female AL appear to be female-specific. Development of the sexually dimorphic glomeruli diverges immediately after the ingrowth of the first olfactory receptor axons, resulting in the formation of these large glomeruli in females and the MGC in males.     

Nitric oxide as a putative messenger molecule in the crayfish olfactory midbrain

NADPH-d histochemistry was used to investigate presumptive nitric oxide synthase (NOS)-containing neurons in the crayfish olfactory midbrain. Three anatomically different types of local olfactory interneurons exhibiting NADPH-d activity were observed: two pairs of large interneurons as well as positively stained globuli cells. Branches derived from the large interneurons were confined to the ipsilateral olfactory lobe and accessory lobe, but only a few branches innervated the olfactory lobe glomeruli. Local field potential recordings on the olfactory lobe showed that administration of SNP or SIN-1 (10-4 M) into the brain had reversible inhibitory effects on electrically-evoked responses of unidentified neuronal cell populations.     

Chemotopic, combinatorial, and noncombinatorial odorant representations in the olfactory bulb revealed using a voltage-sensitive axon tracer

Odor information is first represented in the brain by patterns of input activity across the glomeruli of the olfactory bulb (OB). To examine how odorants are represented at this stage of olfactory processing, we labeled anterogradely the axons of olfactory receptor neurons with the voltage-sensitive dye Di8-ANEPPQ in zebrafish. The activity induced by diverse natural odorants in afferent axons and across the array of glomeruli was then recorded optically. The results show that certain subregions of the OB are preferentially activated by defined chemical odorant classes. Within these subregions, "ordinary" odorants (amino acids, bile acids, and nucleotides) induce overlapping activity patterns involving multiple glomeruli, indicating that they are represented by combinatorial activity patterns. In contrast, two putative pheromone components (prostaglandin F2alpha and 17alpha, 20beta-dihydroxy-4-pregnene-3-one-20-sulfate) each induce a single focus of activity, at least one of which comes from a single, highly specific and sensitive glomerulus. These results indicate that the OB is organized into functional subregions processing classes of odorants. Furthermore, they suggest that individual odorants can be represented by "combinatorial" or "noncombinatorial" (focal) activity patterns and that the latter may serve to process odorants triggering distinct responses such as that of pheromones.     

Topographic bulbar projections and dual neural pathways of the primary olfactory neurons in salmonid fishes

A growing body of evidence indicates spatial patterning of molecular expression and physiological activities in the olfactory epithelium and primary afferent circuits of the vertebrate olfactory bulb. Because our previous studies indicate that olfactory receptors specific for amino acids and a bile acid, taurocholic acid, project to segregated coding centres in the olfactory bulb, we further examined the afferent projections and pathways of the primary neuronal responses to putative pheromones by recording the electroencephalogram from various regions of the olfactory bulb. First, using the electro-olfactogram, we determined olfactory sensitivities of six salmonid species to these odorants. Prostaglandin F2 alpha and 15-keto-prostaglandin F2 alpha were potent olfactory stimulants for all tested salmonids, except rainbow trout (Oncorhynchus mykiss). None of the salmonids responded to 17 alpha,20 beta-dihydroxy-4-pregnen-3-one. However, they were sensitive to etiocholan-3 alpha-ol-17-one glucuronide. In all salmonids examined, electroencephalograms to amino acids and taurocholic acid, applied singly or in combination, projected to two segregated regions, the lateroposterior and mid-olfactory bulb, respectively. Neither prostaglandin F2 alpha, 15-keto-prostaglandin F2 alpha nor etiocholan-3 alpha-ol-17-one glucuronide elicited electroencephalograms. These data indicate that, in salmonids, olfactory neurons responsive to amino acids and taurocholic acid project to spatially segregated regions, and thereby generated signals are encoded spatially and temporarily. The results also suggest that olfactory signals due to hormonal pheromones are processed in a manner distinct from those for amino acids and bile acids, and may possibly be mediated by extrabulbar primary olfactory fibres bypassing the bulb.     

Two novel monoclonal antibodies (1.9.E and 4.11.C) against olfactory bulb ensheathing glia

We produced and characterized two monoclonal antibodies, termed 1.9.E and 4.11.C, that specifically recognize olfactory bulb ensheathing glia. Both antibodies were generated using the olfactory nerve layer (ONL) of newborn rat olfactory bulbs (P0, P1) as immunogens. The specificity of these antibodies was tested by immunofluorescence techniques on tissue sections and cultures of adult and neonatal rat olfactory bulbs, and by Western blot analysis. 1.9.E labeled the ONL and glomerular layer of the olfactory bulb (OB) of adult rats. In newborn rats, 1.9.E immunostained ensheathing cells from the ONL and peripheral olfactory fascicles. Furthermore, 1.9.E reacted with some processes of the radial glia in the periventricular germinal layer of the newborn rat. Although 4.11.C also specifically labeled ensheathing cells in the adult OB, it did not stain any cell type in the ONL of newborn rats. The lack of double labeling with either 1.9.E or 4.11.C and anti-olfactory marker protein (OMP) antibody, a specific marker for olfactory axons, indicated that none of the monoclonals recognized olfactory axons. Double immunostaining of adult OB cultures with 1.9.E or 4.11.C and anti-p75-nerve growth factor receptor revealed that both antibodies specifically recognized ensheathing glia in those cultures. Filaments were strongly labeled throughout the entire cytoplasm of ensheathing cells, suggesting that 1.9.E and 4.11.C immunoreacted with ensheathing glia cytoskeleton. 4.11.C stained a few Schwann cells in adult sciatic nerve sections. Moreover, 4.11.C immunostained cortical astrocyte cultures from newborn rats (P1). In Western blot analysis both antibodies recognized a major component, migrating with an apparent molecular weight of 60 kDa, from olfactory nerve and glomerular layer (ONGL) extracts of adult and neonatal rats. The pattern of immunoreactivity of 1.9.E and 4.11.C antibodies suggest that both antibodies are specific markers for olfactory ensheathing glia in the adult rat central nervous system (CNS).     

The nitric oxide-cGMP pathway may mediate communication between sensory afferents and projection neurons in the antennal lobe of Manduca sexta

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGCalpha1 and MsGCbeta1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca2+-sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with MsNOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NO-sGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.