Evidence for a role of the chemorepellent semaphorin III and its receptor neuropilin-1 in the regeneration of primary olfactory axons

To explore a role for chemorepulsive axon guidance mechanisms in the regeneration of primary olfactory axons, we examined the expression of the chemorepellent semaphorin III (sema III), its receptor neuropilin-1, and collapsin response mediator protein-2 (CRMP-2) during regeneration of the olfactory system. In the intact olfactory system, neuropilin-1 and CRMP-2 mRNA expression define a distinct population of olfactory receptor neurons, corresponding to immature (B-50/GAP-43-positive) and a subset of mature (olfactory marker protein-positive) neurons located in the lower half of the olfactory epithelium. Sema III mRNA is expressed in pial sheet cells and in second-order olfactory neurons that are the target cells of neuropilin-1-positive primary olfactory axons. These data suggest that in the intact olfactory bulb sema III creates a molecular barrier, which helps restrict ingrowing olfactory axons to the nerve and glomerular layers of the bulb. Both axotomy of the primary olfactory nerve and bulbectomy induce the formation of new olfactory receptor neurons expressing neuropilin-1 and CRMP-2 mRNA. After axotomy, sema III mRNA is transiently induced in cells at the site of the lesion. These cells align regenerating bundles of olfactory axons. In contrast to the transient appearance of sema III-positive cells at the lesion site after axotomy, sema III-positive cells increase progressively after bulbectomy, apparently preventing regenerating neuropilin-1-positive nerve bundles from growing deeper into the lesion area. The presence of sema III in scar tissue and the concomitant expression of its receptor neuropilin-1 on regenerating olfactory axons suggests that semaphorin-mediated chemorepulsive signal transduction may contribute to the regenerative failure of these axons after bulbectomy.     

Age-related changes in dividing cells of the olfactory epithelium of the maturing guinea pig

Changes in dividing cells of the olfactory epithelium from guinea pigs of different ages were examined by immunohistochemical staining using anti-proliferating cell nuclear antigen antibody. Numerous dividing cells were scattered diffusely in the basal layer of the olfactory epithelium at 1 and 2 months following birth and then gradually decreased with maturation until 4 months. Findings then remained constant between 4 and 24 months. Subsequently, cell numbers were found to decrease as animals became older. The number of olfactory receptor cells did not vary significantly between 1 and 30 months. Although no correlation could be found between the numbers of dividing cells and olfactory receptor cells, it is still possible that the longevity of the olfactory receptor cells changes to maintain the overall size of the neuronal population.     

Lectin identification of olfactory receptor neuron subclasses with segregated central projections

Our previous studies have demonstrated that the primary olfactory projection in rainbow trout is organized nontopographically; the pattern of termination of olfactory axons in the olfactory bulb is unrelated to the distribution of their cell bodies in the olfactory mucosa. In the present research we have further characterized the organization of this projection by examining the lectin-binding properties of olfactory receptor neurons. The results indicate that in trout, as in mammals, populations of olfactory receptor neurons differ significantly from one another in their carbohydrate "signatures." We have identified subsets of olfactory receptor neurons, specified by unique lectin-binding properties, that are widely distributed and intermingled with the other receptor neurons in the olfactory mucosa and nerve, but that segregate as they enter the olfactory bulb and project to restricted regions of the glomerular layer. This pattern of terminations is bilaterally symmetrical, is remarkably consistent across individuals, and reappears when the primary olfactory projection is reconstituted following transection of the olfactory nerve. As revealed by the carbohydrates on subpopulations of receptor neurons, there is substantial order in the nontopographic projection of olfactory receptor neurons to the olfactory bulb. The functional significance of this organization and the means by which it develops and is maintained remain under investigation.     

Bilateral detection thresholds in dextrals and sinistrals reflect the more sensitive side of the nose, which is not lateralized

Recent studies have shed light on the different relationships between odorant receptor expression and the specification of neural identity in the olfactory systems of vertebrates and invertebrates. In mice, neuronal identity and axon guidance are specified by the single expressed olfactory receptor, whereas in C. elegans, neuronal identity appears to be independent of receptor expression.     

Growth-related and antennular amputation-induced changes in the olfactory centers of crayfish brain

Freshwater crayfish increase in size throughout their lives, and this growth is accompanied by an increase in the length of the appendages and number of mechanoreceptive and chemoreceptive sensilla on them. We find that in the Australian freshwater crayfish Cherax destructor, neuropil volumes of the olfactory centers increase linearly with body size over the entire size range of animals found in their natural habitat. The number of cell somata of two groups of interneurons associated with the olfactory centers (projection neurons and small local neurons) also increases linearly with the size of the animals. In contrast, axon counts of interneurons that represent a nonolfactory input to the olfactory centers show that these reach a total number in the very early adult stages that then remains constant regardless of the size of the animal. Only the axon diameter of these interneurons increases linearly with body size. Amputation of the antennule and olfactory sensilla reduces the number of projection and local interneurons on the amputated side. No change in the size of the olfactory centers occurs on the unamputated side. Amputation of the olfactory receptor neurons in crayfish therefore leads not only to a degeneration of the receptor cell endings in the olfactory lobe but also to a trans-synaptic response in which the number of higher order neurons decreases. Reconstitution of the antennule and olfactory receptor neurons in small adult crayfish is accompanied by the reestablishment of the normal number of interneurons and neuropil volume in the olfactory centers.     

Looking for the answer

Metabotropic glutamate receptors (mGluRs) were identified in olfactory receptor neurons of the channel catfish, Ictalurus punctatus, by polymerase chain reaction. DNA sequence analysis confirmed the presence of two subtypes, mGluR1 and mGluR3, that were coexpressed with each other and with the putative odorant receptors within single olfactory receptor neurons. Immunocytochemical data showed that both mGluR subtypes were expressed in the apical dendrites and some cilia of olfactory neurons. Pharmacological analysis showed that antagonists to each mGluR subtype significantly decreased the electrophysiological response to odorant amino acids. alpha-Methyl-L-CCG1/(2S,3S,4S)-2-methyl-2-(carboxycyclopropyl++ +)glycine (MCCG), a known antagonist to mGluR3, and (S)-4-carboxyphenylglycine (S-4CPG), a specific antagonist to mGluR1, each significantly reduced olfactory receptor responses to L-glutamate. S-4CPG and MCCG reduced the glutamate response to 54% and 56% of control, respectively, which was significantly greater than their effect on a neutral amino acid odorant, methionine. These significant reductions of odorant response by the antagonists, taken with the expression of these receptors throughout the dendritic and ciliated portions of some olfactory receptor neurons, suggest that these mGluRs may be involved in olfactory reception and signal transduction.     

Study of the function of nerve growth factor in the olfactory tract of the mouse

The aim of this study was to examine the function of nerve growth factor (BGF) in the olfactory tract of mice. Using the mice which had received unilateral olfactory bulbectomy and in which antibodies to NGF had been continuously infused with into the contralateral olfactory blub, three kinds of analysis were performed: histological analysis of the olfactory epithelium by HE staining, immunohistochemical analysis of the olfactory epithelium using polyclonal antibodies to trk which forms the NGF receptor, and olfactory-mediated behavioral analysis with cycloheximide. These animals had been sacrificed at day 1, 3, 7, 14, 21 or 28. Several findings were obtained as a result of the above analysis. Degeneration of the olfactory epithelium and trk expression by the olfactory cells were observed on day 7, and the olfactory epithelium was incompletely regenerated on day 28. However, trk expression by the olfactory cell was still recognized and the olfactory function was not restored by day 28. These examinations suggest that NGF produced in the olfactory bulb was transported retrogradely to olfactory cells through the olfactory nerves, and was associated with sustaining the existence of those cells and with regenerating the olfactory tract after injury.     

Recovery of structure and function following transection of the primary olfactory nerves in pigeons.

Studied the function of the pigeon's olfactory system before and after bilateral sectioning of the olfactory nerves. 4 adult pigeons were used in each of 3 experiments; 8 normal controls were used in Exp I. All transected nerves were found to be healed 189-302 days after sectioning. Electrical recording from the regenerated nerves revealed apparently normal receptor function and, indirectly, autonomic reflex responsiveness. Previously untrained Ss learned an olfactory discrimination after reconstitution of the peripheral olfactory system using a conditioned suppression procedure. The olfactory nerves of trained Ss were sectioned and the behavioral response recovered within 16-82 days. The gross sizes of primary olfactory nerves and olfactory bulbs were frequently much less than those of controls, but on the ultrastructural level there was no recognizable morphological deficiency in the receptor cellular organelles or terminal synaptic contacts in bulbar glomeruli. (35 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A modified coaxial compound micropipette for extracellular iontophoresis and intracellular recording: fabrication, performance and theory

Different olfactory cues elicit distinct behaviors such as attraction, avoidance, feeding, or mating. In the nematode C. elegans, these cues are sensed by a small number of olfactory neurons, each of which expresses several different odorant receptors. The type of behavioral response elicited by an odorant could be specified by the olfactory receptor or by the olfactory neuron in which the receptor is activated. The attractive odorant diacetyl is detected by the receptor protein ODR-10, which is normally expressed in the AWA olfactory neurons. The repulsive odorant 2-nonanone is detected by the AWB olfactory neurons. Transgenic animals that express ODR-10 in AWB rather than AWA avoid diacetyl, while maintaining qualitatively normal responses to other attractive and repulsive odorants. Animals that express ODR-10 simultaneously in AWA and AWB have a defective response to diacetyl, possibly because of conflicting olfactory inputs. Thus, an animal's preference for an odor is defined by the sensory neurons that express a given odorant receptor molecule.     

LacZ and OMP are co-expressed during ontogeny and regeneration in olfactory receptor neurons of OMP promoter-lacZ transgenic mice

The ontogeny and cellular specificity of expression of beta-galactosidase activity and olfactory marker protein (OMP) are compared in olfactory tissue of the H-OMP-lacZ-3 line of transgenic mice. In this line the expression of lacZ is driven by a 0.3 kb fragment of the rat OMP promoter. During fetal development, lacZ expression is detectable in olfactory receptor neurons (ORNs) shortly after the initial appearance of endogenous OMP. The beta-galactosidase marker was observed only in mature olfactory receptor neurons where it co-localized with endogenous OMP. It was absent from immature neurons that express the growth associated phosphoprotein B50/GAP43. Lesion of the peripheral olfactory pathway by intranasal irrigation with Triton X-100 eliminated expression of both OMP and lacZ in the olfactory neuroepithelium. Subsequent regeneration of the full complement of olfactory receptor neurons was associated with co-expression of both OMP and beta-galactosidase activity. Neither OMP nor beta-galactosidase activity was induced in any other cell type of the regenerating olfactory mucosa. Thus, as little as 0.3 kb of the OMP promoter has the ability to target lacZ expression to olfactory receptor neurons in a temporally and spatially defined manner. We discuss the potential utility of this transgenic line for future studies of the olfactory system.     

Organization and expression of canine olfactory receptor genes

Four members of the canine olfactory receptor gene family were characterized. The predicted proteins shared 40-64% identity with previously identified olfactory receptors. The four subfamilies identified in Southern hybridization experiments had as few as 2 and as many as 20 members. All four genes were expressed exclusively in olfactory epithelium. Expression of multiple members of the larger subfamilies was detected, suggesting that most if not all of the cross-hybridizing bands in genomic Southern blots represented actively transcribed olfactory receptor genes. Analysis of large DNA fragments using Southern blots of pulsed-field gels indicated that subfamily members were clustered together, and that two of the subfamilies were closely linked in the dog genome. Analysis of the four olfactory receptor gene subfamilies in 26 breeds of dog provided evidence that the number of genes per subfamily was stable in spite of differential selection on the basis of olfactory acuity in scent hounds, sight hounds, and toy breeds.     

Study of the fifth-type cell in the olfactory epithelium

The olfactory epithelium has been shown to contain fifth-type cells and microvillar cells and olfactory receptor cells along with supporting and basal cells. However, the morphological differences among them are unknown, especially between the former two, and most of their functions are still unclear. In this study, the fifth-type cell and the microvillar cell in the olfactory epithelium of mouse were micromorphologically investigated by SEM and TEM. With the aim of elucidation their functions, the morphological change in these cells after resection of olfactory bulb were studied. In addition, the changes in the olfactory epihelium after HRP injections into the postganglionic fiber for the trigeminal nerve and the olfactory bulb were examined. The TEM observation revealed that the fifth-type cell has finger-like microvilli, which are strong and straight. The microvilli were characterized by a specific core structure consisting of microfilament bundles. On the other band, the microvilli of the microvillar cell were meandering and had no core structure, indicating that the two cells are clearly different types. By SEM observation, neither the fifth-type cell nor the microvillar cell was found in the normal olfactory epithelium. When the olfactory cillial mat disappeared as a result of recection of the olfactory bulb, the fifth-type cell became observable. On the 300th-day after the resection, it was found that the olfactory receptor cells had disappeared and the microvilli of the supporting cells were shortened. Thus, the surface structure of the microvillar cell became clearly observable. Then, the microvilli were found to be shorter than those of the fifth-type cell and were distributed radially. Neither the fifth-type cell nor the microvillar cell was not affected by resection of the olfactory bulb. Most of the olfactory vesicles were HRP positive after the HRP injection into the olfactory bulb; however, HRP was not detected in either the fifth-type cells or the microvillar cells. After the HRP injection into the trigeminal postganglionic fiber, it was detectable in part of the nerves in the lamina propria mucosa and the epithelal basement, but not in the fifth-type cell or microvillar cell. These results suggest that the fifth-type cell is a mechanoreceptor for a system other than the olfactory one and the microvillar cell is a kind of supporting cell in an early stage or final stage near death.     

Glycobiology of the olfactory system

The olfactory system is a highly plastic region of the nervous system. Continuous remodeling of neuronal circuits in the olfactory bulb takes place throughout life as a result of constant turnover of primary sensory olfactory neurons in the periphery. Glycoconjugates are very important in olfactory development, regeneration and function. This article deals with different aspects of glycobiology relevant for the olfactory system. Various anatomical, developmental and functional subdivisions of the olfactory system have been labeled with exogenous lectins. The application of reverse lectin histochemistry resulted in the visualization of endogenous lectins, involved in fasciculation of olfactory axons. Numerous glycoproteins, among them members of the immunoglobulin superfamily, the cadherins and integrins as well as different glycolipids and proteoglycans can act as surface adhesion molecules in the olfactory system. The olfactory-specific form of the sialoglycoprotein neural cell adhesion molecule is implicated in olfactory neuronal and axonal guidance. Glycoconjugates including laminin, fibronectin and proteoglycans are abundant components of the olfactory extracellular matrix, influencing neurite outgrowth and cellular migration. Immunohistochemical labeling has revealed occurrence of the carbohydrate differentiation antigen, playing a role in neurulation and morphogenesis of the very early olfactory system. The synaptic vesicle glycoprotein, appearing also early in olfactory development, is used as a marker of olfactory tumors. Finally, membrane and transmembrane glycoconjugates as well as secreted glycoconjugates may act as olfactory receptor molecules.     

The role of surgery in the management of low back pain

Life-history characteristics of female threespine stickleback (Gasterosteus aculeatus) were examined in 12 populations, 11 freshwater and one anadromous, within the Cook Inlet region of Alaska. Because this area has been deglaciated during the last 20 000 years, the freshwater populations are recently derived, probably independently, from the local marine or anadromous stickleback. Freshwater threespine stickleback have undergone considerable morphological evolution within this region, apparently in response to environmental factors including predatory regimes and environmental productivity. Our freshwater study populations were selected to sample this range of morphological variation in order to determine whether life-history traits and morphologies have followed similar evolutionary trajectories. Freshwater populations could be categorized generally into one of three ecomorphotypes: those inhabiting relatively productive lakes having one or more piscivorous fishes present, and in which the stickleback exhibit a fully developed pelvic girdle; those inhabiting low-calcium lakes that lack piscivorous fishes, and in which the pelvic structures are incomplete; those living in streams with piscivorous fishes, in which the stickleback have fully developed pelvic girdles. The anadromous population constituted a fourth ecomorphotype that lives in marine waters, and is robustly armored. The freshwater populations showed considerable variation in all life-history traits assessed, and this variation generally corresponded to our ecomorphological classifications. Nevertheless, within each ecomorphotype there was sufficient variation to suggest that morphological and life-history traits may not always respond in the same manner in response to the same selective regime.Copyright 1998 The Linnean Society of London     

Formulation and evaluation of albuterol metered dose inhalers containing tetrafluoroethane (P134a), a non-CFC propellant

The objectives of this study were to determine: (1) the frequency and distribution of carbonic anhydrase (CA) activity in the bullfrog nasal cavities, and (2) whether inhibition of nasal CA affects the olfactory receptor response to CO2 or other odorants. It was found, using Hansson's staining technique, that some olfactory receptor neurons exhibited CA activity and that these CA-positive receptors were distributed throughout the nasal cavity with peak densities in the dorsal and ventral sensory epithelial regions. To test for the role of CA in olfactory transduction, electro-olfacto-grams (EOGs) were recorded from the surface of the ventral sensory epithelium in response to 2-s pulses of 5% CO2 and amyl acetate before and after topical CA inhibition with acetazolamide (10(-3) mol.l-1). In 52 bullfrogs, 1222 sites on the ventral epithelium were tested resulting in 23 locations that exhibited a response to 5% CO2. Inhibition of CA caused an immediate 65% reduction in the EOG response to CO2 while the response to amyl acetate was not affected. These results, along with the histochemical localization of CA in some olfactory receptor neurons, indicate that CA plays a role in the detection of CO2 in frog olfactory neurons and that only a small population of olfactory receptor neurons are CO2 sensitive.     

Expression of the low affinity neurotrophin receptor, P75NGFR, in the rat forebrain, following unilateral bulbectomy

It has been hypothesized that the main olfactory bulb, with its relatively rich source of neurotrophins, may provide trophic support for neurons that project to the bulb. We monitored expression of the common, low affinity receptor for neurotrophins, p75NGFR, in the olfactory bulb and basal forebrain of unilaterally bulbectomized and sham-treated rats, 1-16 weeks post-surgery, using the monoclonal antibody MAb192. An induction of p75NGFR-immunoreactivity was observed in both the glomerular and olfactory nerve layers of the right, contralateral main olfactory bulb of lesioned animals. The naturally occurring regeneration taking place in the olfactory neuroepithelium is known to be altered by olfactory bulbectomy, with subsequent changes in the sensory input to the remaining bulb. These changes in expression of p75NGFR in the olfactory bulb support the hypothesis we have developed in previous papers, that changes in the extent of the peripheral input from the olfactory neuroepithelium to the main olfactory bulb regulate p75NGFR expression in both the glomerular and the olfactory nerve layers. Expression of p75NGFR in the basal forebrain of bulbectomized animals was found to be no different than sham-treated controls and does not support the hypothesis that the olfactory bulb provides trophic support to this region of the central nervous system.     

Identification of olfactory receptor mRNA sequences from the rat olfactory bulb glomerular layer

In situ hybridization has demonstrated mRNA for olfactory receptors (OR) in the axon terminals of olfactory receptor neurons. Neurons that express the same OR appear to send their axons to two stereotyped glomeruli in the olfactory bulb (OB). Based on these observations, we tested the feasibility of using RT-PCR to isolate and sequence OR mRNA from small samples of the rat OB glomerular layer. Biomagnetic mRNA isolation followed by RT-PCR yielded partial sequences for 21 novel members of the OR family. The results suggest that the topography of OR mRNA can be mapped across the OB, to study synaptic specificity and odor representation in the olfactory system.     

Olfactory receptor proteins. Expression, characterization and partial purification

A rat olfactory epithelium cDNA library was screened for olfactory receptor clones. One of the positively hybridizing cDNA clones was sequenced and found to encode a new member of the olfactory receptor superfamily. This cDNA, termed olp4, was used as a model of olfactory receptor for expression, both in vitro and in vivo. Expression of olp4, as well as of another previously cloned olfactory receptor (F5), was monitored by immunoprecipitation was a monoclonal antibody directed against a Flag peptide epitope tag, inserted at the N-terminus of the open reading frame, and a specific polyclonal antibody against a C-terminal peptide of olp4. Translation in vitro, followed by immunoprecipitation, showed a major olp4-specific band of 27-29 kDa. The olp4 and F5 polypeptides were found to be inserted into microsomal membranes as expected for integral membrane proteins. Expression in vivo of Flag-olp4 in Sf9 insect cells, using the baculovirus expression system, showed a specific polypeptide of the same size as the in vitro species, with an additional band of 34 kDa, which is most likely a glycosylated form. Fluorescence cytometry and immunohistochemical assays demonstrated the localization of the Flag-olp4 product on the cell surface of the infected host Sf9 cells, with the N-terminus and C-terminus in the proper orientation. Affinity chromatography was used for the partial purification of the olp4 polypeptide from infected Sf9 cells. The identification and purification of this expressed olfactory receptor polypeptide could open the way for further characterization and functional studies of the olfactory receptor superfamily members.     

Olfactory ensheathing cells promote neurite extension from embryonic olfactory receptor cells in vitro

The role of ensheathing cells, a macroglial cell type with a unique presence in the olfactory system, in the outgrowth of olfactory receptor cell neurites was explored in vitro. Glial cell cultures harvested from both the olfactory bulb nerve layer and the hippocampus were established and immunocytochemically characterized. The expression of the p75 low-affinity nerve growth factor receptor by ensheathing cells was used to distinguish them from other macroglial subpopulations. Results indicated that ensheathing cell cultures were approximately 80% pure. Olfactory receptor cells were cocultured with ensheathing or hippocampal glial cells or were seeded on laminin or poly-L-lysine as controls. Olfactory receptor cells extended the longest primary neurites when cocultured with ensheathing cells. Neurite extension on hippocampal glia and laminin was less extensive than that observed on ensheathing cells but higher than that on poly-L-lysine. The neurite outgrowth-promoting effect of ensheathing cells was, at least in part, mediated by diffusible factors, because olfactory receptor cell neurite extension could also be facilitated when receptor cells were cultured in ensheathing cell-conditioned media. In contrast, cortical neurons extended neurites of equivalent lengths on ensheathing and hippocampal glia. The results suggest that ensheathing cells may release factors that support the continuous outgrowth of olfactory receptor cell axons and, therefore, the capacity of this pathway to recover from injury.