Luteinizing hormone-releasing hormone in olfactory bulbs of primates

This immunohistochemical study of luteinizing hormone-releasing hormone (LHRH) in the olfactory bulbs in primates was undertaken in order to see whether there was an LHRH innervation in these species similar to that found in rodents. One old world (Macaca fascicularis) and two new world (Saimiri sciureus and Aotus trivirgatus) monkeys were studied. Aotus trivirgatus was of particular interest as it is noctural and so presumably more dependent upon olfactory cues. Animals were perfused with fixative, olfactory bulbs removed and sectioned, and tissues reacted immunocytochemically using LR1 (Benoit) antiserum to LHRH. Some LHRH innervation was found in the olfactory bulbs of all three species, comprising a few LHRH neurons and many fibers that ramified within the bulbs. The accessory bulb (not present as a distinct entity in old world primates) had more LHRH innervation than did the main olfactory bulb. Aotus trivirgatus had the greatest representation of LHRH of the three species. The layer of the olfactory bulb with the greatest number of LHRH fibers was the external plexiform layer. This is also true in rodents. There is evidence that LHRH has a role in the mediation of olfactory cues in reproductive behavior in rodents. It is not known how LHRH functions within the olfactory system in primates. However, the fact that it is distributed similarly in the two groups suggests that it may serve a similar function.     

The postnatal development of the main olfactory bulb of the rat

The postnatal development from birth to 1 year of the main olfactory bulb was examined quantitatively. The volume of the main olfactory bulb increased over seven-fold by day 30 and remained unchanged thereafter. During the same period the volume of the granular layer increased 18-fold and the mean areas of the olfactory glomeruli increased seven-fold. The mean areas of mitral cell perikarya doubled between the neonatal and juvenile periods. The total number of the mitral cells, however, declined during the first three postnatal weeks. In the internal granular layer of the main olfactory bulb, 89% of the granule cells were acquired postnatally. Much of the cellular gain occurred during the first 3 weeks, with the period of maximum acquisition between days 8 and 14. The number of subependymal cells, the precursors of granule cells, reached a peak at 12 days and gradually declined. But some primitive cells could still be found at one year of age and there was an increase in the total number of granule cells beyond day 30. The mean nuber of internal granular layer cells in a single main olfactory bulb of adult rats was about 5 X 10(6); the number of mitral cells about 4 X 10(4). In the animals injected with 3H-thymidine on day 20 and killed 2 h after injection a small but significant proportion of cells was labelled in the subependymal layer but few in the internal granular layer. In the animals killed 20 and 40 days after injection there was a 10--11-fold rise in the proportion of labelled internal granular layer cells. The proportion of labelled internal granular layer cells decreased in longer survival groups but the total number of labelled cells remained the same, even in year-old animals. However, the total number of internal granular layer cells in the sections examined increased with age.     

Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.     

Differential localization of G proteins, Gi and Go, in the olfactory epithelium and the main olfactory bulb of the rat.

The immunohistochemical localization of proteins Gi1 (plus Gi3). Gi2 and Go was studied in the olfactory epithelium and the main olfactory bulb of rats, using purified antibodies to the respective alpha subunits and beta gamma subunits of these G proteins. In the olfactory epithelium, only a restricted population of olfactory cells was immunopositive for Gi2 alpha, but others were not. The immunoreactivity for Gi1 alpha/Gi3 alpha was not observed. The olfactory epithelium was immunopositive for both Go alpha and beta gamma, but its apical surface was immunopositive only for beta gamma. In the main olfactory bulb, all layers were intensely immunopositive for Go alpha and beta gamma but weakly for Gi2 alpha. In contrast to the negative or weak immunostainings in the olfactory nerve fiber layer and glomeruli, the molecular and the internal granular layers were intensely immunopositive for Gi1 alpha/Gi3 alpha. These findings suggest the functional difference among Gi1/Gi3, Gi2 and Go in the signal transduction in the olfactory system.     

Arx homeobox gene is essential for development of mouse olfactory system

The olfactory system provides an excellent model in which to study cell proliferation, migration, differentiation, axon guidance, dendritic morphogenesis, and synapse formation. We report here crucial roles of the Arx homeobox gene in the developing olfactory system by analyzing its mutant phenotypes. Arx protein was expressed strongly in the interneurons and weakly in the radial glia of the olfactory bulb, but in neither the olfactory sensory neurons nor bulbar projection neurons. Arx-deficient mice showed severe anatomical abnormalities in the developing olfactory system: (1) size reduction of the olfactory bulb, (2) reduced proliferation and impaired entry into the olfactory bulb of interneuron progenitors, (3) loss of tyrosine hydroxylase-positive periglomerular cells, (4) disorganization of the layer structure of the olfactory bulb, and (5) abnormal axonal termination of olfactory sensory neurons in an unusual axon-tangled structure, the fibrocellular mass. Thus, Arx is required for not only the proper developmental processes of Arx-expressing interneurons, but also the establishment of functional olfactory neural circuitry by affecting Arx-non-expressing sensory neurons and projection neurons. These findings suggest a likely role of Arx in regulating the expression of putative instructive signals produced in the olfactory bulb for the proper innervation of olfactory sensory axons.     

Spatiotemporal distribution of the insulin-like growth factor receptor in the rat olfactory bulb

Insulin-like growth factor I (IGF-I) and its receptor (IGF-IR) are involved in growth of neurons. In the rat olfactory epithelium, we previously showed IGF-IR immunostaining in subsets of olfactory receptor neurons. We now report that IGF-IR staining was heaviest in the olfactory nerve layer of the rat olfactory bulb at embryonic days 18, and 19 and postnatal day 1, with labeling of protoglomeruli. In the adult, only a few glomeruli were IGF-IR-positive, some of which were unusually small and strongly labeled. Some IGF-IR-positive fibers penetrated deeper into the external plexiform layer, even in adults. In developing tissues, IGF-IR staining co-localized with that for olfactory marker protein and growth associated protein GAP-43, but to a lesser extent with synaptophysin. In the adult, IGF-IR-positive fibers were compartmentalized within glomeruli. IGF-I may play a role in glomerular synaptogenesis and/or plasticity, possibly contributing to development of coding patterns for odor detection or identification.     

Distinct distribution of four Ca2+-dependent subtypes of protein kinase C in rat olfactory bulb; definite expression of betaII-subtype in the accessory olfactory bulb

The localization of four subtypes of Ca2+-dependent protein kinase C (PKC) in the main and accessory olfactory bulb was examined by immunocytochemistry by using specific antibodies against each PKC subtype. In the main olfactory bulb, alpha-PKC was densely localized in a large number of granule cells and in a few tufted cells, and faint immunoreactivity was seen in some periglomerular cells. betaI-PKC was intensely found in periglomerular cells and tufted cells. gamma-PKC immunoreactivity was present in the external plexiform layer, the internal plexiform layer, and the granular layer, but the immunoreactivity was found only in the neuropils. Little, if any, betaII-PKC was seen in the main olfactory bulb. On the other hand, the intense immunoreactivity for betaII-PKC was seen in periglomerular cells of the accessory olfactory bulb. The betaI-PKC and gamma-PKC were also present in periglomerular cells of the accessory olfactory bulb, while alpha-PKC was localized only in granule cells. Double staining study in the accessory olfactory bulb showed that betaII-PKC was present in the GABAergic periglomerular cells, while betaI-PKC localized to the non-GABAergic periglomerular cells; gamma-PKC was expressed in both GABAergic and non-GABAergic cells. These findings suggest that four calcium-dependent subtypes of PKC play different roles in the olfactory bulb and definite expression of betaII-PKC strongly suggested the involvement of this subtype in a specific function in the accessory olfactory bulb.     

Histochemical localization of NADPH-diaphorase in the rat accessory olfactory bulb

The distribution of NADPH-diaphorase activity was examined inthe accessory olfactory bulb of the rat using a direct histochemicaltechnique. Labeled fibers and somata were found in all layersof the accessory olfactory bulb. The entire vomeronasal nerveand all vomeronasal glomeruli were strongly labeled, contraryto the main olfactory bulb, where only dorsomedial olfactoryglomeruli displayed NADPH-diaphorase activity. NADPH-diapborasepositive neurons were identified as periglomerular cells inthe glomerular layer and external plexiform layer, horizontalcells in the internal plexiform layer, and granule cells anddeep short-axon cells in the granule cell layer. The labeleddendrites of the granule cells formed a dense neuropile in thegranule cell layer, internal plexiform layer and external plexiformlayer. The staining pattern in the accessory olfactory bulbwas more complex than what has been previously reported, anddemonstrated both similarities and differences with the distributionof NADPH-diaphorase in the main olfactory bulb.     

Localized and discrete changes in neuropeptide (LHRH and TRH) and neurotransmitter (NE and DA) concentrations within the olfactory bulbs of male mice as a function of social interaction

Individually housed male mice were exposed to either an intact male or an ovariectomized female mouse for 1 min and decapitated at 5, 15, or 60 min to examine the hypothesis whether discrete changes in olfactory bulb neuropeptide (LHRH and TRH) and neurotransmitter (NE and DA) concentrations would occur following onset of exposure. A nonexposed control group (decapitated at time 0) was also included. Bilateral olfactory bulbs were dissected into anterior dorsal (ADOB) and posterior dorsal (PDOB) olfactory bulb fragments and prepared for radioimmunoassays (LHRH and TRH) or radioenzymatic assays (NE and DA). Concentrations of LHRH and NE, but not of TRH and DA, from the PDOB were significantly greater than those of ADOB fragments. Exposure to a male resulted in a significant increase of PDOB LHRH at 5 min following exposure and a significant increase in LHRH at 15 min following female exposure. Norepinephrine within the ADOB and PDOB and DA within the PDOB demonstrated a statistically significant increase at 60 min following exposure to an ovariectomized female. In marked contrast, no statistically significant changes were obtained following male exposure. These results not only demonstrate a preferential localization of neuroregulators within the olfactory bulb of male mice but discrete changes in the concentration of these neuroregulators in response to male or female exposure, suggesting the possibility that some processing and coding of chemical cue information during social encounters already occurs at the level of the olfactory bulb.     

Cellular interactions in the development of the olfactory system: an ablation and homotypic transplantation analysis

In the current study, we addressed two questions: First, is the olfactory placode necessary for the development of the olfactory bulb and the entire telencephalon? Second, does the olfactory placode contribute cells to the olfactory bulb? We addressed these questions by unilaterally ablating the olfactory placode in chick embryos before an olfactory nerve was produced and, in a second series of experiments, by replacing the ablated chick olfactory placode with a quail olfactory placode. Our results indicate that the olfactory placode is critical for olfactory bulb development, but is not necessary for the development of the rest of the telencephalon. Further, our results support the hypothesis that LHRH neurons and olfactory nerve glia originate in the olfactory placode, but do not support an olfactory placodal origin for other cell types within the olfactory bulb.     

Regulation of c-Fos expression in the rat olfactory bulbs during olfactory learning

The distribution of c-Fos-immunopositive neurons was examined in the mitral/tufted and granular cell layers in the medium part of the main olfactory bulbs of 18-day-old rats after they had been trained for propionic acid vapour-guided search for dam in the Y-maze. On the next day these pups exhibited a strong preference for the propionic acid odor as compared to the control pups trained for this task without the odor cue and odor-familiarized pups exposed to propionic acid as a novel neutral stimulus. Exposure to propionic acid produced a moderate activation of c-Fos expression, mainly in the granular layer of the dorsomedial part of the bulb. Training in the Y-maze devoid of odor cues resulted in diffuse increase in the number of c-Fos-positive neurons both in the mitral and granular cell layers in all parts of the olfactory bulb. Maze training with the odor cue produced activation of c-Fos expression (which significantly exceeded the non-odor Y-maze group) in the dorsomedial olfactory bulb. These data suggest that associative olfactory conditioning results in activation of c-Fos expression that combines the effect of diffuse motivational excitation and specific olfactory input to the neurons which process odor cues.     

Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb

In early rat embryos when axons from sensory neurons first contact the olfactory bulb primordium, lactosamine-containing glycans (LCG) are detected on neurons that are broadly distributed within the olfactory epithelium, but that project axons to a very restricted region of the ventromedial olfactory bulb. LCG(+) axons extend through channels defined by the coexpression of galectin-1 and beta2-laminin. These two extracellular matrix molecules are differentially expressed, along with semaphorin 3A, by subsets of ensheathing cells in the ventral nerve layer of the olfactory bulb. The overlapping expression of these molecules creates an axon-sorting domain that is capable of promoting and repelling subsets of olfactory axons. Specifically, LCG(+) axons preferentially grow into the region of the nerve layer that expresses high amounts of galectin-1, beta2-laminin, and semaphorin 3A, whereas neuropilin-1(+) axons grow in a complementary pattern, avoiding the ventral nerve layer and projecting medially and laterally. These studies suggest that initial patterning of olfactory epithelium to olfactory bulb connections is, in part, dependent on extracellular components of the embryonic nerve layer that mediate convergence and divergence of specific axon subsets.     

Age-related changes of parvalbumin immunoreactive neurons in the rat main olfactory bulb

Parvalbumin (PV) is found in the olfactory system, including the main olfactory bulb, and is thought to be one of the neuroactive substances in olfaction. Changes in PV immunoreactivity in the olfactory system during aging have not been examined. We investigated such changes in the main olfactory bulb (MOB) of the rat at postnatal month 1 (PM 1), PM 3, PM 6, PM 12 and PM 24. PV-IR neurons were almost completely restricted to the external plexiform layer. At PM 1 there were only a few PV-IR neurons; at PM 3, the number of PV-IR neurons was at its greatest but they were not well developed morphologically. At PM 6, the number of PV-IR neurons was similar to that at PM 3 and they had satellite somata with well-developed processes with many varicosities. By PM 12 the number of neurons and processes had declined, and by PM 24, they had fallen even further and the remaining processes had lost most of their varicosities. We conclude that age-related degeneration of PV-IR neurons in the MOB may reduce calcium buffering and affect olfactory function in senile species.     

Histochemical and immunocytochemical study of the migration of neurons from the rat olfactory placode

Immunocytochemical and histochemical methods have been used to describe the neuronal population migrating from the rat olfactory placode and to analyze the spatio-temporal evolution of this neuronal migration during development. Several neuronal markers, such as binding to the lectin Ulex europaeus (UEA I) and the presence of neuron-specific enolase (NSE), olfactory marker protein (OMP), and luteinizing hormone-releasing hormone (LHRH), have been tested in order to determine whether migrating neurons originate from both the medial and the lateral parts of the placode and whether they all express LHRH. Our data show that a large population of differentiated migrating neurons can be identified with an antibody against NSE from the 14th day of gestation and with UEA I one day later. Migrating neurons are closely associated with both the vomeronasal axon fascicles emerging from the medial pit and the olfactory axons originating from the lateral pit. However, the neuron migration from the lateral pit appears to be more discrete than that from the medial pit. No LHRH immunoreactivity has been detected among neurons migrating from the lateral pit. Some neurons accompanying the olfactory axon fascicles exhibit a high level of maturation as shown by their OMP-positivity. Numerous neurons positive for both NSE and UEA I have also been observed within the presumptive olfactory nerve layer in early embryonic stages.     

The central pathway of primary olfactory axons is abnormal in mice lacking the N-CAM-180 isoform

Although N-CAM has previously been implicated in the growth and fasciculation of axons, the development of axon tracts in transgenic mice with a targeted deletion of the 180-kD isoform of the neural cell adhesion molecule (N-CAM-180) appears grossly normal in comparison to wild-type mice. We examined the organization of the olfactory nerve projection from the olfactory neuroepithelium to glomeruli in the olfactory bulb of postnatal N-CAM-180 null mutant mice. Immunostaining for olfactory marker protein revealed the normal presence of fully mature primary olfactory neurons within the olfactory neuroepithelium of mutant mice. The axons of these neurons form an olfactory nerve, enter the nerve fiber layer of the olfactory bulb, and terminate in olfactory glomeruli as in wild-type control animals. The olfactory bulb is smaller and the nerve fiber layer is relatively thicker in mutants than in wild-type mice. Previous studies have revealed that the plant lectin Dolichos biflorus agglutinin (DBA) clearly stains the perikarya and axons of a subpopulation of primary olfactory neurons. Thus, DBA staining enabled the morphology of the olfactory nerve pathway to be examined at higher resolution in both control and mutant animals. Despite a normal spatial pattern of DBA-stained neurons within the nasal cavity, there was a distorted axonal projection of these neurons onto the surface of the olfactory bulb in N-CAM-180 null mutants. In particular, DBA-stained axons formed fewer and smaller glomeruli in the olfactory bulbs of mutants in comparison to wild-type mice. Many primary olfactory axons failed to exit the nerve fiber layer and contribute to glomerular formation. These results indicate that N-CAM-180 plays an important role in the growth and fasciculation of primary olfactory axons and is essential for normal development of olfactory glomeruli. © 1997 John Wiley & Sons, Inc. J Neurobiol 32 : 643–658, 1997     

The Temporal Expression Pattern of Alpha-Synuclein Modulates Olfactory Neurogenesis in Transgenic Mice

Background

Adult neurogenesis mirrors the brain´s endogenous capacity to generate new neurons throughout life. In the subventricular zone/ olfactory bulb system adult neurogenesis is linked to physiological olfactory function and has been shown to be impaired in murine models of neuronal alpha-Synuclein overexpression. We analyzed the degree and temporo-spatial dynamics of adult olfactory bulb neurogenesis in transgenic mice expressing human wild-type alpha-Synuclein (WTS) under the murine Thy1 (mThy1) promoter, a model known to have a particularly high tg expression associated with impaired olfaction.

Results

Survival of newly generated neurons (NeuN-positive) in the olfactory bulb was unchanged in mThy1 transgenic animals. Due to decreased dopaminergic differentiation a reduction in new dopaminergic neurons within the olfactory bulb glomerular layer was present. This is in contrast to our previously published data on transgenic animals that express WTS under the control of the human platelet-derived growth factor β (PDGF) promoter, that display a widespread decrease in survival of newly generated neurons in regions of adult neurogenesis, resulting in a much more pronounced neurogenesis deficit. Temporal and quantitative expression analysis using immunofluorescence co-localization analysis and Western blots revealed that in comparison to PDGF transgenic animals, in mThy1 transgenic animals WTS is expressed from later stages of neuronal maturation only but at significantly higher levels both in the olfactory bulb and cortex.

Conclusions

The dissociation between higher absolute expression levels of alpha-Synuclein but less severe impact on adult olfactory neurogenesis in mThy1 transgenic mice highlights the importance of temporal expression characteristics of alpha-Synuclein on the maturation of newborn neurons.     

Negatively calcium-modulated membrane guanylate cyclase signaling system in the rat olfactory bulb

The mechanism by which the individual odor signals are translated into the perception of smell in the brain is unknown. The signal processing occurs in the olfactory system which has three major components: olfactory neuroepithelium, olfactory bulb, and olfactory cortex. The neuroepithelial layer is composed of ciliated sensory neurons interspersed among supportive cells. The sensory neurons are the sites of odor transduction, a process that converts the odor signal into an electrical signal. The electrical signal is subsequently received by the neurons of the olfactory bulb, which process the signal and then relay it to the olfactory cortex in the brain. Apart from information about certain biochemical steps of odor transduction, there is almost no knowledge about the means by which the olfactory bulb and cortical neurons process this information. Through biochemical, functional, and immunohistochemical approaches, this study shows the presence of a Ca(2+)-modulated membrane guanylate cyclase (mGC) transduction system in the bulb portion of the olfactory system. The mGC is ROS-GC1. This is coexpressed with its specific modulator, guanylate cyclase activating protein type 1 (GCAP1), in the mitral cells. Thus, a new facet of the Ca(2+)-modulated GCAP1--ROS-GC1 signaling system, which, until now, was believed to be unique to phototransduction, has been revealed. The findings suggest a novel role for this system in the polarization and depolarization phenomena of mitral cells and also contradict the existing belief that no mGC besides GC-D exists in the olfactory neurons.     

GABA immunoreactivity in the main olfactory bulb of the frog Rana temporaria

Immunoreactivity for gamma-aminobutyric acid (GABA) was localized at the light microscopic level in the main olfactory bulb (MOB) of the frog, Rana temporaria. By means of free-floating peroxidase-antiperoxidase immunocytochemical technique, GABA was found in a large number of neurons in the granular cell layer, in a few small somata in the mitral cell layer and in two different types of cell somata in the glomerular layer. Individual GABA-immunopositive cells were found in the olfactory nerve layer. GABA immunostaining was also localized in cell processes and fiber fragments. There were many immunoreactive puncta in all layers of the MOB. GABA-positive punctate structures often outlined immunonegative cells in the mitral cell and glomerular layers. Rounded tightly packed groups of immunoreactive puncta were found only along ventral border of the glomerular layer. The results are discussed in comparison with data obtained on mammalian MOB in terms of MOB functional organization.     

Transregulation of erbB expression in the mouse olfactory bulb.

Previously, we have shown that erbB-3 expression is restricted to the ensheathing cells of the olfactory nerve layer, while erbB-4 is found in the periglomerular and mitral/tufted cells of the olfactory bulb and in cells coming out from the rostral migratory stream of the subependymal layer. In the present work, we have treated adult mice with zinc sulfate intranasal irrigation and analyzed erbB-3 and erbB-4 expression in the deafferented olfactory bulb. Following treatment, olfactory axons undergo degeneration, as indicated by the loss of OMP expression in the deafferented olfactory bulb. The thickness of the olfactory nerve layer is reduced, but the specific intensity of erbB-3 labeling in the remaining olfactory nerve layer is increased with respect to control. Interestingly, following deafferentation, erbB-4 immunoreactivity decreases specifically in cell types that normally make synaptic contacts with primary olfactory neurons in the glomeruli, i.e. periglomerular and mitral/tufted cells. Partial lesion of the olfactory epithelium allows regenerative axon growth of olfactory neurons to the olfactory bulb. Following olfactory axon regeneration, erbB-3 and erbB-4 immunoreactivity in the olfactory bulb is similar to control. Thus, like tyrosine hydroxylase, the down regulation of erbB-4 expression in the periglomerular cells is reversible.