Calretinin- and parvalbumin-immunoreactive neurons in the rat main olfactory bulb do not express NADPH-diaphorase activity

The presence of nitric oxide synthase (NOS) in neuronal elements expressing the calcium-binding proteins calretinin (CR) and parvalbumin (PV) was studied in the rat main olfactory bulb. CR and PV were detected by using immunocytochemistry and the nitric oxide (NO) -synthesizing cells were identified by means of the reduced nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) direct histochemical method. The possible coexistence of NADPH-diaphorase and each calcium-binding protein marker was determined by sequential histochemical-immunohistochemical double-labeling of the same sections. Specific neuronal populations were positive for these three markers. A subpopulation of olfactory fibers and olfactory glomeruli were positive for either NADPH-diaphorase or CR. In the most superficial layers, groups of juxtaglomerular cells, superficial short-axon cells and Van Gehuchten cells demonstrated staining for all three markers. In the deep regions, abundant granule cells were NADPH-diaphorase- and CR-positive and a few were PV-immunoreactive. Scarce deep short-axon cells demonstrated either CR-, PV-, or NADPH-diaphorase staining. Among all these labeled elements, no neuron expressing CR or PV colocalized NADPH-diaphorase staining. The present data contribute to a more detailed classification of the chemically- and morphologically-defined neuronal types in the rodent olfactory bulb. The neurochemical differences support the existence of physiologically distinct groups within morphologically homogeneous populations. Each of these groups would be involved in different modulatory mechanisms of the olfactory information. In addition, the absence of CR and PV in neuronal groups displaying NADPH-diaphorase, which moreover are calmodulin-negative, indicate that the regulation of NOS activity in calmodulin-negative neurons of the rat olfactory bulb is not mediated by CR or PV.     

An olfactory sensory map develops in the absence of normal projection neurons or GABAergic interneurons

Olfactory sensory neurons expressing a given odorant receptor project to two topographically fixed glomeruli in the olfactory bulb. We have examined the contribution of different cell types in the olfactory bulb to the establishment of this topographic map. Mice with a homozygous deficiency in Tbr-1 lack most projection neurons, whereas mice with a homozygous deficiency in Dlx-1 and Dlx-2 lack most GABAergic interneurons. Mice bearing a P2-IRES-tau-lacZ allele and deficient in either Tbr-1 or Dlx-1/Dlx-2 reveal the convergence of axons to one medial and one lateral site at positions analogous to those observed in wild-type mice. These observations suggest that the establishment of a topographic map is not dependent upon cues provided by, or synapse formation with, the major neuronal cell types in the olfactory bulb.     

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.     

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.     

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.     

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.     

Directed expression of the growth-associated protein B-50/GAP-43 to olfactory neurons in transgenic mice results in changes in axon morphology and extraglomerular fiber growth

B-50/GAP-43, a neural growth-associated phosphoprotein, is thought to play a role in neuronal plasticity and nerve fiber formation since it is expressed at high levels in developing and regenerating neurons and in growth cones. Using a construct containing the coding sequence of B-50/GAP-43 under the control of regulatory elements of the olfactory marker protein (OMP) gene, transgenic mice were generated to study the effect of directed expression of B-50/GAP-43 in a class of neurons that does not normally express B-50/GAP-43, namely, mature OMP-positive olfactory neurons. Olfactory neurons have a limited lifespan and are replaced throughout adulthood by new neurons that migrate into the upper compartment of the epithelium following their formation from stem cells in the basal portion of this neuroepithelium. Thus, the primary olfactory pathway is exquisitely suited to examine a role of B-50/GAP-43 in neuronal migration, lifespan, and nerve fiber growth. We find that B-50/GAP-43 expression in adult olfactory neurons results in numerous primary olfactory axons with enlarged endings preferentially located at the rim of individual glomeruli. Furthermore, ectopic olfactory nerve fibers in between the juxtaglomerular neurons or in close approximation to blood vessels were frequently observed. This suggests that expression of B-50/GAP-43 in mature olfactory neurons alters their response to signals in the bulb. Other parameters examined, that is, migration and lifespan of olfactory neurons are normal in B-50/GAP-43 transgenic mice. These observations provide direct in vivo evidence for a role of B-50/GAP-43 in nerve fiber formation and in the determination of the morphology of axons.     

Modulation of olfactory bulb neuron potassium current by tyrosine phosphorylation

Insulin causes a suppression of whole-cell voltage-dependent outward current in cultured neurons from the rat olfactory bulb. This suppression is time-dependent; it is mimicked by application of Src tyrosine kinase inside the cell via the whole-cell patch electrode or by treatment of the olfactory bulb neurons with the tyrosine phosphatase inhibitor pervanadate. The C-type inactivation properties of the outward current in olfactory bulb neurons resemble those of the cloned Kv1.3 potassium channel. In addition, at picomolar concentrations at which it is specific for Kv1.3, the scorpion toxin margatoxin blocks most of the olfactory bulb neuron outward current. Immunocytochemical analysis demonstrates that Kv1.3 is prominent in the cultured olfactory bulb neurons. To identify specific amino acid residues that might be important for potassium current modulation, we examined the effects of pervanadate and insulin on wild-type and mutant Kv1.3 channels expressed in human embryonic kidney (HEK 293) cells. As shown previously, treatment with either pervanadate or insulin suppresses Kv1.3 current in these cells. Mutational analysis demonstrates that at least two distinct tyrosine residues are required for current suppression by pervanadate. Insulin treatment stimulates the tyrosine phosphorylation of Kv1.3 in HEK 293 cells, and a different combination of tyrosine residues is required for the current suppression by insulin. The results suggest that complex patterns of phosphorylation may be involved in the modulation of neuronal potassium current by receptor and nonreceptor tyrosine kinases.     

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.     

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.     

Olfactory bulb habituation to odor stimuli.

Habituation is a simple form of memory, yet its neurobiological mechanisms are only beginning to be understood in mammals. In the olfactory system, the neural correlates of habituation at a fast experimental timescale involving very short intertrial intervals (tens of seconds) have been shown to depend on synaptic adaptation in olfactory cortex. In contrast, behavioral habituation to odorants on a longer timescale with intertrial intervals of several minutes depends on processes in the olfactory bulb, as demonstrated by pharmacological studies. We here show that behavioral habituation to odorants on this longer timescale has a neuronal activity correlate in the olfactory bulb. Spiking responses of mitral cells in the rat olfactory bulb adapt to, and recover from, repeated odorant stimulation with 5-min intertrial intervals with a time course similar to that of behavioral habituation. Moreover, both the behavioral and neuronal effects of odor habituation require functioning N-methyl-d-aspartic acid receptors in the olfactory bulb. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

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.     

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.     

Segregated distribution of TH-immunoreactivity in olfactory glomeruli

Atypical and typical olfactory glomeruli differ in their primary afferents, centrifugal control and in some chemically identified subpopulations of interneurones. The distribution of tyrosine hydroxylase (TH)-immunopositive neurones in the periglomerular region of both typical and atypical glomeruli has been studied using a double histochemical-immunohistochemical method. A segregated distribution of TH-immunopositive cells was found among both types of glomeruli. TH-immunolabelled cells were more abundant (p < 0.05) in the atypical glomeruli. These data suggest that some neuronal subpopulations are related to specific properties of the glomerular physiology and they have a segregated distribution in different subsets of glomeruli. Thus, catecholamines might be involved in the processing of specific olfactory cues in atypical glomeruli. This study presents new differences in the cellular composition of typical and atypical glomeruli.     

The divergent homeobox gene PBX1 is expressed in the postnatal subventricular zone and interneurons of the olfactory bulb

In the mammalian brain, an important phase of neurogenesis occurs postnatally in the subventricular zone (SVZ). This region consists of a heterogeneous population of cells, some mitotically active, others postmitotic. A subset of mitotically active SVZ precursor cells gives rise to a population of neurons that migrates over a long distance to their final destination, the olfactory bulb. Other SVZ precursor cells continue to proliferate or undergo cell death. The combination of genes that regulates proliferation and cell fate determination of SVZ precursor cells remains to be identified. We have used the rat homolog of the human homeobox gene PBX1 in Northern analysis and in situ hybridization studies to determine the temporal and regional localization of PBX1 expression during embryonic and postnatal rat brain development. PBX1 is expressed embryonically in the telencephalon. In addition, it is expressed at high levels postnatally in the SVZ, in the migratory pathway to the olfactory bulb, and in the layers of the olfactory bulb that are the targets of these migratory neurons. Combining in situ hybridization for PBX1 with immunostaining for markers of cell proliferation (PCNA), postmitotic neurons (class III beta-tubulin), and glia (GFAP), we show that SVZ proliferating cells and their neuronal progeny express rat PBX1 mRNA, whereas glial cells do not express detectable levels of PBX1. The expression of PBX1 in SVZ precursor cells and postmitotic neurons suggests a role for PBX1 in the generation of olfactory bulb interneurons and in mammalian neurogenesis.     

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.     

Sensory information processing in the frog olfactory pathways. Experimental basis for modeling studies

In the frog, unitary electrophysiological recordings have been extensively used to investigate odor processing along the olfactory pathways. By comparing spontaneous and odor-evoked activities of neuroreceptor, mitral and cortical cells, we have collected fundamental data relating to coding abilities of the three olfactory levels, the olfactory mucosa, the bulb and the cortex. Based on a synthesis of our experimental data related to GABAergic and dopaminergic involvement in the olfactory bulb, this paper aims to match this information with computational data and to discuss some questions on bulbar processing. This paper is also devoted to further analyze original results on coding properties of two functionally evidenced neuron subpopulations in the olfactory cortex. Thus, the assumption according to which some cortical neurons may work as temporal integrators while others as coincidence detectors is presented. Moreover, the pertinence that the neural code may be carried by a single spike with varying latency was demonstrated.     

DNA image cytometry on sections as compared with image cytometry on smears and flow cytometry in melanoma

The distribution and morphology of neurocalcin-immunopositive neurons have been studied in the rat accessory olfactory bulb. Different subsets of neurons displaying neurocalcin immunoreactivity were found in the glomerular layer, the external plexiform layer and the internal plexiform layer. The most abundant staining was detected in the glomerular layer where neurocalcin-immunoreactive periglomerular cells and external tufted cells were observed in the lateral glomeruli, whereas the central region of this layer was practically devoid of immunopositive neurons. In the external plexiform layer, medial tufted cells and Van Gehuchten cells displayed neurocalcin immunoreactivity. In the internal plexiform layer, interneurons classified as horizontal cells and vertical cells of Cajal were neurocalcin-immunoreactivity. In the internal plexiform layer, interneurons classified as horizontal cells and vertical cells of Cajal were neurocalcin-immunostained. The staining pattern for neurocalcin in the accessory olfactory bulb showed similarities with the immunostaining described in this brain region for another EF-hand calcium binding protein, calbindin D-28k. However, after double immunohistochemical labeling, colocalization of both proteins in the same neuron was not observed, reflecting a biochemical heterogeneity within morphologically homogeneous neuronal groups.     

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.