Secretagogin expression in the mouse olfactory bulb under sensory impairments.

The interneurons of the olfactory bulb (OB) are characterized by the expression of different calcium-binding proteins, whose specific functions are not fully understood. This is the case of one of the most recently discovered, the secretagogin (SCGN), which is expressed in interneurons of the glomerular and the granule cell layers, but whose function in the olfactory pathway is still unknown. To address this question, we examined the distribution, generation and activity of SCGN-positive interneurons in the OB of two complementary models of olfactory impairments: Purkinje Cell Degeneration (PCD) and olfactory-deprived mice. Our results showed a significant increase in the density of SCGN-positive cells in the inframitral layers of olfactory-deprived mice as compared to control animals. Moreover, BrdU analyses revealed that these additional SCGN-positive cells are not newly formed. Finally, the neuronal activity, estimated by c-Fos expression, increased in preexisting SCGN-positive interneurons of both deprived and PCD mice -being higher in the later- in comparison with control animals. Altogether, our results suggest that the OB possesses different compensatory mechanisms depending on the type of alteration. Particularly, the SCGN expression is dependent of olfactory stimuli and its function may be related to a compensation against a reduction in sensory inputs.

Differential effects of unilateral olfactory deprivation on noradrenergic and cholinergic systems in the main olfactory bulb of the rat.

The lack of environmental olfactory stimulation produced by sensory deprivation causes significant changes in the deprived olfactory bulb. Olfactory transmission in the main olfactory bulb (MOB) is strongly modulated by centrifugal systems. The present report examines the effects of unilateral deprivation on the noradrenergic and cholinergic centrifugal systems innervating the MOB. The morphology, distribution, and density of positive axons were studied in the MOBs of control and deprived rats, using dopamine-beta-hydroxylase (DBH)-immunohistochemistry and acetylcholinesterase (AChE) histochemistry in serial sections. Catecholamine content was compared among the different groups of MOBs (control, contralateral, and ipsilateral to the deprivation) using high-performance liquid chromatography analysis. Sensory deprivation revealed that the noradrenergic system developed adaptive plastic changes after olfactory deprivation, including important modifications in its fiber density and distribution, while no differences in cholinergic innervation were observed under the same conditions. The noradrenergic system underwent an important alteration in the glomerular layer, in which some glomeruli showed a dense noradrenergic innervation that was not detected in control animals. The DBH-positive glomeruli with the highest noradrenergic fiber density were compared with AChE-stained sections and it was observed that the strongly noradrenergic-innervated glomeruli were always atypical glomeruli (characterized by their strong degree of cholinergic innervation). In addition to the morphological findings, our biochemical data revealed that olfactory deprivation caused a decrease in the content of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid in the ipsilateral MOB in comparison to the contralateral and control MOBs, together with an increase in noradrenaline levels in both the ipsilateral and contralateral MOBs. Our results show that regulation of the noradrenergic centrifugal system in the MOB depends on environmental olfactory stimulation and that it is highly reactive to sensory deprivation. By contrast, the cholinergic system is fairly stable and does not exhibit clear changes after the loss of sensory inputs.

Heterogeneous targeting of centrifugal inputs to the glomerular layer of the main olfactory bulb.

The centrifugal systems innervating the olfactory bulb are important elements in the functional regulation of the olfactory pathway. In this study, the selective innervation of specific glomeruli by serotonergic, noradrenergic and cholinergic centrifugal axons was analyzed. Thus, the morphology, distribution and density of positive axons were studied in the glomerular layer of the main olfactory bulb of the rat, using serotonin-, serotonin transporter- and dopamine-beta-hydroxylase-immunohistochemistry and acetylcholinesterase histochemistry in serial sections. Serotonin-, serotonin transporter-immunostaining and acetylcholinesterase-staining revealed a higher heterogeneity in the glomerular layer of the main olfactory bulb than previously reported. In this sense, four types of glomeruli could be identified according to their serotonergic innervation. The main distinctive feature of these four types of glomeruli was their serotonergic fibre density, although they also differed in their size, morphology and relative position throughout the rostro-caudal main olfactory bulb. In this sense, some specific regions of the glomerular layer were occupied by glomeruli with a particular morphology and a characteristic serotonergic innervation pattern that was consistent from animal to animal. Regarding the cholinergic system, we offer a new subclassification of glomeruli based on the distribution of cholinergic fibres in the glomerular structure. Finally, the serotonergic and cholinergic innervation patterns were compared in the glomerular layer. Sexual differences concerning the density of serotonergic fibres were observed in the atypical glomeruli (characterized by their strong cholinergic innervation). The present report provides new data on the heterogeneity of the centrifugal innervation of the glomerular layer that constitutes the morphological substrate supporting the existence of differential modulatory levels among the entire glomerular population.

Calretinin-, neurocalcin-, and parvalbumin-immunoreactive elements in the olfactory bulb of the hedgehog (Erinaceus europaeus).

The distribution pattern and morphology of calretinin-, neurocalcin-, and parvalbumin-immunoreactive neurons were studied in the main and accessory olfactory bulbs of the hedgehog. The detection of these markers was carried out by using monoclonal or polyclonal antibodies and the avidin-biotin-immunoperoxidase method. Specific neuronal populations were positive for these calcium-binding proteins in the hedgehog olfactory bulb, revealing both similarities to and differences from the data reported in the olfactory bulb of rodent species. The distribution pattern of each calcium-binding protein studied in the accessory olfactory bulb was highly similar to that described in other macrosmatic species. However, in the main olfactory bulb, the markers analyzed were expressed in similar interneuronal populations as they are in the rodent olfactory bulb, whereas cell groups categorized as projecting neurons demonstrated striking differences in the expression of these calcium-binding proteins. These results suggest that the expression of calcium-binding proteins in a given brain region is not a constant feature among species despite a similar organization but that different factors could influence their expression. Thus, the accessory olfactory system involved in the processing of specific and similar olfactory cues among species demonstrates a more constant organization among species. By contrast, the functionally important role of the main olfactory system in the hedgehog is accompanied by a more complex organization, which is reflected in an increased diversity of calcium-buffering systems.

Chemical organization of the macaque monkey olfactory bulb: II. Calretinin, calbindin D-28k, parvalbumin, and neurocalcin immunoreactivity.

The distribution and morphologic features of calcium-binding protein- (calbindin D-28k, calretinin, neurocalcin, and parvalbumin) immunoreactive elements were studied in the macaque monkey olfactory bulb by using specific antibodies and the avidin-biotin-immunoperoxidase method. A characteristic laminar pattern of stained elements was observed for each marker. Scarce superficial short-axon cells and superficial stellate cells demonstrated calbindin D-28k immunoreactivity in the outer layers, whereas a moderate number of calbindin D-28k-immunoreactive granule cells and scarce deep short-axon cells were observed in the inner layers. Calretinin-staining demonstrated abundant periglomerular cells and granule cells and a scarce number of other interneuronal populations. Most neurocalcin-immunopositive elements were external and medial tufted cells and periglomerular cells, although other scarcer interneuronal populations were also immunostained. A few superficial and deep short-axon cells as well as small interneurons in the external plexiform layer were the only elements immunoreactive to parvalbumin. The distribution of the immunoreactive elements in the olfactory bulb of the macaque monkey showed a high similarity to that reported in the human, whereas it demonstrated a different and simpler pattern to what has been reported in the olfactory bulb of macrosmatic animals. It suggests more homogeneous calcium-mediated cell responses after stimulation that could be correlated to the lower capability to modulate olfactory signals in microsmatic animals. In addition, these results indicate that experimental models in rodents do not provide an accurate estimation of calcium-binding protein-immunoreactive neuronal populations in the primate olfactory system.

Distribution of parvalbumin immunoreactivity in the brain of the tench (Tinca tinca L., 1758).

The distribution of parvalbumin (PV) immunoreactivity in the tench brain was examined by using the avidin-biotin-peroxidase immunocytochemical method. This protein was detected in neuronal populations throughout all main divisions of the tench brain. In the telencephalic hemispheres, PV-immunopositive neurons were distributed in both the dorsal and ventral areas, being more abundant in the area ventralis telencephali, nucleus ventralis. In the diencephalon, the scarce distribution of PV-containing cells followed a rostrocaudal gradient, and the most evident staining was observed in the nucleus periventricularis tuberculi posterioris and in a few nuclei of the area praetectalis. In the mesencephalon, abundant PV-immunoreactive elements were found in the tectum opticum, torus semicircularis, and tegmentum. In the tectum opticum, PV-immunoreactivity presented a laminar distribution. Three PV-containing neuronal populations were described in the torus semicircularis, whereas in the tegmentum, the PV staining was mainly located in the nucleus tegmentalis rostralis and in the nucleus nervi oculomotorii. In the metencephalon, Purkinje cells were PV-immunopositive in the valvula cerebelli, lobus caudalis cerebelli, and in the corpus cerebelli. In the myelencephalon, PV immunoreactivity was abundant in the nucleus lateralis valvulae, in the nucleus nervi trochlearis, nucleus nervi trigemini, nucleus nervi abducentis, nucleus nervi glossopharyngei, and in the formatio reticularis. Mauthner cells were also PV immunostained. By contrast to other vertebrate groups, only a restricted population of PV-containing neurons was GABA-immunoreactive in the tench, demonstrating that this calcium-binding protein cannot be considered a marker for GABAergic elements in the teleost brain. This study demonstrates a low phylogenetic conservation of the distribution of PV comparing teleosts and tetrapods.

NADPH-diaphorase active and calbindin D-28k-immunoreactive neurons and fibers in the olfactory bulb of the hedgehog (Erinaceus europaeus).

The hedgehog, a macrosomatic insectivore with an extraordinary development of the olfactory structures, has a crucial value for any phylogenetic or comparative study in mammals. The distribution pattern and morphology of NADPH-diaphorase-active and calbindin D-28k-immunoreactive neurons were studied in the main and accessory olfactory bulbs of the hedgehog. NADPH-diaphorase (ND) staining was carried out by a direct histochemical method, and the calbindin D-28k (CaBP) immunoreaction by using a monoclonal antibody and the avidin-biotin-immunoperoxidase method. The possible coexistence of both markers was determined by sequential histochemical-immunohistochemical double labeling of the same sections. Specific neuronal populations were positive for both ND and CaBP markers. No cell colocalized both stains in the hedgehog olfactory bulb. A subpopulation of olfactory fibers, and a subpopulation of olfactory glomeruli, located on the medial side, were positive for ND. Surrounding both the ND-positive and ND-negative glomeruli, there were ND- and CaBP-positive periglomerular cells, the latter group being much more abundant. A subpopulation of superficial short-axon cells was CaBP positive but, contrary to what is observed in rodents, this neuronal type was always ND negative. In addition, three neuronal types were observed in the GL-EPL border after CaBP immunostaining. These neuronal types have not been previously described either in the hedgehog or in the rodent olfactory bulb. Horizontal cells and vertical cells of Cajal were also observed after both ND and CaBP labeling. Distinct groups of ND- and CaBP-positive cells, differing in size, shape, dendritic branching pattern, and staining intensity, were distinguished in the granule cell layer and in the white matter. The large and medium-sized cells were identified as a very heterogeneous population of deep short-axon cells, whereas a subpopulation of granule cells was ND positive. The accessory olfactory bulb showed ND staining in all vomeronasal fibers and glomeruli, and in subpopulations of periglomerular cells, granule cells, and deep short-axon cells. The CaBP immunolabeling was more restricted and located in subpopulations of periglomerular cells and in deep short-axon cells. These results indicate different and more complex ND and CaBP staining patterns in the hedgehog olfactory bulb than those previously described in rodents, including the presence of specific, chemically and morphologically defined new neuronal types.

NADPH-diaphorase in the central nervous system of the tench (Tinca tinca L., 1758).

The distribution and morphological characterization of nicotinamide adenine dinucleotide phosphate-diaphorase (ND)-positive cells and fibers in the tench central nervous system was mapped by using a direct histochemical method. This enzyme was observed in specific cell populations throughout all main divisions of the tench brain. In the telencephalon, we found strongly labeled olfactory fibers, as well as positive cells and fibers in the area ventralis of the telencephalic lobes. Positive staining was observed in the following diencephalic nuclei: nucleus preopticus magnocellularis pars magnocellularis, nucleus recessus lateralis, nucleus recessus posterioris, nucleus posterior tuberis, and nucleus diffusus torus lateralis, as well as small cells with a diffuse distribution surrounding the diencephalic ventricle. In the mesencephalon, heavily stained ND-positive neurons were observed in the nucleus fasciculi longitudinalis medialis, nucleus nervi oculomotorius, and nucleus nervi trochlearis. In the hindbrain the most evident staining was observed as large neurons located in the nuclei of the cranial nerves, scattered positive cells located between the negative fibers of the cranial nerves, and in the nucleus fasciculi solitari. Finally, in the spinal cord, ND-positive cells and fibers were mainly located in the ventral horn. This distribution of ND labeling in the brain of the tench is significantly different from previous data on ND activity in the brain of terrestrial vertebrates and does not correlate with the presence and distribution patterns of several neurotransmitters and neuroactive substances in the teleost brain.

Chemical anatomy of the macaque monkey olfactory bulb: NADPH-diaphorase/nitric oxide synthase activity.

The distribution and the morphology of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase (ND)-active and neuronal nitric oxide synthase (NOS)-immunoreactive neurons and fibers were studied in the olfactory bulb of three species of primates, i.e., the cynomolgus macaque monkey (Macaca fascicularis), the Japanese macaque monkey (Macaca fuscata), and the pig-tail macaque monkey (Macaca nemestrina). The ND staining was carried out by means of a direct histochemical method with beta-NADPH as cosubstrate and nitro blue tetrazolium as chromogen. The NOS immunostaining was carried out by using a polyclonal antibody and the avidin-biotin peroxidase method. Similar results were found in the three species, where a distinct distribution pattern of ND/NOS-stained neurons and fibers was observed. All olfactory fibers demonstrated ND-positive labeling but they were NOS-immunonegative. In the superficial modulatory area of the olfactory bulb, a few weakly ND- and NOS-positive periglomerular cells, stellate cells, and darkly stained superficial short-axon cells were observed. In the inframitral layers, granule cells, deep stellate cells, and deep short-axon cells were distinguished. Short-axon cells had oriented morphologies and spiny dendrites. Many thick, varicose ND/NOS-stained fibers identified as centrifugal fibers were observed in the white matter, granule cell layer, internal plexiform layer, mitral cell layer, and external plexiform layer. This distribution of ND activity and NOS immunoreactivity showed similarities to and differences from what has been reported in the olfactory bulb of macrosmatic mammals including rodents (rat, mouse, and hamster) and insectivores (hedgehog). These data confirm that the complexity of the ND/NOS staining in the olfactory bulb of one species correlates with the importance of olfaction in the biology of such species.

Distribution of acetylcholinesterase and choline acetyltransferase in the main and accessory olfactory bulbs of the hedgehog (Erinaceus europaeus).

The distribution of cholinergic markers was studied in the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) of the western European hedgehog (Erinaceus europaeus) by using choline acetyltransferase (ChAT) immunocytochemistry and acetylcholinesterase (AChE) histochemistry. A dense network of AChE-containing and ChAT-immunoreactive fibers was observed innervating all layers of the MOB except the olfactory nerve layer, where neither AChE- nor ChAT-labeled elements were found. The highest density of AChE- and ChAT-positive axons was found in the glomerular layer (GL)/external plexiform layer (EPL) boundary, and in the internal plexiform layer. This general distribution pattern of ChAT- and AChE-stained axons resembled the distribution pattern found in rodents. Nevertheless, some interspecies differences, such as the lack of atypical glomeruli in the hedgehog, were also found. In addition to fibers, a population of noncholinergic and presumably cholinoceptive AChE-active neurons was observed in the hedgehog. All mitral and tufted cells of the hedgehog MOB showed a dark AChE staining unlike previous observations in the mitral and tufted cells of rodents. As in other species previously reported, subpopulations of external tufted cells and short-axon cells were also AChE-active. Finally, a population of small AChE-containing cells was observed in the EPL of the hedgehog MOB. The size, shape, and location of these cells coincided with those of satellite and perinidal cells, two neuronal types described previously in the EPL of the hedgehog and not present in the rodent MOB. The AOB of the hedgehog showed a distribution of AChE- and ChAT-positive fibers similar to the rodent AOB. Nevertheless, a heterogeneous innervation of vomeronasal glomeruli by bundles of AChE- and ChAT-labeled axons found in the hedgehog has not been previously found in any other species. As in the MOB, all mitral cells in the AOB showed a strong AChE activity. These results demonstrate some similarities but also important differences between the distribution of ChAT and AChE in the MOB and AOB of rodents and this primitive mammalian. These variations may indicate a different organization of the cholinergic modulation of the olfactory information in the insectivores.

Coexpression of neurocalcin with other calcium-binding proteins in the rat main olfactory bulb.

The distribution patterns of four calcium-binding proteins (CaBPs)-calbindin D-28k (CB), calretinin (CR), neurocalcin (NC), and parvalbumin (PV)-in the rat main olfactory bulb were compared, and the degrees ofcolocalization of NC with the other CaBPs were determined by using double immunocytochemical techniques. All investigated CaBPs were detected in groups of periglomerular cells and Van Gehuchten cells, whereas other cell types expressed some of the investigated proteins but not all four. Double-labeling techniques demonstrated the colocalization of NC with CB, CR, or PV in periglomerular cells, whereas each neurochemical group constituted entirely segregated populations in the remaining neuronal types. This is evident in granule cells that demonstrated large but segregated populations immunoreactive to either NC or CR. This study provides a further biochemical characterization of interneuronal types in the rat main olfactory bulb. On the basis of the distinct calcium-binding affinities, each neurochemically defined population may have different responses to calcium influx that would result in the existence of distinct functional subgroups within morphologically defined neuronal types. The expression of the investigated CaBPs in periglomerular cells with both single and colocalized patterns suggests that the local circuits in the glomerular layer are constituted by a complex network of elements with particular calcium requirements.

Bilateral olfactory deprivation reveals a selective noradrenergic regulatory input to the olfactory bulb.

Unilateral olfactory deprivation in the rat induces changes in the catecholaminergic system of the olfactory bulb. Nevertheless, evidence suggests that unilateral deprivation does not fully prevent stimulation of the deprived bulb. The present report analyses the response of the catecholaminergic system of the olfactory bulb in fully deprived rats obtained by bilateral naris occlusion. The complete deprivation produces more rapid and dramatic changes in both the intrinsic and extrinsic catecholaminergic systems of the olfactory bulb. Intrinsic responses involve a rapid decrease in dopamine-containing cells to about 25% of controls, correlated with a decreased Fos expression in juxtaglomerular cells of all olfactory glomeruli, with the only exception of those of the atypical glomeruli which maintain unaltered expression of both markers. In parallel with these events, there is a progressive increase in the density of extrinsic noradrenergic axons arising from neurons in the locus coeruleus, which shows, in parallel, a progressive increase in Fos expression. This model demonstrates plastic changes in the catecholaminergic system of the olfactory bulb forming a valid morphological substrate for lowering thresholds in the processing of olfactory information. In addition to this generalized response, there is another one, directed to a specific subset of olfactory glomeruli (atypical glomeruli) involved in the processing of odor pheromone-like cues related to behavioral responses, that could be responsible for keeping active this reduced and selected group of glomeruli carrying crucial olfactory information. These results indicate the existence of adaptive changes in the catecholaminergic system of the olfactory bulb as a response to the lack of afferent peripheral stimulation. These changes involve dopamine- and noradrenaline-immunoreactive elements, in a strategy presumably directed at maintaining to the highest possible level the ability to detect olfactory signals.

Nonspecific labeling of myelin with secondary antisera and high concentrations of Triton X-100.

Triton X-100 is used in immunohistochemistry to make tissue permeable, to present certain antigens to antisera, and to prevent certain nonspecific interactions. This detergent is routinely dissolved in buffers at concentrations of 0.01-0.2%. Using high concentrations of Triton X-100 (0.2-2%) and anti-immunoglobulins G (anti-IgGs), labeling of myelin and microglia was detected in fixed brain tissue by indirect fluorescence and avidin-biotin-immunoperoxidase techniques. Differences were found between the species studied (mouse and rat), the type of anti-IgG (anti-mouse, anti-rabbit, anti-sheep, anti-rat, or anti-guinea pig), the detergent concentration, and whether Triton X-100 was included in the incubation media or applied as a pretreatment. Mouse brain displayed strong myelin labeling with all anti-IgGs but rat brain only with anti-rabbit or anti-sheep IgGs. Staining of ramified microglia occurred only in mouse tissue when anti-mouse IgG was used. Nonspecific staining of myelin was also intense in paraffin-embedded tissue and in human brain frozen sections. These results are significant for the prevention of undesirable staining in routine immunolabeling and they also provide a comparatively inexpensive, easy to perform strong labeling of myelin. In addition, the double marker signal (peroxidase and fluorescence) is useful for double labeling studies. (J Histochem Cytochem 46:109-117, 1998)

Distribution of the calcium-binding proteins parvalbumin, calbindin D-28k and calretinin in the retina of two teleosts.

Using monoclonal antibodies against parvalbumin (PV) and calbindin (CB), and a polyclonal antiserum against calretinin (CR), the expression patterns of these proteins in the retina of the tench and rainbow trout were studied at light microscopic level in in toto preparations and radial sections. Parvalbumin was present in subpopulations of small amacrine cells in both species, but these cells were more abundant and had a clear centre-periphery gradient distribution in the tench. Using the McAB 300 monoclonal antibody against CB, glial cells such as Müller cells, astrocytes in the nerve fibre layer, and sparse large cells close to the entrance of the optic nerve were observed in both species. Moreover, this antibody strongly labelled H1 horizontal cells and their thick axon terminals in the tench retina, whereas only a small population of amacrine cells was stained in the trout. Calretinin was expressed in different types of ganglion cells and numerous neurones located in the inner plexiform layer in both species, but was more abundant and more strongly stained in the trout retina, where some bipolar cells were easily distinguishable. A comparison to current results in other vertebrate species is offered.

Calbindin D-28K and NADPH-diaphorase activity are localized in different populations of periglomerular cells in the rat olfactory bulb.

Calbindin D-28k (CaBP) immunocytochemistry and NADPH-diaphorase (ND) histochemistry have been combined in the rat olfactory bulb by successive incubations of the same sections. The outer strata showed a similar neuronal staining pattern for both markers with positive periglomerular neurons (although the CaBP-stained periglomerular cells were six-fold more abundant than the ND-active ones) and larger neurons scattered in the glomerular and external plexiform layers. Both populations of periglomerular cells were distinct but they did not show specific morphological characteristics nor a predominant distribution around ND-positive and negative glomeruli. The colocalization study demonstrates that the larger ND and CaBP-stained juxtaglomerular cells, identified according to their size, location and processes branching patterns as two types of short axon cells (superficial short-axon and Van Gehuchten Cells) were also independent populations.

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.

Colocalization of NADPH-diaphorase and acetylcholinesterase in the rat olfactory bulb.

The colocalization of NADPH-diaphorase and acetylcholinesterase activities in the rat main and accessory olfactory bulbs has been studied by successive double histochemical staining of the same sections. In the main olfactory bulb, three patterns of glomerular labeling were found: typical/NADPH-diaphorase-positive, typical/NADPH-diaphorase-negative, and atypical/NADPH-diaphorase-negative glomeruli. Although both enzymatic activities were present in periglomerular cells and superficial short-axon cells, colocalization of NADPH-diaphorase and acetylcholinesterase was not observed in these neuronal types. By contrast, both enzymes were colocalized in a small subpopulation (less than 3% of NADPH-diaphorase- or acetylcholinesterase-positive cells) of short-axon cells located in the external plexiform layer, internal plexiform layer, granule cell layer, and white matter. In the accessory olfactory bulb, deep short-axon cells were the only neurons where both enzymes were present, and colocalization of both markers was observed in some of these cells located in the granule cell layer.

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.

NADPH-diaphorase/nitric oxide synthase-positive elements in the human olfactory bulb.

The distribution and morphology of nitric oxide-synthesizing elements in the human olfactory bulb were studied using NADPH-diaphorase histochemistry and nitric oxide synthase immunohistochemistry. NADPH-diaphorase was detected in all olfactory fibers and groups of superficial short-axon cells, deep short-axon cells, stellate cells and abundant centrifugal fibers. Similar cell types were nitric oxide synthase immunoreactive but olfactory fibers were immunonegative. The distribution patterns of nitric oxide-synthesizing elements showed significant differences from what has been reported in the olfactory bulb of macrosmatic mammals including rodents and insectivores. These differences are likely to correlate with interspecies differences in the processing of olfactory information.

Calcium-binding proteins in the periglomerular region of typical and typical olfactory glomeruli.

The distribution of chemically identified neuronal populations was studied in the glomerular layer of the rat olfactory bulb using calcium-binding protein immunocytochemistry combined with acetylcholinesterase histochemistry. Four calcium-binding proteins (calbindin D-28k, parvalbumin, calretinin, and neurocalcin) were analyzed in the periglomerular region of two different glomerular subsets; typical and atypical glomeruli. Atypical glomeruli were clearly distinguishable from typical ones by their dense network of acetylcholinesterase-positive centrifugal fibers. Each calcium-binding protein studied showed a specific distribution pattern in the rat olfactory bulb. Calbindin D-28k-, calretinin-, and neurocalcin-immunoreactive neurons were specially abundant in the glomerular layer. These three calcium-binding proteins had their main expressions in neuronal subpopulations directly involved in the glomerular circuitries of the rat olfactory bulb. Specific populations of periglomerular cells were stained for calbindin D-28k, parvalbumin, calretinin, or neurocalcin, whereas external tufted cells were only immunoreactive to neurocalcin. Both neuronal types, periglomerular cells and external tufted cells, were found in the periglomerular region of both glomerular subsets. Nevertheless, a homogeneous distribution of calbindin D-28k- or calretinin-immunopositive periglomerular cells were found between typical and atypical glomeruli, whereas the neurocalcin-immunostained external tufted cells were statistically more abundant in typical glomeruli than atypical ones (P < 0.001). These data suggest that some neuronal subpopulations are related with general properties of the glomerular physiology, and they have a homogeneous distribution in different subsets of glomeruli, whereas other chemically identified populations are related with a finer tuning of the olfactory processing, and they are segregately distributed in relation to particular glomerular subsets. In addition, this work adds new differences in the cellular composition of typical and atypical glomeruli.