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.

Olfactory bulb plasticity ensures proper olfaction after severe impairment in postnatal neurogenesis.

The olfactory bulb (OB) neurons establish a complex network that ensures the correct processing of the olfactory inputs. Moreover, the OB presents a lifelong addition of new neurons into its existing circuitry. This neurogenesis is considered essential for the OB function. However, its functional impact on physiology and behavior is still unclear. Here, we investigate the mechanisms of OB plasticity that underlie bulbar physiology in relation to severe damage of neurogenesis. The neurogenesis of young mice was altered by ionizing radiation. Afterwards, both multi-channel olfactometry and electrophysiological studies were performed. Furthermore, neurogenesis and differentiation of the newly formed cells were assessed using bromodeoxyuridine labeling combined with a wide battery of neuronal markers. Our results demonstrate a reduction in both neurogenesis and volume of the OB in irradiated animals. The number of neuroblasts reaching the OB was reduced and their differentiation rate into interneurons selectively changed; some populations were noticeably affected whereas others remained preserved. Surprisingly, both olfactory detection and discrimination as well as electrophysiology presented almost no alterations in irradiated mice. Our findings suggest that after damaging postnatal neurogenesis, the neurochemical fate of some interneurons changes within a new biological scenario, while maintaining homeostasis and olfaction.

The olfactory system as a puzzle: playing with its pieces.

The mammalian olfactory bulb (OB) has all the features of a whole mammalian brain but in a more reduced space: neuronal lamination, sensory inputs, afferences, or efferences to other centers of the central nervous system, or a contribution of new neural elements. Therefore, it is widely considered as "a brain inside the brain." Although this rostral region has the same origin and general layering as the other cerebral cortices, some distinctive features make it very profitable in experimentation in neurobiology: the sensory inputs are driven directly on its surface, the main output can be accessed anatomically, and new elements appear in it throughout adult life. These three morphological characteristics have been manipulated to analyze further the response of the whole OB. The present review offers a general outlook into the consequences of such experimentation in the anatomy, connectivity and neurochemistry of the OB after (a) sensory deprivation, mainly by naris occlusion; (b) olfactory deinnervation by means of olfactory epithelium damage, olfactory nerve interruption, or even olfactory tract disruption; (c) the removal of the principal neurons of the OB; and (d) management of the arrival of newborn interneurons from the rostral migratory stream. These experiments were performed using surgical or chemical methods, but also by means of the analysis of genetic models, some of whose olfactory components are missing, colorless or mismatching within the wild-type scenario of odor processing.

Changes in the serotonergic system and in brain-derived neurotrophic factor distribution in the main olfactory bulb of pcd mice before and after mitral cell loss.

The serotonergic centrifugal system innervating the main olfactory bulb (MOB) plays a key role in the modulation of olfactory processing. We have previously demonstrated that this system suffers adaptive changes under conditions of a lack of olfactory input. The present work examines the response of this centrifugal system after mitral cell loss in the Purkinje cell degeneration (pcd) mutant mice. The distribution and density of serotonergic centrifugal axons were studied in the MOB of control and pcd mice, both before and after the loss of mitral cells, using serotonin (5-HT) and 5-HT transporter immunohistochemistry. Studies of the amount of 5-HT and its metabolite, 5-hydroxyindole acetic acid (5-HIAA), were performed by means of high-performance liquid chromatography (HPLC), and the relative amounts of brain-derived neurotrophin factor, BDNF, and its major receptor, tropomyosin-related kinase B (TrkB), were measured by Western blot. Our study revealed that the serotonergic system develops adaptive changes after, but not before, mitral cell loss. The lack of the main bulbar projection cells causes a decrease in the serotonergic input received by the MOB, whereas the number of serotonergic cells in the raphe nuclei remains constant. In addition, one of the molecules directly involved in serotonergic sprouting, the neurotrophin BDNF and its main receptor TrkB, underwent alterations in the MOBs of the pcd animals even before the loss of mitral cells. These data indicate that serotonergic function in the MOB is closely related to olfactory activity and that mitral cell loss induces serotonergic plastic responses.

Long-lasting changes in the anatomy of the olfactory bulb after ionizing irradiation and bone marrow transplantation.

The adult brain is considered to be a radioresistant organ since it is mainly composed of non-dividing cells. However, in adult animals there are a few neurogenic brain areas that are affected by ionizing radiation whose plasticity and capacity for recovery are still unclear. Here, mice were irradiated with a minimal lethal dose of radiation in order to determine its effects on the subventricular zone (SVZ), the rostral migratory stream (RMS), and the olfactory bulb (OB). These regions underwent a dramatic reduction in cell proliferation and ensuing morphological alterations, accompanied by a patent reactive gliosis. Bone marrow stem cell (BMSC) transplants were also performed after the radiation treatment to allow the mouse survival with a view to analyzing long-term effects. Normal proliferation rates were not recovered over time and although bone marrow-derived cells reached the brain, they were not incorporated into the SVZ-RMS-OB pathway in an attempt to rescue the damaged regions. Since neurogenesis produces new interneurones in the OB, thus feeding cell turnover, the volume and lamination of the OB were analyzed. The volume of the OB proved to be dramatically reduced at postnatal day 300 (P300), and this shrinkage affected the periependymal white matter, the granule cell layer, the external plexiform layer, and the glomerular layer. These results should be taken into account in cell therapies employing BMSC, since such cells reach the encephalon, although they cannot restore the damage produced in neurogenic areas. This study thus provides new insight into the long-term effects of ionizing radiation, widely employed in animal experimentation and even in clinical therapies for human beings.

Sexual dimorphic stages affect both proliferation and serotonergic innervation in the adult rostral migratory stream.

One of the sexual dimorphic differences in adult rodents is neural proliferation. Here we demonstrate that physiological hormone stages can modulate this proliferation in the adult forebrain. Female mice, both pregnant and synchronized in oestrus, exhibited higher proliferating cell percentages than males in both the rostral migratory stream (RMS) and the olfactory bulb (OB). Moreover, although the hormonal component also influenced the subventricular zone (SVZ), no differences in proliferation were observed in this region. In addition, both groups of females had higher numbers of serotonergic fibres in these regions. Serotonin may therefore be related to the mechanism of action by which hormones can affect cell proliferation of this brain region. We also evaluated cell death in the SVZ in males and females, finding that this was higher in the former. Taken together, our results support the idea that in female rodents more neuroblasts are able to reach the RMS and then proliferate, apoptosis being an additional mechanism affecting the low proliferation of cells in the RMS and OB in males. Thus, proliferation in the RMS is influenced by sexual dimorphism.

Distribution of neurocalcin-containing neurons reveals sexual dimorphism in the mouse olfactory bulb.

Olfactory sexual dimorphism has mainly been described in the vomeronasal system, in relation to reproductive behavior, while evidence of sexual dimorphism in the main olfactory bulb (OB) remains scarce. There are no data indicating sex-related differences in the neurochemistry of intrinsic olfactory elements. Neurocalcin (NC) is a calcium-binding protein that is expressed in specific neuronal populations of the central nervous system. Here we analyzed by immunohistochemistry the NC-containing neurons in the mouse main OB, comparing both their quantities and their locations between male and female animals. NC cell density was higher in males than in females in specific locations of the glomerular layer, the external plexiform layer, the mitral cell layer, and the internal plexiform layer. This divergence in the numbers of NC cells was especially patent in central rostrocaudal levels. The NC-containing neurons exhibiting sexual divergence were identified as both juxtaglomerular and short-axon cells. This is the first description of sexual dimorphism regarding neurons belonging to the mouse main OB. According to their distribution in the OB, neurocalcin-immunoreactive interneurons could reflect a sexually dimorphic regulation of specific odorants.

Changes in cell migration and survival in the olfactory bulb of the pcd/pcd mouse.

Postnatally, the Purkinje cell degeneration mutant mice lose the main projecting neurons of the main olfactory bulb (OB): mitral cells (MC). In adult animals, progenitor cells from the rostral migratory stream (RMS) differentiate into bulbar interneurons that modulate MC activity. In the present work, we studied changes in proliferation, tangential migration, radial migration patterns, and the survival of these newly generated neurons in this neurodegeneration animal model. The animals were injected with bromodeoxyuridine 2 weeks or 2 months before killing in order to label neuroblast incorporation into the OB and to analyze the survival of these cells after differentiation, respectively. Both the organization and cellular composition of the RMS and the differentiation of the newly generated neurons in the OB were studied using specific markers of glial cells, neuroblasts, and mature neurons. No changes were observed in the cell proliferation rate nor in their tangential migration through the RMS, indicating that migrating neuroblasts are only weakly responsive to the alteration in their target region, the OB. However, the absence of MC does elicit differences in the final destination of the newly generated interneurons. Moreover, the loss of MC also produces changes in the survival of the newly generated interneurons, in accordance with the dramatic decrease in the number of synaptic targets available.

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.

Dopaminergic modulation of nNOS expression in the pituitary gland of male rat.

Nitric oxide is an unconventional transmitter since it is not transported and released by exocytosis. In the pituitary gland, nitric oxide is locally synthesised by gonadotroph and folliculo-stellate cells. Dopamine, the principal central inhibitory signal in prolactin release, may exert its inhibitory effects by stimulation of nitric oxide production. However, the effects of dopaminergic modulation on nitric oxide-producing pituitary cells have not been analysed. Therefore, we examined the effects of intraventricular administration of the dopamine antagonist haloperidol (40 microg) on the pituitary expression of neuronal nitric oxide synthase (nNOS) in male adult rats. In untreated and control animals, nNOS-positive cells were very similar. Two types of nNOS-positive cells appeared in the pars distalis: round or polygonal cells and stellate cells. Although some isolated cells were found, the nNOS-positive cells commonly appeared grouped in clusters close to blood vessels. nNOS immunoreactivity appeared as a uniform staining throughout the cytoplasm, including cell prolongations. The number and size of nNOS-expressing cells in the pituitary gland decreased significantly after treatment with haloperidol (p<0.01). To evaluate the potential direct effect of dopamine on pituitary cells, pituitary monolayer cultures were treated with dopamine during a time-course of 12 h. Our in vitro studies revealed that dopamine increases the percentage of nNOS-positive cells and augments cellular area (p<0.05). These results demonstrate that: (1) treatment of rats in vivo with a dopamine antagonist significantly decreases expression of nNOS in the pituitary and (2) in vitro dopamine exerts a direct effect on pituitary cultures by increasing nNOS-positive cells. Thus, these findings suggest that dopamine may function as a physiological stimulator of nNOS expression in the rat pituitary gland.

Changes in immunoreactivity to calcium-binding proteins in the anterior olfactory nucleus of the rat after neonatal olfactory deprivation.

The effects of olfactory deprivation on the density of neuronal populations expressing the calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin in the anterior olfactory nucleus of the rat were studied immunohistochemically in 60-day-old rats subjected to unilateral naris closure on the day of birth. The neuronal populations were characterized morphologically and topologically, and the density of each cell type was calculated in each subdivision of the anterior olfactory nucleus at seven rostrocaudal levels. Data were gathered into three groups: data from either the ipsilateral or contralateral anterior olfactory nucleus of experimental animals and data from control animals. Statistical analysis indicated that disruption of the normal afferent activity to one olfactory bulb affects the expression of the calcium-binding proteins investigated in the anterior olfactory nucleus, as revealed by variations in the density of certain neuronal populations. The observed effects were very heterogeneous and could not be related to any specific neuronal type, location, or to the expression of a given calcium-binding protein. Nevertheless, as a general rule the most affected neuronal populations were those expressing calbindin D-28k located in the rostral subdivisions. These subdivisions are the latest to develop in mammals and are those that receive the largest amount of inputs from the olfactory bulb.

Effects of axotomy on the expression of NADPH-diaphorase in the visual pathway of the tench.

The distribution of NADPH-diaphorase (ND) positive elements was analyzed throughout the visual pathway of the tench in normal conditions and after optic nerve transection. In the control retina, ND-labeled elements were observed in the photoreceptor, inner nuclear, outer nuclear and ganglion cell layers. In the optic nerve of control animals, small and numerous ND-positive glial cells that were identified as presumably astrocyte-like cells were observed. In the optic tracts and optic tectum, a different type of ND-positive glial cell was detected. Axotomy induced severe changes in the ND staining pattern in the visual pathway. A decrease in the number of ND-stained cells was detected in the retina. In the optic nerve of lesioned animals, the number of small cells gradually decreased, whereas the number of large cells did not change. Two new ND-positive cell populations were observed after the lesion: microglial-like cells appeared close to the lesioned area from 24 h to 7 days after transection, and astrocyte-like cells were found throughout the optic nerve from 14 days up to at least 120 days. The total number of ND-stained glial cells increased at 30 and 60 days and returned to control parameters at 120 days. In addition, the number of ND-positive cells increased at the same survival times in the optic tracts and in the retinorecipient strata of the optic tectum with respect to control animals. Thus, degenerative/regenerative processes in the fish visual pathway are accompanied by an increase in the number of ND-positive cells. Synthesis of nitric oxide is elicited in microglial-like cells as a response to axon injury, whereas the expression in astrocyte-like cells seems to be associated with both normal processes under physiological conditions and with the regenerative phase after the lesion.

Volumetric changes in the anterior olfactory nucleus of the rat after neonatal olfactory deprivation.

The effect of olfactory deprivation in the postnatal development of the anterior olfactory nucleus (AON) was studied in 60-day-old rats which underwent unilateral naris closure after birth (postnatal day 1). Volumetric and morphometric analyses of the AON ipsilateral and contralateral to the closed naris were performed and data were statistically compared among them and with those of control animals. The volumes of the AONs and those of their subdivisions were calculated by the Cavalieri method and the area of the subdivisions was measured at seven established rostrocaudal levels. Whereas no statistically significant differences were detected between the ipsilateral and the contralateral AONs, comparison of these with controls revealed significant reductions in the volumes and dimensions of most AON subdivisions. The reduction was larger in the ipsilateral than in the contralateral AON and more pronounced in the rostralmost subdivisions (external and lateral) than in the caudal ones, the dorsal subdivision not being affected. These data demonstrate that the disruption of the normal afferent activity to one olfactory bulb has effects on the postnatal development of both the ipsilateral and the contralateral AONs. In addition, the most affected subdivisions were those that develop later and that receive the bulk of projections from the olfactory bulb, suggesting that the degree of maturity is an important factor in susceptibility to changes induced by reduced afferent activity. Finally, the results indicate that, contrary to the olfactory bulb, the contralateral AON cannot be used as a control structure in deprivation studies.

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.

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.

A sexually dimorphic group of atypical glomeruli in the mouse olfactory bulb.

Atypical glomeruli (AtG) are clearly distinguishable from typical ones because of their strong cholinergic innervation. AtG are located in defined positions in the caudal half of the main olfactory bulb of rodents. The AtG partially overlap with other specialized olfactory subsystems, such as the modified glomerular complex, which is close to the accessory olfactory bulb. So far, possible sex differences in these specialised olfactory systems have not been investigated. In this work we have identified AtG in the mouse by means of acetylcholinesterase histochemistry and compared the number and size of these glomeruli between the sexes and also between the two strains that demonstrate intraglomerular synaptic differences, i.e. BALB/c and CD-1 mice. First, we divided the AtG into three types according to their position (I, rostral-most; II, around the accessory olfactory bulb; III, caudal-most) or their reactivity to acetylcholinesterase histochemistry (AtG type II being the least reactive glomeruli). ANOVA analyses revealed differences in the maximum diameter of glomeruli among the three types, but not in their sectional areas, indicating that all three types have different shapes. Moreover, both morphoplanimetric parameters were seen to be different between the two strains studied and also between the sexes: male mice and BALB/c animals had the largest glomeruli. The number of AtG was also significantly different between the sexes and strains, although these factors presented a strong interaction. Thus, the males had higher numbers of AtG in the CD-1 strain whereas in the BALB/c mice males demonstrated fewer AtG than females. These differences in number were largely due to AtG type II. The present work is evidence that AtG type II is a sexually dimorphic group of specialized glomeruli located in the main olfactory bulb.

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.

Co-localization of cart peptide immunoreactivity and nitric oxide synthase activity in rat hypothalamus.

Because of the reported presence of both CART peptide and NOS activity in the same hypothalamic nuclei, their colocalization was examined. Eighteen percent of the neurons in the supraoptic nuclei, and 16% of the neurons in the paraventricular nucleus contained both CART immunoreactivity and NOS activity. Many other neurons in these regions stained for only one marker although they were often close by. Thus, CART peptides and NO may interact in these regions.

Expression of neuronal nitric oxide synthase/NADPH-diaphorase during olfactory deafferentation and regeneration.

Neuronal nitric oxide synthase (nNOS) expression can be regulated under natural or experimental conditions. This work aims at elucidating whether the expression of nNOS or its related NADPH-diaphorase (ND) activity are modified by manipulation of the normal inputs to neurons. We used the olfactory bulbs from two mouse strains, BALB and CD1, because they show divergences in their synapse patterns, and these differences affect periglomerular cells, interneurons expressing tyrosine hydroxylase or nNOS/ND. The olfactory inputs to these neurons can be disrupted by inhalation of methyl bromide. The effect of this gas on olfactory axons, as well as the synaptic features in both mouse strains, were studied using electron microscopy. The changes in expression were analysed qualitatively and quantitatively at different times after lesion to nine topographical regions of the olfactory bulb. Methyl bromide inhalation induced a degeneration of olfactory axons in both strains, but had different effects on the expression of nNOS/ND and tyrosine hydroxylase. In BALB mice, where periglomerular cells do not receive direct inputs from olfactory axons, no changes were detected in tyrosine hydroxylase or in ND expression. In CD1 periglomerular cells, where olfactory axons establish direct synapses, a significant down-regulation of both markers was observed. These changes were observed differentially across the olfactory bulb, being more pronounced in rostral regions and more acute for ND than for tyrosine hydroxylase. Our results indicate that the synaptic inputs influence the expression of ND activity related to nNOS and that the activation of the enzyme is more severely affected than its protein expression.

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.