Calcium-binding protein parvalbumin-immunoreactive neurons in the rat olfactory bulb

The laminar distribution and morphological features of parvalbumin-immunoreactive [PV(+l)] neurons, one of the subpopulations of GABAergic neurons, were studied in the rat olfactory bulb at a light microscopic level. In the main olfactory bulb of adult rats, PV(+) neurons were mainly located in the external plexiform layer (EPL), and a few were scattered in the glomerular layer (GL), mitral cell layer (ML), and granule cell layer (GRL); whereas PV(+) neurons were rarely seen in the accessory olfactory bulb. The inner and outer sublayers of the EPL (ISL and OSL) appeared to be somewhat different in the distribution of PV(+) somata and features of PV(+) processes. PV(+) somata were located throughout the OSL, and PV(+) processes intermingled with one another, making a dense meshwork in the OSL; whereas, in the ISL, PV(+) somata were mainly located near the inner border of the EPL, and PV(+) processes made a sparser meshwork than that in the OSL. PV(+) neurons in the EPL were apparently heterogeneous in their structural features and appeared to be classifiable into several groups. Among them there appeared five distinctive types of PV(+) neurons. The most prominent group of PV(+) neurons in the OSL were superficial short-axon cells, located in the superficial portion of this sublayer and giving rise to relatively thick processes, in horizontal or oblique directions, which usually bore spines and varicosities. Another prominent group of PV(+) neurons extended several short, branched dendrites with spines and varicosities, which appeared to intermingle with one another, making a relatively small, spherical or ovoid dendritic field around the cell bodies; most of them resembled Van Gehuchten cells reported in previous Golgi studies. A third distinctive and most numerous group of PV(+) neurons were of the multipolar type; their somata and processes were located throughout the EPL. Their relatively smooth processes with frequent varicosities and a few spines were extended horizontally or diagonally throughout the EPL. A fourth group, which could be a subtype of the multipolar type, were located in or just above th ML and extended several thin, smooth dendrites in the EPL, some of which appeared to reach the border between the GL and EPL. Occasionally, axonlike processes arose from their cell bodies and extended into the ML. This fourth type of PV(+) neuron was named inner short-axon cells. A fifth group of neuron was located in the ML; processes of these neurons were extended horizontally, so they were named inner horizontal cells. PV(+) processes from the fourth and the fifth group of cells appeared to make contacts on mitral cell somata. In the GL some presumably periglomerular cells were also PV(+). In the GRL, PV(+) neurons were small in number, but they were also heterogeneous in their structural features; Some were identified as Golgi cells. This study shows a tremendous heterogeneity in morphological features of a chemically defined subpopulation of GABAergic interneurons in the olfactory bulb.     

Proteinase-activated receptors 1 and 2 in rat olfactory system: layer-specific regulation of multiple signaling pathways in the main olfactory bulb and induction of neurite retraction in olfactory sensory neurons

Proteinase-activated receptors (PARs) are a family of four G protein-coupled receptors that are widely distributed in the CNS and involved in neural cell proliferation, differentiation and survival. The olfactory system undergoes continuous neurogenesis throughout life and may represent a critical target of PAR cellular actions. In the present study we investigated the functional activity of PAR1 and PAR2 in microdissected tissue preparations of olfactory nerve-glomerular layer (ON-GL), external plexiform layer (EPL) and granule cell layer (GRL) of the rat main olfactory bulb and in primary cultures of olfactory neuroepithelial cells. Activation of either PAR1 or PAR2 regulated multiple signaling pathways, including activation of pertussis-toxin sensitive Gi/o proteins, inhibition of cyclic AMP formation, stimulation of Gq/11-mediated phosphoinositide (PI) hydrolysis, phosphorylation of Ca2+/calmodulin-dependent protein kinase II and activation of the monomeric G protein Rho, predominantly in ON-GL, whereas only activation of Rho was detected in the deeper layers. Olfactory nerve lesion by nasal irrigation with ZnSO4 induced a marked decrease of PAR signaling in ON-GL. In primary cultures of olfactory neurons, double immunofluorescence analysis showed the localization of PAR1 and PAR2 in cells positive for olfactory-marker protein and neuron-specific enolase. Cell exposure to either nanomolar concentrations of thrombin and trypsin or PAR-activating peptides caused rapid neurite retraction. This study provides the first characterization of the laminar distribution of PAR1 and PAR2 signaling in rat olfactory bulb, demonstrates the presence of the receptors in olfactory sensory neurons and suggests a role of PARs in olfactory sensory neuron neuritogenesis.     

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat main and accessory olfactory bulbs

Immunocytochemical localization of GABA neurons and dopamine neurons in the rat olfactory bulb was obtained with sheep antiserum to glutamate decarboxylase (GAD) and rabbit antiserum to tyrosine hydroxylase (TH). GAD-positive neurons include periglomerular cells, granule cells, superficial and deep short axon cells. TH-positive neurons represent periglomerular cells. Two-color immunocytochemistry shows that GABA and dopamine periglomerular cells are separate populations. The accessory olfactory bulb has rare dopamine cells and few superficial short axon cells. Radial gradients of GAD-immunostaining are evident in the main but not in the accessory olfactory bulb.     

Somatostatin-14-like immunoreactive neurons and fibres in the human olfactory bulb

Summary This study describes the morphological features and the distribution pattern of neurons in the human olfactory bulb which are immunoreactive for an antiserum against the neuropeptide somatostatin-14.Immunoreactive nerve cell bodies were mainly found in the white matter surrounding the cell clusters of the anterior olfactory nucleus. Some immunoreactive neurons were also found scattered throughout the anterior olfactory nucleus and the deeper parts of the inner granule cell layer. Only a few immunoreactive neurons were localized in the glomerular layer and the outer granule cell layer.Immunoreactive fibres were found in all layers of the olfactory bulb. In addition, an impressive number of coiled and kinked immunoreactive fibres were localized within the anterior olfactory nucleus forming a dense plexus. Accumulations of twisted and coiled branches of immunoreactive fibres were rarely found either surrounding or within the olfactory glomerula.The characteristics of somatostatin-14 immunoreactive neurons as seen in the combined pigment-Nissl preparation were studied after decolourizing the chromogen and restaining the preparations with aldehydefuchsin in order to demonstrate the lipofuscin pigment and gallocyanin chrome alum for Nissl material. About 90% of the immunoreactive neurons studied in this manner turned out to be devoid of lipofuscin granules. The remaining 10% displayed different patterns of pigmentation.These findings suggest the presence of different types of somatostatin-14-like immunoreactive neurons in the olfactory bulb of the human adult.     

Odorant modulation of neuronal activity and local field potential in sensory-deprived olfactory bulb

Neuronal discharge and local field potential (LFP) oscillations in the olfactory bulb (OB) are modulated by odorant stimulation. The LFP oscillations have been proposed as the mechanism that facilitates synchronization of OB output neurons and the representation of similar odorants. Gamma LFP oscillations depend on the OB inhibitory network and early sensory deprivation modifies this inhibitory network. However, little is known about the LFP oscillations and neuronal discharge in the deprived OB. We examined the mitral/tufted (MT) cells' oscillatory activity and LFP oscillations in both sensory-deprived and normal OBs in urethane anesthetized rats. We found that MT cells in deprived and normal OBs have similar basal mean firing rate; 44% of the recorded cells in deprived OB and only 8% of the cells in normal OB showed firing rate modulation by odorants, both exhibiting a similar ratio of excitatory to inhibitory responses. A fraction of MT cells exhibited oscillatory discharge centered on gamma (60–70 Hz) and beta (20 Hz) frequencies, although this feature was not consistently dependent on odorant stimulation. Odorants decreased the LFP oscillatory power in the gamma band (35–90 Hz) and increased the power in the beta band (12–30 Hz). The modulation of LFP oscillations by odorants was also predominant in the deprived (53%) compared to the normal OB (17%). In contrast, a higher fraction of MT cells' discharge was locked to the gamma LFP cycle in the normal OB. These results suggest that early unilateral olfactory deprivation increases the OB sensitivity to odorants and reduce the temporal synchrony between unitary activity and gamma LFP oscillations without altering the basal neuronal discharge.     

Differential effect of kainic acid on somatostatin, GABAergic and cholinergic neurons in the rat striatum

Kainic acid was injected into the rat striatum and its effects on presumptive striatal cholinergic, GABAergic and somatostatin-containing neurons were examined with three histochemical staining methods. Presumptive cholinergic and GABAergic neurons are damaged to a similar extent, but somatostatin neurons are more sensitive to the neurotoxic effect of kainic acid and are more severely affected.     

Release of adenosine 5′-triphosphate from synaptosomes from different regions of rat brain

The release of adenosine 5′-triphosphate by elevated extracellular concentrations of KC1 and by veratridine was determined in synaptosomal fractions prepared from different regions of rat brain. Following correction for yields of synaptosomes from the various regions, the relative distribution of K⁺-induced release was corpus striatum > cerebral cortex > medulla > hypothalamus > cerebellum. In contrast, the relative distribution of veratridine-induced release of adenosine 5′-triphosphate was medulla > corpus striatum > hypothalamus > cerebral cortex > cerebellum.From these findings, it was concluded that (1) depolarization-induced release of adenosine 5′-triphosphate was not distributed uniformly throughout the brain but varied from region to region, (2) the K⁺-induced release of adenosine 5′-triphosphate which is Ca²⁺-dependent, had a different regional distribution than the veratridine-induced release, which is greatest in Ca²⁺-free medium, and (3) the distribution of K⁺-induced release of adenosiae 5′-triphosphate did not correlate well with the known distribution of noradrenaline concentrations in rat brain, but did correlate to some extent with the distributions of 5-hydroxytryptamine, dopamine and especially acetylcholine, so that co-release of adenosine 5′-triphosphate with these transmitters may possibly occur.     

Convergence of main and accessory olfactory pathways onto single neurons in the hamster amygdala

Summary Chemoreceptor pathways from the vomeronasal organ (VNO), and main olfactory system are known to be separate as they pass into the brain, at least until the level of the amygdala. In the amygdala, vomeronasal pathways project to the posteromedial cortical nucleus (PMCN), and medial nucleus (MN). The main olfactory pathways have terminations in the posterolateral cortical nucleus (PLCN), and anterior cortical nucleus (ACN), both of which project to the PMCN and MN. The anatomy thus suggests that the PMCN and MN are sites for convergence of input from the main and accessory olfactory pathways. We have recorded single units in the amygdala and found that electrical stimulation of either the main olfactory bulbs or the VNO could drive some of the same units in the PMCN. Units were also found that were driven by one system but not the other, and units in which activity driven by one system was suppressed by stimulation of the other system.     

Spontaneous activity of single neurons in the olfactory bulb of the rainbow trout (Salmo gairdneri) and its modulation by olfactory stimulation with amino acids

Summary Spontaneous action potentials recorded from 126 neurons in the olfactory bulb of Salmo gairdneri show a higher tendency towards bursting patterns of activity than those shown in previous reports about other fish. Granule cells and periglomerular cells are more likely to fire in bursts than mitral cells. Natural chemical stimulation of the olfactory mucosa with amino acid solutions produced a unique pattern of excitatory and inhibitory responses across all units. Chi-square values were calculated for stimulatory effectiveness between forty-five pairs of odours. L-serine and L-alanine consistently showed a high degree of similarity with several other odours. The converse was true for GABA and L-histidine, although this pair had a high chi-square value when mutually compared. Enantiomeric pairs of amino acids were often found to have opposite stimulatory effects on bulbar units. These results are discussed in relation to the possible properties and configurations of odorant receptor sites for amino acids in the fish olfactory mucosa.     

GABAergic basal forebrain afferents innervate selectively GABAergic targets in the main olfactory bulb

In this work we have analyzed the targets of the GABAergic afferents to the main olfactory bulb originating in the basal forebrain of the rat. We combined anterograde tracing of 10 kD biotinylated dextran amine (BDA) injected in the region of the horizontal limb of the diagonal band of Broca that projects to the main olfactory bulb, with immunocytochemical detection of GABA under electron microscopy or vesicular GABA transporter (vGABAt) under confocal fluorescent microscopy. GABAergic afferents were identified as double labeled BDA-GABA boutons. Their targets were identified by their ultrastructure and GABA content. We found that GABAergic afferents from the basal forebrain were distributed all over the bulbar lamination, but were more abundant in the glomerular and inframitral layers (i.e. internal plexiform layer and granule cell layer). The fibers had thick varicosities with abundant mitochondria and large perforated synaptic specializations. They contacted exclusively GABAergic cells, corresponding to type 1 periglomerular cells in the glomerular layer, and to granule cells in inframitral layers. This innervation will synchronize the bulbar inhibition and consequently the response of the principal cells to the olfactory input. The effect of the activation of this pathway will produce a disinhibition of the bulbar principal cells. This facilitation might occur at two separate levels: first in the terminal tufts of mitral and tufted cells via inhibition of type 1 periglomerular cells; second at the level of the firing of the principal cells via inhibition of granule cells. The GABAergic projection from the basal forebrain ends selectively on interneurons, specifically on type 1 periglomerular cells and granule cells, and is likely to control the activity of the olfactory bulb via disinhibition of principal cells. Possible similarities of this pathway with the septo-hippocampal loop are discussed.     

Gender and age related changes in number of dopaminergic neurons in adult human olfactory bulb

IntroductionDopamine is one of the major brain neurotransmitters, and the loss of dopaminergic neurons in basal ganglia cause motor deficits in Parkinson's disease. We proposed that the difficulty in olfaction observed in the elderly may be due to an alteration in the number of dopaminergic neurons.Materials and methodsSections were taken from olfactory bulbs of post-mortem tissue specimens of 13 humans, males and females, aged from 19 to 63 years (≤35 and ≥50 years), with no history of neurological disorders. The tissues were fixed, embedded, cut on a freezing microtome, and prepared for immunohistochemical analysis using tyrosine hydroxylase (TH) and aromatic l-amino acid decarboxylase (AADC) antibodies. The number of positive neurons was counted.ResultsTH- and AADC-positive cells were present in the glomerular layer. There was no significant difference between the numbers of TH- and AADC-positive cells, in males and females, and in young and elderly individuals. The quantitative analysis revealed that the number of TH- and AADC-positive neurons were significantly higher in males than in females (P < 0.05). Moreover, there was a significant increase in the number of TH- and AADC-positive neurons in the olfactory bulbs of the elderly compared with young individuals (P < 0.05).ConclusionFactors such as gender and age may affect the number of dopaminergic neurons, and there is a correlation between increased dopaminergic neurons and olfactory performance. Moreover, the increase in dopaminergic cells in the olfactory bulb of the elderly may indicate the existence of rostral migratory stream in adult humans.     

NADPH-diaphorase distribution in the rabbit superior colliculus and co-localization with calcium-binding proteins

Nitric oxide (NO) and calcium‐binding proteins (CaBP) are important neuromodulators implicated in brain plasticity and brain disease. In addition, the mammalian superior colliculus (SC) has one of the highest concentrations of NO within the brain. The present study was designed to determine the distribution of nitric oxide‐synthesizing neurons in the SC of the rabbit by enzyme histochemistry for reduced nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d), and its degree of co‐localization with CaBP, parvalbumin (PV) and calbindin (CB). NADPH‐d‐labelled fibres formed dense patches of terminal buttons within the intermediate grey layer and streams of fibres within the deepest layers of SC. Cells expressing NOS constitute a subpopulation of neurons in which practically all cell types are represented. Combined PV/NADPH‐d experiments showed a complete lack of co‐localization within individual neurons and fibres. On the contrary, double‐labelled neurons appeared in CB/NADPH‐d‐stained sections, only in the superficial layers, and mostly in the SGS and SO. These cells, which were intermingled with other neurons containing either NADPH‐d or CB, appear to be a subtype of narrow‐field and wide‐field vertical cells, and display an anterior–posterior gradient of density. Owing to the involvement of the superficial layers of the SC in the organization and integration of the visual information, it is suggested that these neurons may play a concrete role within the visual circuits. Our data indicate a clear selectivity in the expression of NADPH‐d, PV and CB in the SC, and that NO and CB probably serve as co‐modulators and/or co‐transmitters in the connectivity of the superficial layers of this midbrain structure.     

Localization of C-PON immunoreactivity in the rat main olfactory bulb. Demonstration that the population of neurons containing endogenous C-PON display NADPH-diaphorase activity

The presence of the neuropeptide C-terminal flanking peptide of neuropeptide-Y, C-PON, has been investigated in the main olfactory bulb of the rat using conventional fluorescence and peroxidase-antiperoxidase immunocytochemical techniques. The distribution of immunoreactive structures to C-PON was examined in both horizontal and coronal sections. Endogenous C-PON was localized within two types of short-axon cells including (1) superficial short-axon cells in the glomerular layer and (2) deep short-axon cells lying in the deepest portion of the granule cell layer and in the adjacent white matter. In addition, varicose immunoreactive processes were detected in all layers, although they were more numerous in the deepest portion of the granule cell layer. Immunoreactive cell bodies and processes were also observed in the nucleus olfactorius anterior and in the intrabulbar portion of the anterior commissure. Nevertheless, immunoreactive structures were not localized in the lateral olfactory tract. The indirect immunofluorescence technique to detect endogenous C-PON in combination with the enzyme histochemical demonstration of NADPH-diaphorase activity, in single sections, showed that the NADPH-diaphorase procedure is a reliable marker for these C-PON positive cells. Also, indirectly, that, in the rat main olfactory bulb, C-PON and neuropeptide-Y are contained in the same cell types. Many glomeruli were stained following the NADPH-diaphorase procedure, but they were not C-PON immunoreactives. Results of this study provide evidence suggesting that C-PON may influence polysynaptically the function of mitral cells and, therefore, the olfactory bulb output.     

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.