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

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

Immunocytochemical identification of GABAergic neurons in the main olfactory bulb of the rat

With the aid of a sheep antiserum against rat brain glutamate decarboxylase (GAD), the endogenous marker for GABAergic neurons, we have labeled immunocytochemically various types of nerve cells in the main olfactory bulb of rats, with and without topic injections of colchicine. The peroxidase-antiperoxidase procedure was applied to floating Vibratome and frozen sections. A large part of the periglomerular cell population and practically all granule cells in the deep layers contain GAD-like immunoreactivity in untreated rats, while tufted and mitral cells (the projection neurons) are unstained. This observation confirms a previous study with a rabbit antiserum against mouse brain GAD, which suggested that GABAergic neurons with presynaptic dendrites contain high somatal concentrations of GAD. We show, however, that immunostaining of granule cell bodies decreases progressively from the internal plexiform layer to the deep portion of the granule cell layer. Many cell processes in the glomeruli are densely stained. They presumably represent synaptic gemmules of the numerous GAD-positive periglomerular cells, which thus could provide initial, inhibitory modulation of the afferent input. In the external plexiform layer immunostaining of the neuropil is substantially denser in the superficial half than in the deep half. This may reflect a corresponding gradient of inhibition related to unequal frequency of occurrence of synaptic gemmules of granule cell dendrites. Alternatively such a graded immunostaining of cell processes could be related to the corresponding gradient in the density of immunostaining of granule cell bodies in the deep layers, in accordance with recent data indicating that superficial and deep granule cells project their ascending dendrites respectively to superficial and deep portions of the external plexiform layer. Furthermore, we have demonstrated the presence of additional classes of GAD-positive neurons, microneurons in the external plexiform layer, small neurons in the periglomerular region, the external plexiform layer, the mitral cell layer, the internal plexiform layer, and medium-size neurons in the granule layer and the white matter. The small- and medium-size GAD-positive neurons appear weakly immunoreactive in untreated rats, but become densely stained after topic colchicine injection. Such cells presumably lack presynaptic dendrites and may correspond to different types of short axon cells demonstrated by the Golgi method.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regional distribution of protein kinases in normal and odor-deprived mouse olfactory bulbs

Unilateral naris closure produced dramatic down-regulation of tyrosine hydroxylase (TH) gene expression in periglomerular dopaminergic neurons in the olfactory bulb. To explore molecular mechanisms of TH gene regulation, the present study investigated the regional distribution of protein kinase A (PKAalpha), protein kinase C (PKCalpha), and CaM kinases II (CaMKIIalpha, beta) and IV (CaMKIV) in the normal olfactory bulb and in response to odor deprivation. Strong PKAalpha immunostaining was found in the glomerular, granule cell, external plexiform and olfactory nerve layers. PKCalpha staining was strong in granule cell and external plexiform layers but weak in the glomerular layer. Whereas CaMKIV was primarily found in granule cells, CaMKII was present in the glomerular, external plexiform, mitral cell and granule cell layers. No change in immunoreactivities of these kinases occurred in the olfactory bulb ipsilateral to naris closure. The expression of PKAalpha, PKCalpha and CaMKII, but not CaMKIV, in periglomerular cells suggests that these three kinases may play a role in TH gene regulation in the olfactory bulb. The lack of change in kinase protein levels after naris closure also suggests that any involvement of these kinases in TH gene expression in the olfactory bulb must be through altered kinase activity and not protein levels.     

Calbindin D-28k-positive neurons in the rat olfactory bulb

Summary We have studied the distribution of calbindin D-28k immunoreactivity in the rat olfactory bulb using specific monoclonal antibodies and the avidin-biotin-immunoperoxidase method. The largest number of positive neurons was located in the periglomerular layer. These neurons were identified as periglomerular cells; they have been described also by other authors as calbindin-positive elements. Close to these neurons, a second population of nerve cells was identified as superficial shortaxon neurons. The remaining layers showed a smaller number of stained elements. Other labeled neurons were located along the external border of the external plexiform layer; the scarce neurons marking its internal border were identified as van Gehuchten cells. No immunoreactive structures were found in the mitral cell layer, although we observed another population of immunostained short-axon cells at its internal border. Some reactive structures, identified by us as horizontal and vertical cells of Cajal, were located in the boundary zone between the internal plexiform layer and the granule layer. In the white matter, we found a neuronal type characterized by its large size and oriented arborization of varicose dendrites.     

Autoradiographic localization of high affinity uptake sites for 3H-D-aspartate in the rat olfactory bulb

In order to reveal excitatory amino acid-ergic neuronal connections in the rat olfactory bulb, uptake sites for the tritiated D-aspartic acid were analyzed by high resolution autoradiography. Light microscopy revealed both cellular and terminal-like uptake. Based on electron microscopy, overwhelming majority of the cellular uptake was assigned to glial cells. A fairly high number of labelled terminals appeared in the surroundings of the mitral cell somata, within the deepest portion of the external plexiform layer, in the internal plexiform layer and in the outer half of the granule cell layer. Labelled terminals synapsed onto likely granule cell dendrites or spines, at asymmetric membrane thickenings. These results suggest that, although the output neurons may not utilize glutamic or aspartic acid as their transmitters, these amino acids may, however, contribute to the bulbar neurotransmission, as mediator substances of a subgroup of centrifugal fibers to the olfactory bulb.     

Simultaneous recordings from two physiologically different types of relay neurons, mitral cells and ruffed cells, in the olfactory bulb of goldfish.

Anatomical differences characterizing mitral cells and ruffed cells were published by Kosaka and Hama in three teleost species. Physiological responses from both different types of relay neurons were recorded extracellularly and simultaneously in the plexiform layer using a single tungsten microelectrode. During interstimulus intervals mitral cells responded with higher, frequently burst-like impulse rates triggered by the activity of epithelial receptor neurons. The mitral cell activity could be totally suppressed during local anesthesia of the olfactory epithelium. Ruffed cell impulse rates were low, and each action potential triggered a long-lasting (3-5 ms), continuously variable, summed up granule cell potential. In contrast to mitral cells, blockade of epithelial receptor cells significantly increased the activity of ruffed cells. I.e., the ruffed cells, which have no input from the olfactory epithelium, are spontaneously active, and are laterally inhibited by granule cells activated by mitral cells. During olfactory stimulation contrasting interactions between mitral cells and ruffed cells resulting in a drastic intensification of centrally transmitted information, frequently were recorded. An excitation of mitral cells activity via granule cells laterally inhibited the ruffed cells activity, and an inhibition of mitral cells activity simultaneously "released" an excitation of ruffed cells. This is the first physiological determination of different types of relay neurons in the olfactory bulb of fish.     

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

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

Blanes cells mediate persistent feedforward inhibition onto granule cells in the olfactory bulb

Inhibitory local circuits in the olfactory bulb play a critical role in determining the firing patterns of output neurons. However, little is known about the circuitry in the major plexiform layers of the olfactory bulb that regulate this output. Here we report the first electrophysiological recordings from Blanes cells, large stellate-shaped interneurons located in the granule cell layer. We find that Blanes cells are GABAergic and generate large I(CAN)-mediated afterdepolarizations following bursts of action potentials. Using paired two-photon guided intracellular recordings, we show that Blanes cells have a presumptive axon and monosynaptically inhibit granule cells. Sensory axon stimulation evokes barrages of EPSPs in Blanes cells that trigger long epochs of persistent spiking; this firing mode was reset by hyperpolarizing membrane potential steps. Persistent firing in Blanes cells may represent a novel mechanism for encoding short-term olfactory information through modulation of tonic inhibitory synaptic input onto bulbar neurons.     

Neuronal organization of the accessory olfactory bulb of the lizard Podarcis hispanica: Golgi study

The neuronal organization of the accessory olfactory bulb (AOB), which receives sensory information from the vomeronasal organ, was described in a squamate reptile (Podarcis hispanica) by means of light microscopy. Using the Golgi-impregnation method, seven neuronal types could be distinguished: Periglomerular cells constitute a morphologically heterogeneous population of small neurons located between and around the glomeruli. The mitral cells are diffusely distributed in the AOB. Their cell bodies are usually located within the mitral cell layer, but some of them could be also observed in the plexiform layers. Mitral cells were classified into three subgroups on the basis of their sizes and dendritic tree morphologies. Thus, the “outer mitral cells” have the biggest cell bodies, and their distal secondary dendrites are mainly distributed rostrocaudally in the external plexiform layer. The “inner mitral cells” have large cell bodies, and their secondary dendrites are distributed dorsoventrally and are located deeper than those of the other two subgroups. The third type, the “small mitral cells,” is the smallest one among mitral cells in the AOB, and from their cell bodies, only two main dendritic trunks arise. The granule cells are composed of several categories based on their different cell body locations and dendritic tree morphologies. Thus, the “superficial granule cells” are located exclusively in the external plexiform layer and have small dendritic fields. The “middle granule cells” have fusiform cell bodies—situated in the internal plexiform layer—and present a wide dendritic projection area. Finally, the “deep granule cells” are distributed throughout the granule cell layer and include a great variety of dendritic tree morphologies. The distribution and morphological features of all neuronal types constituting the AOB of Podarcis were compared with those reported on other vertebrates. The results suggest that the lamination pattern and neuronal organization of the AOB in lizards are more similar to that of mammals than to that of the remaining vertebrates.     

Age-related Changes in Calbindin-D28k,Parvalbumin, and Calretinin Immunoreactivity in the Dog Main Olfactory Bulb

Expression and age-related changes of calbindin-D28k (CB), parvalbumin (PV), and calretinin (CR) in the main olfactory bulb of the dog were investigated by immunohistochemistry and western blot analysis. Neurons that expressed these calcium-binding proteins showed a characteristic laminar distribution. Most of CB-immunoreactive neurons were observed in the glomerular layer (GL) and the inner sublayer of the external plexiform layer (EPL). Most of PV-immunoreactive neurons were observed in the outer sublayer of the EPL. CR-immunoreactive neurons were mainly distributed in the GL and the granule cell layer. With regard to age-related changes, CB-immunoreactive neurons in the GL were stable among all age groups; however, in the EPL they decreased with age. PV-immunoreactive neurons decreased in middle-aged and aged groups. However, CR-immunoreactive neurons were not decreased in middle-aged and aged groups. These results suggest that CB-immunoreactive neurons in the EPL were most sensitive to aging, and that their reduction may be related to aging in the dog.     

Photoperiod Mediated Changes in Olfactory Bulb Neurogenesis and Olfactory Behavior in Male White-Footed Mice (Peromyscus leucopus)

Brain plasticity, in relation to new adult mammalian neurons generated in the subgranular zone of the hippocampus, has been well described. However, the functional outcome of new adult olfactory neurons born in the subventricular zone of the lateral ventricles is not clearly defined, as manipulating neurogenesis through various methods has given inconsistent and conflicting results in lab mice. Several small rodent species, including Peromyscus leucopus, display seasonal (photoperiodic) brain plasticity in brain volume, hippocampal function, and hippocampus-dependent behaviors; plasticity in the olfactory system of photoperiodic rodents remains largely uninvestigated. We exposed adult male P. leucopus to long day lengths (LD) and short day lengths (SD) for 10 to 15 weeks and then examined olfactory bulb cell proliferation and survival using the thymidine analog BrdU, olfactory bulb granule cell morphology using Golgi-Cox staining, and behavioral investigation of same-sex conspecific urine. SD mice did not differ from LD counterparts in granular cell morphology of the dendrites or in dendritic spine density. Although there were no differences due to photoperiod in habituation to water odor, SD mice rapidly habituated to male urine, whereas LD mice did not. In addition, short day induced changes in olfactory behavior were associated with increased neurogenesis in the caudal plexiform and granule cell layers of the olfactory bulb, an area known to preferentially respond to water-soluble odorants. Taken together, these data demonstrate that photoperiod, without altering olfactory bulb neuronal morphology, alters olfactory bulb neurogenesis and olfactory behavior in Peromyscus leucopus.     

The effect of some drugs on the mitral cell odor-evoked responses in the gecko olfactory bulb

The activity of odor-evoked olfactory mitral cell response of the gecko was recorded extracellularly by glass microelectrodes. The activities of the mitral cell observed during the presentation of the odor (n-amyl acetate) could be described as excitation, suppression or zero. The present experiments were undertaken to study the neural activities of the mitral cell in the olfactory bulb by perfusion application of some drugs (cobalt chloride, carnosine, norepinephrine, GABA and D-L-homocysteate) on the olfactory bulb surface or iontophoretic application of some drugs (carnosine, norepinephrine, GABA and D-L-homocysteate) to the glomerulus and the external plexiform layer to change the physiological environment. The effect of the drugs suggested that the synaptic neurons on the mitral cell have different chemical characteristics.     

Volumetric and horseradish peroxidase tracing analysis of rat olfactory bulb following reversible olfactory nerve lesions

Olfactory receptor neurons can regenerate from basal stem cells. Receptor neuron lesion causes degenerative changes in the olfactory bulb followed by regeneration as new olfactory receptor axons innervate the olfactory bulb. To our knowledge, parametric analyses of morphometric changes in the olfactory bulb during degeneration and regeneration do not exist except in abstract form. To better characterize olfactory bulb response, we performed morphometric analysis in rats following reversible olfactory nerve lesion with diethyldithiocarbamate. We also performed anterograde tracing of the olfactory nerve with wheatgerm agglutinin linked to horseradish peroxidase. Results of morphometry and tracing were complementary. The glomerular layer and external plexiform layer showed shrinkage of 45 and 26%, respectively, at 9 days. No significant shrinkage occurred in any other layer. Individual glomeruli shrank by 40-50% at 3 and 9 days following lesion. These data show that degenerative changes occur both in the glomeruli and transneuronally in the external plexiform layer. Olfactory nerve regeneration (identified by WGA-HRP transport) paralleled volumetric recovery. Recovery occurred first in ventral and lateral glomeruli between 9 and 16 days followed by recovery in medial and dorsal glomeruli. These data indicate substantial transynaptic degeneration in the olfactory bulb and a heretofore unrecognized gradient in olfactory nerve regeneration that can be used to systematically study recovery of a cortical structure.     

Distribution of GluR1 is altered in the olfactory bulb following neonatal naris occlusion

The olfactory system is well suited for studies of glutamate receptor plasticity. The sensory neurons are glutamatergic, and they turn over throughout life, and the olfactory bulb neurons that process their inputs express many of the known glutamate receptor subunits. Neonatal naris occlusion alters olfactory bulb development and the expression of certain neuroactive substances and receptors, at least in part due to loss of the sensory inputs. We therefore postulated that neonatal naris occlusion might alter glutamate receptor expression during postnatal development. Single nares of newborn mice were occluded on postnatal days 1-2, and the distribution of glutamate receptor subunits was evaluated using immunoperoxidase methods. Light microscopic examination on postnatal day 6 failed to reveal adult-like staining of neuronal cell bodies in the olfactory bulbs. By day 12, cell bodies that were immunoreactive (-IR) for the GluR1 subunit were visible in the external plexiform layer (EPL) of both sides. By day 18, many of the GluR1-IR cell bodies could be identified as cell types that had previously been reported to express homomeric GluR1 receptors. Analysis of single, mid-dorsal sections from 18-25-day-old mice showed that the medial EPL of the occluded side had a significantly lower density of these cell bodies. The GluR1 staining of the adjacent mitral cell layer (MCL) was also heavier on the occluded side, but no gross differences in staining for other glutamate receptor subunits were observed. Neonatal naris occlusion therefore appears to provide a new model for studying expression of GluR1 receptors during the development of a discrete population of olfactory bulb neurons.     

Postnatal developmental expression of calbindin, calretinin and parvalbumin in mouse main olfactory bulb

The distribution of calbindin, calretinin and parvalbumin during the development of the mouse main olfactory bulb (MOB) was studied using immunohistochemistry techniques. The results are as follows:(1) calbindin-immunoreactive profiles were mainly located in the glomerular layer, and few large calbindin-immunoreactive cells were found in the subependymal layer of postnatal day 10 (P10) to postnatal day 40 (P40) mice; (2) no calbindin was detected in the mitral cell layer at any stage; (3) calretinin-immunoreactive profiles were present in all layers of the main olfactory bulb at all stages, especially in the olfactory nerve layer, glomerular layer and granule cell layer; (4) parvalbumin-immunoreactive profiles were mainly located in the external plexiform layer (except for P10 mice); (5) weakly stained parvalbumin-immunoreactive profiles were present in the glomerular layer at all stages; and (6) no parvalbumin was detected in the mitral cell layer at any stage.     

Odour discrimination in the olfactory bulb of goldfish: contrasting interactions between mitral cells and ruffed cells

Anatomical differences characterizing mitral cells and ruffed cells have been published by T. Kosaka and K. Hama in three teleost species. Physiological responses from both types of relay neurons were recorded extracellularly and simultaneously in the plexiform layer, using a single tungsten microelectrode. During interstimulus intervals mitral cells responded with higher, frequently burst-like impulse rates triggered by the activity of epithelial receptor neurons. Mitral cell activity could be totally suppressed by local anaesthesia of the olfactory epithelium. Ruffed cell impulse rates were low, and each action potential triggered a long-lasting (3-5 ms), continuously varying, summed granule cell potential. During olfactory stimulation with non-familiar stimuli and important biological stimuli such as amino acids, preovulatory and ovulatory pheromones, and a probable alarm pheromone, contrasting interactions between mitral cells and ruffed cells were recorded frequently, which resulted in a drastic intensification of centrally transmitted information. An excitation of mitral cells' activity via granule cells laterally inhibited the ruffed cells' activity, and an inhibition of mitral cells' activity simultaneously 'released' an excitation of ruffed cells.     

Comparison of Changes in GAD65 and GAD67 Immunoreactivity and Levels in the Gerbil Main Olfactory Bulb Induced by Transient Ischemia

In the present study, we investigated changes in glutamate decarboxylase 65 (GAD65) and GAD67 immunoreactivity and protein levels in the main olfactory bulb (MOB) after 5 min of transient forebrain ischemia in gerbils. GAD65 immunoreactivity in the sham-operated group was shown in neurons and neuropil except for the somata of granule cells. GAD65 immunoreactivity was increased in neurons in the external plexiform layer 60 days after ischemia, and in mitral cells 30 and 60 days after ischemia. GAD67 immunoreactivity in the sham-operated group was shown in periglomerular cells, neuron in the external plexiform layer and granule cells with neuropil. GAD67 immunoreactivity in periglomerular cells was increased 10, 45 and 60 days after ischemia. GAD67 immunoreactivity in neurons in the external plexiform layer was increased 10 and 15 days after ischemia. Mitral cells showed strong GAD67 immunoreactivity 10 days after ischemia. However, GAD67 immunoreactivity in the granule cells was not changed with time after ischemia. In Western blot analysis for GAD65 and GAD67 protein levels in the ischemic gerbil MOB, GAD65 level was not changed after ischemia; GAD67 level was increased 10 days after ischemia. These results suggest that transient ischemia causes changes in GAD65 and GAD67 immunoreactivity in the gerbil MOB, and this change may induce a malfunction in olfaction after an ischemic insult. Ki-Yeon Yoo and In Koo Hwang equally contributed to this article.     

Short-term Effects of Endothelins on Tyrosine Hydroxylase Activity and Expression in the Olfactory Bulb of Normotensive Rats

The olfactory system in rats is part of the limbic region with extensive afferent connections with brain areas involved in the regulation of behaviour and autonomic responses. The existence of the endothelin system and catecholaminergic neurons in the olfactory bulb suggests that endothelins may modulate noradrenergic transmission and diverse olfactory mediated processes. In the present work we studied the effect of endothelin-1 and -3 on neuronal norepinephrine release and the short-term regulation of tyrosine hydroxylase in the olfactory bulb. Results showed that both endothelins increased tyrosine hydroxylase activity through the activation of a non-conventional endothelin G-protein coupled receptor, coupled to the stimulation of protein kinase A and C, as well as Ca²⁺/calmodulin-dependent protein kinase II. On the other hand, neither endothelin-1 nor endothelin-3 modified tyrosine hydroxylase total protein levels, but both peptides increased the phosphorylation of serine residues of the enzyme at sites 19 and 40. Furthermore, endothelins enhanced norepinephrine release in olfactory neurons suggesting that this event may contribute to increased tyrosine hydroxylase activity by reducing the feedback inhibition. Taken together present findings show a clear interaction between the endothelin system, and the catecholaminergic transmission in the olfactory bulb. Additional studies are required to evaluate the physiological functions regulated by endothelins at this brain level.