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.     

Ketamine and MK801 attenuate paired pulse inhibition in the olfactory bulb of the rat

Summary We have investigated the effects of the phencyclidine like-compounds ketamine and MK801 on the evoked field potentials of rat olfactory bulb. Low doses of ketamine (3–6 mg/kg) blocked the inhibition of mitral cells by granule cells evoked by stimulation of lateral olfactory tract fibres or by stimulation of olfactory nerve. This blockade was not accompanied by a decrease in granule cell excitation as revealed by field potential recording. MK801 had a similar effect on the inhibition of mitral cells evoked by stimulation of the lateral olfactory tract. As ketamine does not influence the inhibitory action of GABA (Anis et al. 1983) these results suggest that both ketamine and MK801 block inhibition by an action on intrinsic excitatory feed-back circuits in the olfactory bulb.     

A centrifugal respiratory modulation of olfactory bulb unit activity: a study on acute rat preparation

Summary In behaving rats, unit activity in the mitral and granule cell layers of the olfactory bulb (OB) can be modulated by respiration. In order to determine whether central influences could take part in this phenomenon, respiratory rhythm and the activity of OB units were recorded in the present experiment and analyzed temporally in 18 anaesthetized tracheotomized rats. In spite of the interrupted nasal airflow, 30 of the 80 cells recorded in the mitral and granule cell layers, still displayed a significant respiratory patterning of their activity. Maximal neuronal discharges were time-locked with different phases of the respiratory cycle, most often synchronized with the end of expiration. This is in contrast with previous observations in intact animals. Possible underlying mechanisms are discussed.     

Reactions of olfactory bulb neurons to different stimulus intensities in laboratory mice

Summary The activity of 219 cells in the olfactory bulb of waking, freely breathing mice was analysed, and it was found that their spontaneous discharge rate varied between 0.3 and 33 AP/s. Both butyric acid and geraniol elicited responses of four types: 38% of the 98 responses were of type 1 (excitation), 43% of type 2 (inhibition), 10% of type 3 (complex structure), and 8% were characterized by a change in the temporal pattern of the activity. Response duration varied from less than 500 ms to more than 1 min. 52 secondary neurons were stimulated with four different concentrations of the odor substances. All of the responsive cells showed a clear ability to discriminate concentration. That is, response magnitude varied with intensity, producing non-monotonie curves. Most of the neurons responded only in a region of a more or less limited concentration, and in no case was a saturation curve observed. Approx. 40% of the neurons responded to the lowest concentration tested (10^–8 vol. % of butyric acid or geraniol). The strongest stimuli, 10^–2 vol.%, were relatively ineffective.The work was supported by the Deutsche Forschungsgemeinschaft (Schm 322/7).     

Odor representation and discrimination in mitral/tufted cells of the rat olfactory bulb

Extracellular single-unit responses to odorants with various properties were recorded from mitral/tufted cells over large areas of the olfactory bulb of anesthetized rats. Each cell was exposed to one stimulus set consisting of five different odorants each at five concentrations. The resulting concentration-response profiles were compared. All mitral/tufted cells examined responded to two or more odorants, and the largest proportion of the cells were sensitive to all five odorants. Cells unresponsive to all five odorants regardless of concentration were not observed. Mitral/tufted cells sensitive to all three of the odorants that are known to evoke maximal electro-olfactograms in different regions of the olfactory epithelium were distributed widely throughout the olfactory bulb. There were no significant differences in latencies of odor responses either across recording sites or across odorants. A comparison of the concentration-response profiles suggested that all of the mitral/tufted cells were equally capable of responding to any odorant with their own distinctive pattern, but that the cells tended to show an identical pattern rather than variable pattern of response to different odorants. Five mitral/tufted cells isolated within 800 m of one electrode track showed different concentration-response profiles. Of 18 simultaneously recorded spike pairs with different amplitudes and discharge patterns recorded incidentally through one electrode at different sites, 10 had different and 8 had identical response patterns to odorants. These results suggest that: (1) mitral/tufted cells are sensitive to a broad spectrum of odorants, but respond with their own patterns to odorants; (2) odor discrimination is not uniform in neighboring cells, and a discrimination unit is comprised of a single cell.     

A light and electron microscopic study of taurine-like immunoreactivity in the main olfactory bulb of frogs

The distribution of taurine in the frog olfactory bulb was studied using light and electron microscopic immunohistochemical techniques. At the light microscopic level, taurine-like immunoreactivity (taurine-LI) was found in (i) fibers coursing from the olfactory nerve layer to the glomerular layer, (ii) cell bodies and processes primarily located in the caudal part of the granule cell layer (GCL), and (iii) puncta outlining unstained somata of mitral cells and cells in the GCL. In consecutive sections processed for taurine or GABA, numerous cells of the caudal GCL displayed taurine-LI and GABA-like immunoreactivity (GABA-LI). A bimodal distribution of the cross-sectional cell area for GABA-LI cells implied their morphological diversity, and the peak for larger GABA-LI cells coincided with the maximum for taurine-LI cells. At the electron microscopic level, single immunogold labeling showed that GABA-LI, but not taurine-LI, is present in granule cells, whereas both taurine-LI and GABA-LI were localized in a ‘non-granule’ type of cell. The double labeling procedure demonstrated coexistence of taurine-LI and GABA-LI in neurons of a ‘non-granule’ type. These cells had some ultrastructural features typical of short axon cells in the GCL of the mammalian olfactory bulb and were tentatively considered as short axon-like cells. Results suggest that, in the frog olfactory bulb, taurine is contained in primary olfactory afferents and short axon-like cells of the GCL co-localizing GABA and taurine.     

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.     

传入神经阻滞对成年大鼠嗅球钙视网膜蛋白的影响

目的：研究传入神经阻滞对成年SD大鼠嗅球中钙视网膜蛋白(CR)的影响，探讨阻滞的影响是否与动物年龄相关。方法： 成年SD大鼠，以ZnSO4灌流单边鼻孔，用免疫组化方法，在损伤后10 d、20 d、30 d、60 d，检测嗅球中酪氨酸羟化酶(TH)和CR的表达，以TH表达下降作为传入神经阻滞的标志。结果： 损伤后10 d、20 d、30 d和60 d，与对照组相比，损伤组嗅球的TH阳性细胞密度和染色强度显著下降，并且从10 d、20 d到30 d逐渐下降，损伤后60 d的结果与30 d接近。而在所观察的各个阶段，损伤组嗅球的CR阳性细胞数目和染色强度与对照组相比，均无显著差异。结论： 大鼠嗅球中CR的表达不受传入神经阻滞的影响，且阻滞对CR表达的影响与动物年龄无关。     

Capillaries in aging rat olfactory bulb: A quantitative light and electron microscopic analysis

Olfactory bulbs from Charles River (Crl) rats from 3 to 36 months have been examined with light and electron microscopy. Total capillary length, surface, and volume, as well as number of endothelial cells, increases during the twofold increase in olfactory bulb volume from 3 to 18 months, but the relative density of these parameters shows no change during this time; from 18 to 36 months when neuronal cell body and dendrites are decreasing markedly in size, the relative density of capillaries shows only a modest decrease. Capillary lumen size and capillary wall thickness remain the same throughout life, but basal lamina thickness doubles from 3 to 24 months and then remains constant from 24 to 36 months. The incidence of several unusual ultrastructural features of the outer capillary basal lamina has been shown to increase with age.     

传入神经阻滞对成年大鼠嗅球calbindin表达的影响

目的：研究传入神经阻滞对成年SD大鼠嗅球中calbindin（CB）表达的影响。 方法： 成年SD大鼠，ZnSO4 灌流单边鼻孔，10、20、30和60 d后用免疫组化方法检测嗅球的CB表达。 结果： 损伤后10、20、30和60 d，与对照组相比，损伤组嗅球的CB阳性细胞密度和染色强度显著下降，并且从10、20到30 d逐渐下降，损伤后60 d的结果与30 d接近。 结论： 大鼠嗅球中CB的表达受传入神经阻滞的影响。     

A potential reservoir of immature dopaminergic replacement neurons in the adult mammalian olfactory bulb

A significant fraction of the interneurons added in adulthood to the glomerular layer (GL) of the olfactory bulb (OB) are dopaminergic (DA). In the OB, DA neurons are restricted to the GL, but using transgenic mice expressing eGFP under the tyrosine hydroxylase (TH) promoter, we also detected the presence of TH-GFP+ cells in the mitral and external plexiform layers. We hypothesized that these could be adult-generated neurons committed to become DA but not yet entirely differentiated. Accordingly, TH-GFP+ cells outside the GL exhibit functional properties (appearance of pacemaker currents, synaptic connection with the olfactory nerve, intracellular chloride concentration, and other) marking a gradient of maturity toward the dopaminergic phenotype along the mitral–glomerular axis. Finally, we propose that the establishment of a synaptic contact with the olfactory nerve is the key event allowing these cells to complete their differentiation toward the DA phenotype and to reach their final destination.     

The projection from the olfactory epithelium to the olfactory bulb in the salamander, Ambystoma tigrinum

Summary Odor quality may be represented as a topographic code of responses of receptor cells throughout the olfactory epithelium, with this code conveyed to the central nervous system by a topographic projection from the olfactory epithelium to the olfactory bulb. There is good evidence for topographic differences in odor-induced receptor cell activity in the tiger salamander but there is no evidence for a topographic epithelium-to-bulb projection in this species. In the present study ³H-leucine autoradiography was used to trace the projections of olfactory receptor neurons in the tiger salamander. Thirteen animals received small injections of tritiated leucine into different regions of the dorsal or the ventral olfactory epithelium, or into the ventrolateral, vomeronasal organ. The results show that the anterior-to-posterior axes in the dorsal and ventral epithelia are represented along the ventral-to-dorsal axis in the rostral end of the olfactory bulb. The vomeronasal organ projects to the caudal end of the bulb. We conclude that the central projection of the olfactory epithelium in the tiger salamander is topographically organised only along the antero-posterior axis and not the medio-lateral axis. Thus epithelial receptor cell activity along the antero-posterior axis would be represented in the glomerular layer of the bulb by activity along its ventro-dorsal axis.     

Quantitative analysis of GABA-like-immunoreactive and parvalbumin-containing neurons in the CA1 region of the rat hippocampus using a stereological method,the disector

The numerical density of neurons in the CA1 region of the rat dorsal hippocampus has been estimated by a stereological method, the disector, using pairs of video images of toluidine blue-stained, plastic-embedded, 0.5-m-thick sections, 3 m distant from each other. The chemical properties of those disector-counted cells were further analyzed by postembedding immunocytochemical methods on adjacent, semithin sections using antibodies against gamma-aminobutyric acid (GABA) and a specific calcium-binding protein, parvalbumin (PV). The density of neurons in the CA1 region was 35.2 × 10³/mm³; numerical densities in the stratum oriens (SO), stratum pyramidale (SP), and strata radiatum-lacunosum-moleculare (SRLM) were 11.3 × 10³/mm³, 272.4 × 10³/mm³, and 1.9 × 10³/mm³, respectively. The numerical densities of GABA-like immunoreactive (GABA-LIR) and PV-immunoreactive (PV-IR) neurons were 2.1 × 10³/mm³ and 1.1 × 10³/mm³, respectively, which were 5.8% and 3.2% of all neurons, respectively. In the CA1 region only about 60% of PV-positive neurons were GABA-LIR. However, taking the previous observation into consideration that almost all hippocampal PV-positive neurons were immunoreactive for the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD), neurons that were immunoreactive to either GABA or PV or both (GABA+ and/or PV + neurons) were regarded as a better representative of GABAergic neurons in this region; thus, the numerical density of these GABA + and/or PV + neurons was 2.5 × 10³/mm³ and they were 7.0% of all neurons in the CA1 region. Lamellar analysis showed that the numerical densities of GABA+ and/or PV+, GABA-LIR, and PV-IR neurons were highest in the SP, where they were 8.2 × 10³/mm³, 6.2 × 10³/mm³, and 5.4 × 10³/mm³, respectively. The results of the present study indicate that the proportions of GABAergic neurons and a subpopulation of them, PV-containing GABAergic neurons, to other presumable non-GABAergic neurons are far smaller in the CA1 region of the hippocampus than in several neocortical regions previously reported.     

Effects of olfactory bulb removal on fear responses and passive avoidance in the rat

Following complete bulbectomies, male hooded rats showed an increase in irritability and difficulty of handling, but a decrease in timidity or fear responses. After rats had learned to drink in an open field, a cat was confined in the center, and fear was defined by the behavior of controls, viz., almost total suppression of drinking and long periods of freezing, broken by brief bursts of high-speed activity. Bulb animals could not have differed more radically. Bulbs showed neither freezing nor suppression of drinking. The present results could not be attributed to differences in shock reactivity; nor could they be attributed to differences in learning or retention. Bulb animals also showed an impairment on the acquisition of step-down passive avoidance but no difference in step-downs after acquisition. These results argue against an impairment of response inhibition. The present study suggests that bulbectomy increases irritability and decreases timidity.     

Responses of olfactory bulb neurones to the dipeptide carnosine

Summary Carnosine has been applied by microiontophoresis to identified neurones in the olfactory bulb of the rat from solutions of different pH. Although mainly without effect when compared with conventional excitatory and inhibitory amino acids, the dipeptide tended to be depressive when ejected as a cation and excitant when ejected as an anion. The results obtained are not in favour of this substance being an excitatory transmitter in the primary olfactory pathway.     

Postnatal X-ray irradiation effects on glomerular layer of rat olfactory bulb: quantitative and immunocytochemical analysis

Summary In the rat olfactory bulb, the majority of interneurons in the glomerular layer (GL) are supposed to be generated during first postnatal week. Low and repeated doses of X-rays (200 rad x 4 and 200 rad x 6) were used during this period to impair the development of interneurons. The resulting effects on olfactory bulb neurons were examined stereologically and immunocytochemically in animals of 4 and 12 weeks of age. Quantitative analysis showed that, 1) the volume of the GL decreased to 55% (1200 rad) – 70% (800 rad) of control, 2) numerical cell densities in GL decreased to 40% (1200 rad) – 60% (800 rad) of control, thus resulting in 3) a decrease of the total cell number in GL to 20% (1200 rad) – 40% (800 rad) of control in irradiated olfactory bulbs of animals 4 weeks old. In comparison, mitral cells, which are generated prenatally, were much less affected (total cell number: 70–80% of control), indicating a selective loss of cells generated during the first postnatal week in GL. Effects on somata and processes immunoreactive for GABA, tyrosine hydroxylase (TH), calbindin D-28K and parvalbumin (PV) were examined in irradiated bulbs of both 4 and 12 week-old rats. All of these immunoreactive elements showed a drastic decrease in all layers. Semiquantitative analysis showed that in the GL, calbindin D-28K immunoreactive (calbindin D-28K(+)) neurons decreased more extensively than TH immunoreactive (TH(+)) and GABA-like immunoreactive (GABA(+)) neurons; that is, TH(+) and GABA(+) neurons decreased to 20% (1200 rad) – 40% (800 rad) of control, whereas calbindin D-28K(+) neurons decreased to 10% (1200 rad) – 30% (800 rad) of control in the GL of irradiated bulbs. These findings indicated that larger proportions of calbindin D-28K(+) neurons might be generated during the first postnatal week than those of GABA(+) and TH(+) neurons. Furthermore, in irradiated bulbs the proportion of GABA(-)TH(+) cells in TH(+) cells increased to about twice of control, and the estimated total numbers of GABA(-)TH(+) cells in irradiated rats were 95% (800 rad) and 40% (1200 rad) of control. These observations suggest that the majority of GABA(-)TH(+) neurons were less affected by X-ray irradiation during the first postnatal week and thus that they might be generated in the prenatal period. Since during the first 2 postnatal weeks, neurons showing GABA(-)TH(+) were not seen in GL (Kosaka et al. 1987a), the majority of GABA(-)TH(+) neurons in adult olfactory bulb were assumed to change their phenotype at some postnatal developmental period.     

Sex hormone binding globulin in the rat olfactory system

Ovarian steroids are known to act on the olfactory system. Their mode of action, however, is mostly unclear to date since nuclear receptors are lacking in sensory neurons. Here we used immunocytochemistry and RT-PCR to study expression and distribution of sex hormone binding globulin (SHBG) in the rat olfactory system. Single sensory cells in the olfactory mucosa and their projections in the olfactory bulb showed specific SHBG immunostaining as determined by double immunofluorescence with olfactory marker protein OMP. Larger groups of SHBG stained sensory cells occurred in the vomeronasal organ (VNO). A portion of the olfactory glomeruli in the accessory olfactory bulb showed large networks of SHBG positive nerve fibres. Some of the mitral cells showed SHBG immune fluorescence. RT-PCR revealed SHBG encoding mRNA in the olfactory mucosa, in the VNO and in the olfactory bulbs indicating intrinsic expression of the binding globulin. The VNO and its related projections within the limbic system are known to be sensitive to gonadal steroid hormones. We conclude that SHBG may be of functional importance for rapid effects of olfactory steroids on limbic functions including the control of reproductive behaviours through pheromones.     

Morphological characteristics of dopaminergic immunoreactive neurons in the olfactory bulb of the common marmoset monkey (Callithrix jacchus)

The present study describes the distribution of tyrosine hydroxylase (TH)-immunoreactive (IR) elements in the olfactory bulb of the common marmoset monkey (Callithrix jacchus), a primate species by immunohistochemistry. We identified six layers of the olfactory bulb of the common marmoset monkey in sections stained with cresyl violet. The majority of TH-IR cells were found in the glomerular layer. A few TH-IR cells were present in the external plexiform and granule cell layers. TH-IR fibers were identified in all layers of the olfactory bulb. The density of these nerve fibers was high in the internal plexiform and granule cell layers. The results in the olfactory bulb of the common marmoset monkey are generally similar to previous reports in some mammals. These data suggest that TH in the olfactory bulb of the common marmoset monkey may play a role in olfactory transmission via the glomeruli like in other mammals.     

Responses of olfactory bulb neurons to repeated odor stimulations in awake freely-breathing rabbits

Thirty-one olfactory bulb neurons were recorded in the olfactory bulbs of unanaesthetized rabbits during repeated stimulations. Their single-unit activity associated with the inspiratory phases of the respiratory cycles and that associated with the expiratory phases were processed separately. When responses were classified into 3 types, i.e., excitation, inhibition and null, it was found that a large number of neurons presented variable responses to repeated stimulations with the same stimulus. However, the passage from one type to another was found to be limited: responses by excitation or inhibition to the first stimulation turned into null responses only; null responses turned into either excitation or inhibition. Inspiration- and expiration-related responses were also subjected to a principal component analysis in order to determine whether changes in responses were compatible with a reliable coding of the qualitative properties of a stimulus. The results indicated that the repeated presentations of an odorant induced fairly similar profiles of activity across the set of neurons while different odorants induced clearly discriminable profiles. It is concluded that repeated stimulations do not blur the characteristic features of the across-neuron profile of response of an odorant in the olfactory bulb despite the variability of the responses of the neurons which compose the profile.     

Substance P and tyrosine hydroxylase are localized in different neurons of the hamster olfactory bulb

Summary Both tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, and substance P were localized previously to juxtaglomerular neurons in the hamster main olfactory bulb. These neurons had similar features and distribution suggesting that the enzyme, a marker for dopaminergic neurons, and the peptide transmitter, respectively, might coexist in the same cells. To determine if the two antigens occurred in the same neurons specific antibodies and a two color double label technique were utilized. Co-localization of the transmitters was not observed in any region of the olfactory bulb. In addition, interspecies differences were observed in the distribution of both TH and substance P labeled neurons which support the lack of co-localization in the double label studies. The data suggest that olfactory bulb neurons of similar morphology and distribution may synthesize different transmitters.