A morphological study of the vomeronasal organ and the accessory olfactory bulb in the Korean roe deer, Capreolus pygargus

The vomeronasal organ (VNO) and accessory olfactory bulb (AOB) of the Korean roe deer (Capreolus pygargus) were studied histologically to evaluate their morphological characteristics. Grossly, the VNO, encased by cartilage, has a paired tubular structure with a caudal blind end and a rostral connection through incisive ducts on the hard palate. In the VNO, the vomeronasal sensory epithelium (VSE) consists of galectin-3-positive supporting cells, protein gene product (PGP) 9.5-positive receptor cells, and basal cells. The vomeronasal respiratory epithelium (VRE) consists of a pseudostratified epithelium. The AOB strata included a vomeronasal nerve layer (VNL), a glomerular layer (GL), a mitral/tufted cell layer, and a granular cell layer. All lectins used in this study, including Bandeiraea simplicifolia agglutinin isolectin B4 (BSI-B4), soybean agglutinin (SBA), Ulex europaeus agglutinin I (UEA-I), and Triticum vulgaris wheat germ agglutinin (WGA), labeled the VSE with varying intensity. In the AOB, both the VNL and the GL reacted with BSI-B4, SBA, and WGA with varying intensity, but not with UEA-I. This is the first morphological study of the VNO and AOB of the Korean roe deer, which are similar to those of goats.     

Olfactory epithelia differentially express neuronal markers

All three olfactory epithelia, the olfactory epithelium proper (OE), the septal organ of Masera (SO), and the vomeronasal organ of Jacobson (VNO) originate from the olfactory placode. Here, their diverse neurochemical phenotypes were analyzed using the immunohistochemical expression pattern of different neuronal markers. The olfactory bulb (OB) served as neuronal control. Neuronal Nuclei Marker (NeuN) is neither expressed in sensory neurons in any of the three olfactory epithelia, nor in relay neurons (mitral/tufted cells) of the OB. However, OB interneurons (periglomerular/granule cells) labeled, as did supranuclear structures of VNO supporting cells and VNO glands. Protein Gene Product 9.5 (PGP9.5 = C-terminal ubiquitin hydrolase L1 = UCHL1) expression is exactly the opposite: all olfactory sensory neurons express PGP9.5 as do OB mitral/tufted cells but not interneurons. Neuron Specific Enolase (NSE) expression is highest in the most apically located OE and SO sensory neurons and patchy in VNO. In contrast, the cytoplasm of the most basally located neurons of OE and SO immunoreacted for Growth Associated Protein 43 (GAP-43/B50). In VNO neurons GAP-43 labeling is also nuclear. In the cytoplasm, Olfactory Marker Protein (OMP) is most intensely expressed in SO, followed by OE and least in VNO neurons; further, OMP is also expressed in the nucleus of basally located VNO neurons. OB mitral/tufted cells express OMP at low levels. Neurons closer to respiratory epithelium often expressed a higher level of neuronal markers, suggesting a role of those markers for neuronal protection against take-over. Within the VNO the neurons show clear apical–basal expression diversity, as they do for factors of the signal transduction cascade. Overall, expression patterns of the investigated neuronal markers suggest that OE and SO are more similar to each other than to VNO.     

Morphological Analysis for Neuron‐Like Cells in the Vomeronasal Organ of Human Fetuses at the Middle of Gestation

The vomeronasal organ (VNO) of 5‐month‐old fetuses was examined immunohistochemically by the use of an antiserum to protein gene product 9.5 (PGP). The purpose was to identify if the human fetal VNO is lined by neuroepithelium. The PGP antiserum labeled abundant cells within the vomeronasal epithelium (VE), nerve fiber bundles in its lamina propria, and cells associated with these bundles. PGP‐immunoreactive (ir) vomeronasal epithelial cells were classified into three subtypes. Type I cells, about 44% of the total cells observed, did not have any processes and tended to be located in the basal layer of the VE. Type II cells, about 37% had a single apical process that projected toward the lumen, ending at the epithelial surface. Type III cells sent a prominent process mainly toward the basement membrane, and occupied about 19% of the total cells observed. In the lamina propria, a considerable number of PGP‐ir cells was observed. Some of them were present in nerve fiber bundles and contained processes parallel to the bundles. In addition, PGP‐ir nerve fiber bundles and cells associated with them were even present in the portion of the nasal septal mucosa that was very close to the brain. The present results strongly suggested that the VE in human fetuses at mid‐gestation is a neuroepithelium and that the VE may produce migrating cells toward the brain. Anat Rec, 299:88–97, 2016. © 2015 Wiley Periodicals, Inc.     

Cholinergic innervation of the main and the accessory olfactory bulbs of the rat as revealed by a monoclonal antibody against choline acetyltransferase

Summary The main and accessory olfactory bulbs (MOB and AOB) of the rat were immunohistochemically stained with a monoclonal antibody against choline acetyltransferase (ChAT) in order to know the difference in the distribution patterns of cholinergic fibers between these two structures. A few ChAT-immunoreactive cell bodies were found in the superficial and middle parts of the external plexiform layer (EPL) of the MOB, in the granule cell layer (GCL) of the MOB, and in the GCL of the AOB. The frequency in appearance of these cells was 0.9 cells/section in the MOB and 0.3 cells/section in the AOB. While the glomerular layer (GL) and the superficial part of the EPL were most densely innervated in the MOB, the internal plexiform layer received the richest innervation in the AOB. There were no immunoreactive structures in the olfactory nerve layer of the MOB and in the vomeronasal nerve layer and glomerular layer of the AOB. In addition to a relatively homogenous distribution of cholinergic fibers in the MOB and AOB, there were several foci of very dense network of immunoreactive fibers at the posterior level of the OB. These foci formed a part of the modified glomerular complex that was recently identified using 2-deoxyglucose method and was presumed to be related to suckling behaviour in the neonatal rat.     

Sustained acceleration of colonic transit following chronic homotypic stress in oxytocin knockout mice

A sex difference has been reported in the responsiveness of the vomeronasal (VN) system to pheromones. In the present study, to clarify a direct and acute influence of 17β-estradiol (E2) on the accessory olfactory bulb (AOB) neurons, we investigated the effect of E2 on dendritic spines in cultured AOB cells derived from male and female neonatal rats. After 17-18 days in vitro (DIV), cultured AOB cells were transfected with GFP expression vectors. At 21-23 DIV, cells were treated with E2, and time-lapse images of transfected AOB neurons identified as granule cells were taken under a confocal laser scanning microscope for 3h. The dendritic spine head area of granule cells was quantitatively evaluated, and spine heads were classified into larger (≥ 1 μm2) and smaller (<1 μm2) ones before E2-treatment (0 h). In cultured cells derived from both sexes, the larger spines were not significantly changed at 1, 2 and 3 h after E2-treatment. In contrast, E2-treatment significantly enlarged the head area of the smaller spines of granule cells derived from the female, whereas E2 did not cause any significant effects on those from the male. Our results provide evidence for the sexually-dimorphic effect of E2 on spine development in AOB granule cells.     

Can social behaviour drive accessory olfactory bulb asymmetries? Sister species of caviomorph rodents as a case in point

In mammals, the accessory olfactory or vomeronasal system exhibits a wide variety of anatomical arrangements. In caviomorph rodents, the accessory olfactory bulb (AOB) exhibits a dichotomic conformation, in which two subdomains, the anterior (aAOB) and the posterior (pAOB), can be readily distinguished. Interestingly, different species of this group exhibit bias of different sign between the AOB subdomains (aAOB larger than pAOB or vice versa). Such species-specific biases have been related with contrasting differences in the habitat of the different species (e.g. arid vs. humid environments). Aiming to deepen these observations, we performed a morphometric comparison of the AOB subdomains between two sister species of octodontid rodents, Octodon lunatus and Octodon degus. These species are interesting for comparative purposes, as they inhabit similar landscapes but exhibit contrasting social habits. Previous reports have shown that O. degus, a highly social species, exhibits a greatly asymmetric AOB, in which the aAOB has twice the size of the pAOB and features more and larger glomeruli in its glomerular layer (GL). We found that the same as in O. degus, the far less social O. lunatus also exhibits a bias, albeit less pronounced, to a larger aAOB. In both species, this bias was also evident for the mitral/tufted cells number. But unlike in O. degus, in O. lunatus this bias was not present at the GL. In comparison with O. degus, in O. lunatus the aAOB GL was significantly reduced in volume, while the pAOB GL displayed a similar volume. We conclude that these sister species exhibit a very sharp difference in the anatomical conformation of the AOB, namely, the relative size of the GL of the aAOB subdomain, which is larger in O. degus than in O. lunatus. We discuss these results in the context of the differences in the lifestyle of these species, highlighting the differences in social behaviour as a possible factor driving to distinct AOB morphometries.     

A Golgi study on the accessory olfactory bulb in the snake, Elaphe quadrivirgata

The intrinsic organization of the accessory olfactory bulb (AOB) in the snake was studied using the rapid Golgi method. A distinct laminar organization was observed in the snake AOB. Beginning with the most superficial surface, the following layers were distinguished: the layer of the vomeronasal fibers, the olfactory glomeruli, the mitral cells, the deep fiber plexus, the granule cells and the ependymal cells. While the general organizational pattern of the snake AOB resembles that of the main olfactory bulb (MOB) and the AOB reported in various vertebrate species, the present study shows that: (1) the external and internal plexiform layers cannot be identified as independent layers and are considered to be included in the mitral cell layer; (2) the afferent and efferent paths, which are disseminated in the granule cell layer in the mammalian MOB, accumulate external to the granule cell layer to form the layer of the deep fiber plexus: and (3) as a result of accumulation of the afferent and efferent paths in the layer of the deep fiber plexus, the granule cell layer is very fiber-sparse. These structural patterns are quite similar to those of the snake MOB.     

Distribution of GAP-43 nerve fibers in the skin of the adult human hand

Skin is an important region of somatic sensory input, and is one of the most innervated areas of the human body. In this study, we investigated in human hand skin the distribution of nervous structures immunoreactive for the growth-associated protein 43 (GAP-43) and the protein gene product 9.5 (PGP 9.5). GAP-43 is a neuronal presynaptic membrane protein that is generally considered to be a marker of neuronal plasticity. PGP 9.5 is a neuron-specific soluble protein that is widely used as general marker for the peripheral nervous system. The entire neural network of the dermis and epidermis was stained with antibody to PGP 9.5. In the dermis, there were fewer GAP-43-immunostained nerve fibers than PGP 9.5-immunostained nerve fibers, whereas in the epidermis the numbers were equal. Only some Merkel cells and Meissner corpuscles were GAP-43-immunoreactive. In conclusion, our results show that GAP-43 protein is expressed in a subset of PGP 9.5-immunoreactive nerve structures.     

Characterization of Meibomian gland innervation in the cynomolgus monkey (Macaca fascicularis)

To characterize the innervation of the cynomolgus monkey (Macaca fascicularis) Meibomian (tarsal) glands, upper lids of six cynomolgus monkeys were investigated with electronmicroscopical and double-labeling immunocytochemical methods. Antibodies against calcitonin gene-related peptide (CGRP), dopamine-β-hydroxylase (DBH), neuropeptide Y (NPY), nitric oxide synthase (NOS), protein gene product 9.5 (PGP 9.5), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. In addition, sections were processed for NADPH-diaphorase (NADPH-d) histochemistry. Staining for PGP 9.5 and electron microscopy showed that Meibomian gland acini were surrounded by a network of unmyelinated nerves and terminal varicose axons. The terminals contained small agranular (30–60 nm) and large granular vesicles (65–110 nm), and were observed in close contact with the basal lamina of the acini, but never internally to the basal lamina. Meibomian axons showed like-immunore-activity (LI) for the neuropeptides SP, CGRP, NPY, and VIP. In addition, the axons stained for TH, DBH, NOS, and NADPH-d. VIP-LI, NOS- and NADPH-d-positive axons appeared to be more numerous, TH- and DBH-positive axons more rare than others. Most SP-LI axons were double-labelled for CGRP-LI, some for VIP-LI or NPY-LI. In addition, some VIP-LI axons were double-labeled for NPY-LI. NPY/VIP-LI and NPY/SP-LI axons were only observed close to the Meibomian acini. Conversely, NPY-LI colocalized with TH-IR or DBH-IR predominated in perivascular nerves of Meibomian gland vasculature. The close association of varicose axons with the acini of Meibomian glands indicates that nervous signals modulate meibomian secretion. Meibomian gland nerve fibers in the cynomolgus monkey appear to utilize various neuropeptides, catecholamines and nitric oxide as transmitter substances, and seem to derive from the pterygopalatine, superior cervical and trigeminal ganglion respectively.     

Anatomical and immunohistological demonstration of the primary neural connections of the vomeronasal organ in the dog.

Macro- and microdissection methods together with conventional histology and lectin immunohistochemistry have been used to identify the course of the vomeronasal nerves and their site of termination (accessory olfactory bulb; AOB) in the dog. The AOB in this species is small and variable in size, situated on the medial surface of the main olfactory bulb, and has an anatomical structure unlike that described for other mammals. The vomeronasal nerves and their terminal glomeruli in the AOB are easily identifiable by selective immunohistochemical staining using Ulex europeus agglutinin I.     

Anatomical and immunohistological demonstration of the primary neural connections of the vomeronasal organ in the dog

Macro- and microdissection methods together with conventional histology and lectin immunohistochemistry have been used to identify the course of the vomeronasal nerves and their site of termination (accessory olfactory bulb; AOB) in the dog. The AOB in this species is small and variable in size, situated on the medial surface of the main olfactory bulb, and has an anatomical structure unlike that described for other mammals. The vomeronasal nerves and their terminal glomeruli in the AOB are easily identifiable by selective immunohistochemical staining using Ulex europeus agglutinin I. © 1992 Wiley-Liss, Inc.     

Characterization of Meibomian gland innervation in the cynomolgus monkey (Macaca fascicularis)

To characterize the innervation of the cynomolgus monkey (Macaca fascicularis) Meibomian (tarsal) glands, upper lids of six cynomolgus monkeys were investigated with electronmicroscopical and double-labeling immunocytochemical methods. Antibodies against calcitonin gene-related peptide (CGRP), dopamine--hydroxylase (DBH), neuropeptide Y (NPY), nitric oxide synthase (NOS), protein gene product 9.5 (PGP 9.5), substance P (SP), tyrosine hydroxylase (TH), and vasoactive intestinal peptide (VIP) were used. In addition, sections were processed for NADPH-diaphorase (NADPH-d) histochemistry. Staining for PGP 9.5 and electron microscopy showed that Meibomian gland acini were surrounded by a network of unmyelinated nerves and terminal varicose axons. The terminals contained small agranular (30–60 nm) and large granular vesicles (65–110 nm), and were observed in close contact with the basal lamina of the acini, but never internally to the basal lamina. Meibomian axons showed like-immunore-activity (LI) for the neuropeptides SP, CGRP, NPY, and VIP. In addition, the axons stained for TH, DBH, NOS, and NADPH-d. VIP-LI, NOS- and NADPH-d-positive axons appeared to be more numerous, TH- and DBH-positive axons more rare than others. Most SP-LI axons were double-labelled for CGRP-LI, some for VIP-LI or NPY-LI. In addition, some VIP-LI axons were double-labeled for NPY-LI. NPY/VIP-LI and NPY/SP-LI axons were only observed close to the Meibomian acini. Conversely, NPY-LI colocalized with TH-IR or DBH-IR predominated in perivascular nerves of Meibomian gland vasculature. The close association of varicose axons with the acini of Meibomian glands indicates that nervous signals modulate meibomian secretion. Meibomian gland nerve fibers in the cynomolgus monkey appear to utilize various neuropeptides, catecholamines and nitric oxide as transmitter substances, and seem to derive from the pterygopalatine, superior cervical and trigeminal ganglion respectively.     

Sex steroids modulate the signals from volatile female odors in the accessory olfactory bulb of male mice

We previously reported that male mice detect volatile female odors via the accessory olfactory system, and that these odors activate granule cells in the accessory olfactory bulb (AOB) with a characteristic pattern. We also reported that sex steroids modulate the attraction of male mice to volatile female odors. The present study investigated hormonal modulation of signals from volatile female odors in the AOB with c-Fos immunostaining. After intact male mice were exposed to volatile female odors, there were more c-Fos positive cells in the caudal granule cell layer (GCL) than in the rostral GCL of the AOB. This effect was observed 3 days but not 7 days after castration, suggesting that hormonal deficiency causes the reorganization of the AOB after 3 days. There was no difference in the number of c-Fos positive cells between the rostral and caudal GCL of castrated male mice treated with 17 beta-estradiol (E). In contrast, there were more c-Fos positive cells in the caudal GCL than in the rostral GCL of castrated male mice treated with dihydrotestosterone (DHT). In both DHT- and E-treated castrated male mice, there was no difference in the number of c-Fos positive cells between the rostral GCL and caudal GCL. This finding suggests that E disrupts the effect of DHT, and that androgen is required for maintaining the intact neuronal network of the AOB. The present study suggests that sex steroids modulate the signals from volatile female odors in the AOB of male mice.     

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.     

The existence of Merkel cells in the lingual connective tissue of the Surinam caiman, Caiman crocodilus crocodilus (order Crocodilia)

The tongue of the Surinam caiman (a reptilian species) was studied by light microscopy including immunohistochemistry for protein gene product 9.5 (PGP 9.5), and transmission electron microscopy. The connective tissue immediately under taste buds housed a cluster of cells immunoreactive for PGP 9.5. These cells synapsed on nerves, and their cytoplasm contained characteristic granules of 90 nm in the mean diameter, glycogen particles, and bundles of intermediate filaments. In light of these ultrastructural features, they were identified as Merkel cells. The Merkel cells were also surrounded by Schwann cells. These findings indicate that the present Merkel cell-neurite-Schwann cell complex is comparable to the avian Merkel corpuscle. On the basis of the granule localization in the cytoplasm, the caiman Merkel cell was presumed to be involved in not only mechanoreception but also endocrine or paracrine functions.     

Immunocytochemical expression of protein gene product (PGP) 9.5 in the cat bronchopulmonary neuroendocrine cells and nerves

Variously fixed, wax-embedded lung and gastrointestinal serial tissue sections from newborn to adult cats were stained with hematoxylin-eosin (H&E), Grimelius' silver, and immunohistochemical techniques using antisera to protein gene product (PGP) 9.5, a neuron-specific protein under strong evolutionary constraints. PGP 9.5 is revealed as a pan-neuroendocrine marker useful for tracing the pulmonary diffuse neuroendocrine system (PDNES) and studying the relationships between neuronal and neuroendocrine elements at various stages of life. Its occurrence is also compared in the pulmonary and the gastrointestinal tract. In spite of a close resemblance to already described neuroepithelial bodies (NEB) of other mammals, cat NEB feature typical constitutional and distributional difference, illustrating interspecies differences. The number of PGP 9.5 immunopositive pulmonary neuroendocrine cells declines gradually after 3 weeks and throughout adult life. Immunoreactivity in neuronal elements is lost after 1 week of age. In gastrointestinal tissues, only neuronal lelements immunostain, suggesting functional variations or a separate embryological origin for enteroendocrine cells. © 1993 Wiley-Liss, Inc.     

Axonal growth of newly formed vomeronasal receptor neurons after nerve transection

Chemosensory neurons in the vomeronasal epithelium (vomeronasal neurons) regenerate following experimentally induced degeneration. Transection of the vomeronasal nerves leads to retrograde degeneration of vomeronasal neurons followed by replacement of the cell population. The projection of the axons of regenerated vomeronasal neurons was examined by horseradish peroxidase(HRP) histochemistry and electron microscopy. HRP-wheat germ agglutinin (WGA) was placed on the surface of the vomeronasal organ of the rat. Dense distribution of HRP-labeled fibers was observed in the vomeronasal nerve and glomerular layers in the accessory olfactory bulb (AOB) of the intact rat. At one week after transection, HRP-labeled fibers were not found in the AOB, and no labeled fibers could be observed on the medial surface of the olfactory bulb where the vomeronasal nerve traversed. Three weeks after transection, labeled fiber bundles were observed on the medial surface of the olfactory bulb in all animals. No labeled fibers were detected in the AOB. From 12 to 32 weeks after transection, projection of HRP-labeled fibers was identified in the AOB in 8 out of 26 rats (the incidence of projection was 30%). But the number of projection fibers on the operated side was much smaller than on the control side. Electron microscopy confirmed that the HRP-labeled terminals make synaptic contacts with neurons in the AOB.     

Immunolocalization of VEGF/VEGFR system in human fetal vomeronasal organ during early development

The vomeronasal system (VNS) is an accessory olfactory structure present in most mammals adhibited to the detection of specific chemosignals implied in social and reproductive behavior. The VNS comprises the vomeronasal organ (VNO), vomeronasal nerve and accessory olfactory bulb. VNO is characterized by a neuroepithelium constituted by bipolar neurons and supporting and stem/progenitor cells. In humans, VNO is present during fetal life and is supposed to possess chemoreceptor activity and participate in gonadotropin-releasing hormone neuronal precursor migration toward the hypothalamus. Instead, the existence and functions of VNO in postnatal life is debated. Vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) have been demonstrated to play fundamental roles in various neurogenic events. However, there are no data regarding the localization and possible function of VEGF/VEGFRs in human fetal VNO. Therefore, this study was conceived to investigate the expression of VEGF/VEGFRs in human VNO in an early developmental period (9–12 weeks of gestation), when this organ appears well structured. Coronal sections of maxillofacial specimens were subjected to peroxidase-based immunohistochemistry for VEGF, VEGFR-1 and VEGFR-2. Double immunofluorescence for VEGF, VEGFR-1 or VEGFR-2 and the neuronal marker protein gene product 9.5 (PGP 9.5) was also performed. VEGF expression was evident in the entire VNO epithelium, with particularly strong reactivity in the middle layer. Strongly VEGF-immunostained cells with aspect similar to bipolar neurons and/or their presumable precursors were detected in the middle and basal layers. Cells detaching from the basal epithelial layer and detached cell groups in the surrounding lamina propria showed moderate/strong VEGF expression. The strongest VEGFR-1 and VEGFR-2 expression was detected in the apical epithelial layer. Cells with aspect similar to bipolar neurons and/or their presumable precursors located in the middle and basal layers and the detaching/detached cells displayed a VEGFR-1 and VEGFR-2 reactivity similar to that of VEGF. The basal epithelial layer exhibited stronger staining for VEGFRs than for VEGF. Cells with morphology and VEGF/VEGFR expression similar to those of the detaching/detached cells were also detected in the middle and basal VNO epithelial layers. Double immunofluorescence using anti-PGP 9.5 antibodies demonstrated that most of the VEGF/VEGFR-immunoreactive cells were neuronal cells. Collectively, our findings suggest that during early fetal development the VEGF/VEGFR system might be involved in the presumptive VNO chemoreceptor activity and neuronal precursor migration.     

Neuronal gap junctions in the mouse main olfactory bulb: morphological analyses on transgenic mice

In the present study we analyzed the structural features of extraglomerular gap junction-forming processes in mouse olfactory bulb electron microscopically. This work complements a previous study in which we analyzed the structural features of neuronal gap junction-forming processes within the glomerulus itself. Furthermore we examined connexin 36 expressing cells in the mouse olfactory bulb by analyzing transgenic mice in which the connexin 36 coding sequence was replaced with histological reporters. In extraglomerular regions, the mitral/tufted cell somata, dendrites and axon hillocks made gap junctions and mixed synapses with interneuronal processes. These gap junctions and synapses were associated with various types of interneuronal processes, including a particular type of sheet-like or calyx-like process contacting the somata or large dendrites of mitral/tufted cells. In the olfactory bulbs of the transgenic mice, connexin 36 was expressed in mitral cells, tufted cells, presumed granule cells and periglomerular cells. Multiple immunofluorescent labelings further revealed that presumed interneurons expressing connexin 36 in the periglomerular region rarely expressed calbindin, calretinin or tyrosine hydroxylase and are likely to comprise a chemically uncharacterized class of neurons. Similarly, interneurons expressing connexin 36 in the granule cell layer were rarely positive for calretinin, which was expressed in numerous presumed granule cells in the mouse main olfactory bulb. In summary, these findings revealed that mitral/tufted cells make gap junctions with diverse types of neurons; in the glomeruli gap junction-forming interneuronal processes originated from some types of periglomerular cells but others from a hitherto uncharacterized neuron type(s), and in the extraglomerular region gap-junction forming processes originate mainly from a subset of cells within the granule cell layer.     

Protein gene product 9.5-immunoreactive neurons in the retina of striped dolphin (Stenella coeruleoalba) and Fraser dolphin (Lagenodelphis hosei).

This study demonstrates immunocytochemically that protein gene product 9.5 (PGP 9.5), a neuronal marker, is expressed by various populations of retinal cells in Stenella coeruleoalba (striped dolphin) and Lagenodelphis hosei (Fraser dolphin): one in the retinal ganglion cells and the other in the inner nuclear layer, resembling horizontal and amacrine cells. The specific distribution of PGP 9.5 in a dolphin closely resembles that in rodents and carnivores; however, some differences arise among these animals. In a dolphin's retina, for example, only a few of giant ganglion cells are immunoreacted while almost all the small ganglion cells are stained strongly. The processes of horizontal cells, identified according to their localization, appear not to connect entirely in a dolphin. Instead, PGP 9.5 positive cells are widely distributed in the small to moderate ganglion cells and have distinct processes which are ramified extensively in the outer plexiform layer in rodents and carnivores. The high levels of PGP 9.5 expressing in the inner part of dolphin retina, including ganglion cells and their axons as well as distinct sublamination in the inner plexiform layer, indicate that this molecule markedly influences the retinal system, possibly in visual connection. Although mammals have various visual behavior, i.e., living marine vs. terrestrial environment, and active during daytime vs. in the night, the retina is a common model to characterize the neurochemical properties.