The Efferent Connections of the Olfactory Bulb and Accessory Olfactory Bulb in the Snakes,Thamnophis sirtalis and Thamnophis radix

The efferent connections of the olfactory bulb and accessory olfactory bulb of two species of garter snakes, Thamnophis sirtalis and T. radix were studied with experimental anterograde degeneration techniques. Axons of cells located in the olfactory bulb terminate ipsilaterally in all parts of the anterior olfactory nucleus, olfactory tubercle and lateral pallium. In addition, some axons enter the ipsilateral stria medullaris thalami, cross the midline in the habenular commissure, enter the contralateral stria medullaris thalami and terminate in the contralateral lateral pallium. The axons of cells in the accessory olfactory bulb course through the telencephalon completely separated from the fibers of olfactory bulb origin and terminate predominantly in the nucleus sphericus. These results confirm previous reports of the separation between the central projections of the olfactory and vomeronasal systems in a variety of vertebrates. The totality of the separation between these two systems coupled with the extensive development of the vomeronasal-accessory bulb system in these snakes suggests that they may be ideal subjects for further research on the functional significance of the vomeronasal system.     

Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes

Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal neurons. The present results are the first clear biochemical and electrophysiological evidence for a vomeronasal epithelium response to a purified nonvolatile odorant, and this makes the garter snake vomeronasal system ideal for studying the mechanisms of chemosignal transduction.     

Histochemical identification of carbohydrate moieties in the accessory olfactory bulb of the mouse using a panel of lectins

Lectin binding patterns in the olfactory bulb of the mouse were investigated using 12 biotinylated lectins. Three, with specificities for galactose, N-acetylgalactosamine and L-fucose, stained only the nervous and glomerular layers of the accessory olfactory bulb; four, with specificities for galactose or N-acetylglucosamine, stained these layers in both the accessory and the main olfactory bulbs; three, with specificities for N-acetylgalactosamine or L-fucose, effected general staining with little contrast between the background and the accessory olfactory bulb or other structures; the remaining two, both of them specific for mannose, stained no part of the tissue studied. In the nervous and glomerular layers of the accessory olfactory bulb six lectins stained the anterior and posterior halves with different intensities and two of these six similarly differentiated between rostral and caudal regions of the posterior half. We conclude that: (i) three lectins binding to different monosaccharides are specific stains for the vomeronasal system when used in this area of the mouse brain; (ii) it may be appropriate to distinguish three parts in the mouse accessory olfactory bulb, instead of the hitherto generally accepted two.     

The prenatal maturity of the accessory olfactory bulb in pigs

The morphological development of the accessory olfactory bulb of the fetal pig was studied by classical and histo-chemical methods, and the vomeronasal organ and nasal septum were studied histochemically. Specimens were obtained from an abattoir and their ages estimated from their crown-to-rump length. The accessory olfactory bulb was structurally mature in fetuses of crown-to-rump length 21-23 cm, by which time the lectin Lycopersicum esculentum agglutinin stained the same structures as in adults (in particular, the entire sensory epithelium of the vomeronasal organ, the vomeronasal nerves, and the nervous and glomerular layers of the accessory olfactory bulb). These results suggest that the vomeronasal system of the pig may, like that of vertebrates such as snakes, be functional at birth.     

Embryonic and neonatal development of the vomeronasal and olfactory systems in garter snakes (Thamnophis spp.)

Newborn, unfed garter snakes (Thamnophis spp.) respond preferentially to aqueous extracts of natural prey items, and these responses are mediated by the vomeronasal system (VNS). Since the VNS, and possibly the olfactory system (OS), are functional at birth, we examined the ontogeny of VNS and OS structures in four embryonic stages and two postnatal ages in garter snakes. The results of this study show 1) significant changes in thickness of the receptor epithelia for both systems; 2) temporal differences in the innervation of the telencephalon for each system; and 3) concurrent development of primary and secondary projection sites in both systems. Possible interactions between different cell populations and their significance for morphogenesis are discussed.     

A map of pheromone receptor activation in the mammalian brain

In mammals, the detection of pheromones is mediated by the vomeronasal system. We have employed gene targeting to visualize the pattern of projections of axons from vomeronasal sensory neurons in the accessory olfactory bulb. Neurons expressing a specific receptor project to multiple glomeruli that reside within spatially restricted domains. The formation of this sensory map in the accessory olfactory bulb and the survival of vomeronasal organ sensory neurons require the expression of pheromone receptors. In addition, we observe individual glomeruli in the accessory olfactory bulb that receive input from more than one type of sensory neuron. These observations indicate that the organization of the vomeronasal sensory afferents is dramatically different from that of the main olfactory system, and these differences have important implications for the logic of olfactory coding in the vomeronasal organ.     

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.     

The rodent accessory olfactory system

The accessory olfactory system contributes to the perception of chemical stimuli in the environment. This review summarizes the structure of the accessory olfactory system, the stimuli that activate it, and the responses elicited in the receptor cells and in the brain. The accessory olfactory system consists of a sensory organ, the vomeronasal organ, and its central projection areas: the accessory olfactory bulb, which is connected to the amygdala and hypothalamus, and also to the cortex. In the vomeronasal organ, several receptors—in contrast to the main olfactory receptors—are sensitive to volatile or nonvolatile molecules. In a similar manner to the main olfactory epithelium, the vomeronasal organ is sensitive to common odorants and pheromones. Each accessory olfactory bulb receives input from the ipsilateral vomeronasal organ, but its activity is modulated by centrifugal projections arising from other brain areas. The processing of vomeronasal stimuli in the amygdala involves contributions from the main olfactory system, and results in long-lasting responses that may be related to the activation of the hypothalamic–hypophyseal axis over a prolonged timeframe. Different brain areas receive inputs from both the main and the accessory olfactory systems, possibly merging the stimulation of the two sensory organs to originate a more complex and integrated chemosensory perception.     

Mating Increases Neuronal Tyrosine Hydroxylase Expression and Selectively Gates Transmission of Male Chemosensory Information in Female Mice

Exposure to chemosensory signals from unfamiliar males can terminate pregnancy in recently mated female mice. The number of tyrosine hydroxylase-positive neurons in the main olfactory bulb has been found to increase following mating and has been implicated in preventing male-induced pregnancy block during the post-implantation period. In contrast, pre-implantation pregnancy block is mediated by the vomeronasal system, and is thought to be prevented by selective inhibition of the mate’s pregnancy blocking chemosignals, at the level of the accessory olfactory bulb. The objectives of this study were firstly to identify the level of the vomeronasal pathway at which selective inhibition of the mate’s pregnancy blocking chemosignals occurs. Secondly, to determine whether a post-mating increase in tyrosine hydroxylase-positive neurons is observed in the vomeronasal system, which could play a role in preventing pre-implantation pregnancy block. Immunohistochemical staining revealed that mating induced an increase in tyrosine-hydroxylase positive neurons in the arcuate hypothalamus of BALB/c females, and suppressed c-Fos expression in these neurons in response to mating male chemosignals. This selective suppression of c-Fos response to mating male chemosignals was not apparent at earlier levels of the pregnancy-blocking neural pathway in the accessory olfactory bulb or corticomedial amygdala. Immunohistochemical staining revealed an increase in the number of tyrosine hydroxylase-positive neurons in the accessory olfactory bulb of BALB/c female mice following mating. However, increased dopamine-mediated inhibition in the accessory olfactory bulb is unlikely to account for the prevention of pregnancy block to the mating male, as tyrosine hydroxylase expression did not increase in females of the C57BL/6 strain, which show normal mate recognition. These findings reveal an association of mating with increased dopaminergic modulation in the pregnancy block pathway and support the hypothesis that mate recognition prevents pregnancy block by suppressing the activation of arcuate dopamine release.     

Distinct axonal projections from two types of olfactory receptor neurons in the middle chamber epithelium of <Emphasis Type="BoldItalic">Xenopus laevis</Emphasis>

Most vertebrates have two olfactory organs, the olfactory epithelium (OE) and the vomeronasal organ. African clawed frog, Xenopus laevis, which spends their entire life in water, have three types of olfactory sensory epithelia: the OE, the middle chamber epithelium (MCE) and the vomeronasal epithelium (VNE). The axons from these epithelia project to the dorsal part of the main olfactory bulb (d-MOB), the ventral part of the MOB (v-MOB) and the accessory olfactory bulb, respectively. In the MCE, which is thought to function in water, two types of receptor neurons (RNs) are intermingled and express one of two types of G-proteins, Golf and Go, respectively. However, axonal projections from these RNs to the v-MOB are not fully understood. In this study, we examined the expression of G-proteins by immunohistochemistry to reveal the projection pattern of olfactory RNs of Xenopus laevis, especially those in the MCE. The somata of Golf- and Go-positive RNs were separately situated in the upper and lower layers of the MCE. The former were equipped with cilia and the latter with microvilli on their apical surface. These RNs are suggested to project to the rostromedial and the caudolateral regions of the v-MOB, respectively. Such segregation patterns observed in the MCE and v-MOB are also present in the OE and olfactory bulbs of most bony fish. Thus, Xenopus laevis is a very interesting model to understand the evolution of vertebrate olfactory systems because they have a primitive, fish-type olfactory system in addition to the mammalian-type olfactory system.     

Zonal organization of the mammalian main and accessory olfactory systems

Zonal organization is one of the characteristic features observed in both main and accessory olfactory systems. In the main olfactory system, most of the odorant receptors are classified into four groups according to their zonal expression patterns in the olfactory epithelium. Each group of odorant receptors is expressed by sensory neurons distributed within one of four circumscribed zones. Olfactory sensory neurons in a given zone of the epithelium project their axons to the glomeruli in a corresponding zone of the main olfactory bulb. Glomeruli in the same zone tend to represent similar odorant receptors having similar tuning specificity to odorants. Vomeronasal receptors (or pheromone receptors) are classified into two groups in the accessory olfactory system. Each group of receptors is expressed by vomeronasal sensory neurons in either the apical or basal zone of the vomeronasal epithelium. Sensory neurons in the apical zone project their axons to the rostral zone of the accessory olfactory bulb and form synaptic connections with mitral tufted cells belonging to the rostral zone. Signals originated from basal zone sensory neurons are sent to mitral tufted cells in the caudal zone of the accessory olfactory bulb. We discuss functional implications of the zonal organization in both main and accessory olfactory systems.     

Lectin-binding patterns in the olfactory system of the lizard, Physignathus lesueurii

Lectin binding histochemistry was performed on the olfactory system of Physignathus lesueurii to investigate the distribution and density of defined carbohydrate terminals on the cell-surface glycoproteins of the olfactory and vomeronasal receptor cells and their terminals in the olfactory bulbs. The lectin staining patterns indicate that the vomeronasal and olfactory receptor cells are characterized by glycoconjugates containing alpha-D-galactose and N-acetyl-D-glucosamine terminal residues. The presence of specific glycoproteins, whose terminal sugars are detected by lectin binding, might be related to the chemoreception and transduction of the odorous message into a nervous signal or to the histogenesis and development of the olfactory system. The olfactory and vomeronasal receptor cells are vertebrate neurons that undergo a continual cycle of proliferation not only during development but also in mature animals.     

Genetic manipulation of blood group carbohydrates alters development and pathfinding of primary sensory axons of the olfactory systems

Primary sensory neurons in the vertebrate olfactory systems are characterised by the differential expression of distinct cell surface carbohydrates. We show here that the histo-blood group H carbohydrate is expressed by primary sensory neurons in both the main and accessory olfactory systems while the blood group A carbohydrate is expressed by a subset of vomeronasal neurons in the developing accessory olfactory system. We have used both loss-of-function and gain-of-function approaches to manipulate expression of these carbohydrates in the olfactory system. In null mutant mice lacking the alpha(1,2)fucosyltransferase FUT1, the absence of blood group H carbohydrate resulted in the delayed maturation of the glomerular layer of the main olfactory bulb. In addition, ubiquitous expression of blood group A on olfactory axons in gain-of-function transgenic mice caused mis-routing of axons in the glomerular layer of the main olfactory bulb and led to exuberant growth of vomeronasal axons in the accessory olfactory bulb. These results provide in vivo evidence for a role of specific cell surface carbohydrates during development of the olfactory nerve pathways.     

gamma-Aminobutyric acid activity in the olfactory bulb of the rat during the sexual cycle and response to olfactory stimuli.

Glutamic acid decarboxylase activity in the main and accessory olfactory bulbs throughout the sexual cycle of the rat was studied. The effect of male pheromonal secretion on enzyme activity during proestrus and estrus day was also tested. The enzyme activity showed circadian rhythm during the estrous cycle. This rhythm was disrupted during diestrus-2 afternoon in the main bulb and came back during proestrus afternoon. A different pattern of enzyme activity was present in the accessory bulb, since the circadian rhythm was altered during proestrus morning, returning during estrus afternoon. Male odor exposition did not change enzyme profile activity during proestrus day and during estrus morning in the main bulb. In contrast, in the accessory bulb the olfactory stimuli induced opposite changes to that found in rats from the vivarium during proestrus. Comparison of enzyme activity in olfactory stimuli-deprived rats with that of pheromone-stimulated rats during proestrus showed that male odor exposure specifically affects accessory bulb enzyme activity. It is concluded that the changes of the olfactory bulb GABAergic system during proestrus and estrus day, or that evoked by odor stimuli, demonstrate the discriminative response of this system between the accessory olfactory bulb and the main olfactory bulb.     

Convergent vomeronasal system reduction in mammals coincides with convergent losses of calcium signalling and odorant‐degrading genes

The vomeronasal system (VNS) serves crucial functions for detecting olfactory clues often related to social and sexual behaviour. Intriguingly, two of the main components of the VNS, the vomeronasal organ (VNO) and the accessory olfactory bulb, are regressed in aquatic mammals, several bats and primates, likely due to adaptations to different ecological niches. To detect genomic changes that are associated with the convergent reduction of the VNS, we performed the first systematic screen for convergently inactivated protein‐coding genes associated with convergent VNS reduction, considering 106 mammalian genomes. Extending previous studies, our results support that Trpc2, a cation channel that is important for calcium signalling in the VNO, is a predictive molecular marker for the presence of a VNS. Our screen also detected the convergent inactivation of the calcium‐binding protein S100z, the aldehyde oxidase Aox2 that is involved in odorant degradation, and the uncharacterized Mslnl gene that is expressed in the VNO and olfactory epithelium. Furthermore, we found that Trpc2 and S100z or Aox2 are also inactivated in otters and Phocid seals for which no morphological data about the VNS are available yet. This predicts a VNS reduction in these semi‐aquatic mammals. By examining the genomes of 115 species in total, our study provides a detailed picture of how the convergent reduction of the VNS coincides with gene inactivation in placental mammals. These inactivated genes provide experimental targets for studying the evolution and biological significance of the olfactory system under different environmental conditions.     

Luteinizing hormone-releasing hormone in olfactory bulbs of primates

This immunohistochemical study of luteinizing hormone-releasing hormone (LHRH) in the olfactory bulbs in primates was undertaken in order to see whether there was an LHRH innervation in these species similar to that found in rodents. One old world (Macaca fascicularis) and two new world (Saimiri sciureus and Aotus trivirgatus) monkeys were studied. Aotus trivirgatus was of particular interest as it is noctural and so presumably more dependent upon olfactory cues. Animals were perfused with fixative, olfactory bulbs removed and sectioned, and tissues reacted immunocytochemically using LR1 (Benoit) antiserum to LHRH. Some LHRH innervation was found in the olfactory bulbs of all three species, comprising a few LHRH neurons and many fibers that ramified within the bulbs. The accessory bulb (not present as a distinct entity in old world primates) had more LHRH innervation than did the main olfactory bulb. Aotus trivirgatus had the greatest representation of LHRH of the three species. The layer of the olfactory bulb with the greatest number of LHRH fibers was the external plexiform layer. This is also true in rodents. There is evidence that LHRH has a role in the mediation of olfactory cues in reproductive behavior in rodents. It is not known how LHRH functions within the olfactory system in primates. However, the fact that it is distributed similarly in the two groups suggests that it may serve a similar function.     

Neuropilin-2 mediates axonal fasciculation, zonal segregation, but not axonal convergence, of primary accessory olfactory neurons

The mechanisms that underlie axonal pathfinding of vomeronasal neurons from the vomeronasal organ (VNO) in the periphery to select glomeruli in the accessory olfactory bulb (AOB) are not well understood. Neuropilin-2, a receptor for secreted semaphorins, is expressed in V1R- and V3R-expressing, but not V2R-expressing, postnatal vomeronasal neurons. Analysis of the vomeronasal nerve in neuropilin-2 (npn-2) mutant mice reveals pathfinding defects at multiple choice points. Vomeronasal sensory axons are severely defasciculated and a subset innervates the main olfactory bulb (MOB). While most axons of V1R-expressing neurons reach the AOB and converge into distinct glomeruli in stereotypic locations, they are no longer restricted to their normal anterior AOB target zone. Thus, Npn-2 and candidate pheromone receptors play distinct and complementary roles in promoting the wiring and patterning of sensory neurons in the accessory olfactory system.     

The sorting behaviour of olfactory and vomeronasal axons during regeneration

Summary In order to begin to understand how primary olfactory and vomeronasal organ (VNO) axons target specific regions of the olfactory bulb, we examined the sorting behaviour of these axons following neonatal unilateral olfactory bulbectomy. Bulbectomy induced widespread ipsilateral death of the primary olfactory and VNO neurons. After 4 weeks, many new sensory axons had re-grown into the cranial cavity and established a prominent plexus with evidence of dense tufts that were similar in gross appearance to glomeruli. Axons expressing the cell adhesion molecule OCAM, which normally innervate the ventrolateral and rostral halves of the main and accessory olfactory bulbs, respectively, sorted out and segregated from those axons not expressing this molecule within the plexus. In addition, VNO axons formed large discrete bundles that segregated from main olfactory axons within the plexus. Thus, VNO and primary olfactory axons as well as discrete subpopulations of both are able to sort out and remain segregated in the absence of the olfactory bulb. Sorting and convergence of axons therefore occur independently of the olfactory bulb and are probably attributable either to inherent properties of the axons themselves or to interactions between the axons and accompanying glial ensheathing cells.     

哺乳动物主要嗅觉系统和犁鼻系统信息识别的编码模式

哺乳动物具有两套嗅觉系统, 即主要嗅觉系统和犁鼻系统。前者对环境中的大多数挥发性化学物质进行识别, 后者对同种个体释放的信息素进行识别。本文从嗅觉感受器、嗅球、嗅球以上脑区三个水平综述了这两种嗅觉系统对化学信息识别的编码模式。犁鼻器用较窄的调谐识别信息素成分, 不同于嗅上皮用分类性合并受体的方式识别气味; 副嗅球以接受相同受体输入的肾丝球所在区域为单位整合信息, 而主嗅球通过对肾丝球模块的特异性合并编码信息; 在犁鼻系统, 信息素的信号更多地作用于下丘脑区域, 引起特定的行为和神经内分泌反应。而在主要嗅觉系统, 嗅皮层可能采用时间模式编码神经元群, 对气味的最终感受与脑的不同区域有关。犁鼻系统较主要嗅觉系统的编码简单, 可能与其执行的功能较少有关。