Regeneration of vomeronasal nerves into the main olfactory bulb in the mouse

Vomeronasal neurosensory cells which are continuously formed in adult mice have been shown to possess axons running in the vomeronasal nerves, since they undergo a reaction of retrograde cell death after vomeronasal nerve transection. Retrograde axonal transport of horseradish peroxidase has been combined with [³H]thymidine labelling of dividing cells to show that the axons of the newly-formed neurosensory cells reach their appropriate target, the accessory olfactory bulb.     

Immunochemical identification of subgroups of vomeronasal nerve fibers and their segregated terminations in the accessory olfactory bulb

Vomeronasal nerve (VNN) fibers and their terminations in the accessory olfactory bulb (AOB) were studied immunohistochemically using 3 monoclonal antibodies (MAbs). One MAb (R2D5) labeled all VNN fibers. Another MAb (R4B12) labeled a subgroup of the VNN fibers which terminated in the rostrolateral glomeruli in the AOB. The third MAb (R5A10) recognized a complementary subgroup of the VNN which terminated in the caudomedial portion of the AOB. These results for the first time show occurrence of subtypes in the VNN axons with segregated terminations in the AOB.     

Vomeronasal and olfactory nerves of adult and larval bullfrogs: II. Axon terminations and synaptic contacts in the accessory olfactory bulb

The ultrastructure of the accessory olfactory bulb (AOB) of the bullfrog tadpole and adult was examined, and the main difference between tadpole and adult is that the latter is more compact and shows more synapses. Except for vomeronasal (VMN) glomeruli, the AOB is not highly organized, with mitral cell neurons scattered throughout the neuropil. VMN axon terminals form asymmetric synapses with mitral cell dendrites in glomeruli; in VMN axon terminals, dense-cored vesicles are seen along with the more abundant lucent vesicles 40-50 nm in diameter. Counts indicated that more than 90% of the dendro-dendritic synapses between mitral cells and presumed granule cells are of the asymmetrical type, and reciprocal asymmetrical-symmetrical synapses are not common. Lucent vesicles with round or slightly ellipsoidal profiles and less abundant dense-cored vesicles 60-90 nm in diameter are found in pre- and postsynaptic dendrites; sometimes the dense-cored vesicles lie against or near the presynaptic membrane. Microtubules were often seen to be closely associated with pre- and postsynaptic elements of dendro-dendritic synapses. The most characteristic feature of mitral cell bodies, apart from their large size, is an extensive Golgi system that may extend well into their major dendritic extensions. Dense-cored vesicles are associated with Golgi membranes, from which they probably originate. Centrioles are associated with the Golgi system, and some become basal bodies and give rise to cilia in some mitral cells.     

Changes in electrophysiological activity in the accessory olfactory bulb and medial amygdala associated with mate recognition in mice

The ability of female mice to recognize their mate's pheromonal identity is critical for the maintenance of their pregnancy and is hypothesized to involve increases in the inhibitory control of mitral/tufted projection neurons in the accessory olfactory bulb. Local field potential recordings from this region of freely behaving female mice showed oscillating neural activity over a wide range of frequencies, which was affected by chemosensory input and prior experience. Mating caused lasting increases in the baseline neural activity in the accessory olfactory bulb, with large increases in the amplitude of local field potential oscillations across a range of frequencies. Exposure to the mate's urinary cues remained effective in increasing the power of these oscillations following mating, but urinary cues from an unfamiliar male were ineffective. A differential response to the familiar and unfamiliar chemosignals was also observed at the level of the amygdala following mating. Individual neurons in the medial amygdala responded more strongly to urine from an unfamiliar male than from the mating male. These findings are consistent with the selective enhancement of inhibition of the familiar pheromonal signal at the level of the accessory olfactory bulb, which is proposed to underlie recognition of the mating male.     

Estimation of numbers of vomeronasal synapses in the glomerular layer of the accessory olfactory bulb of the mouse at different ages

The total number of vomeronasal nerve synapses was estimated in a series of mice from 1 to 12 months of age. The volume of the glomerular layer of the accessory olfactory bulb was derived from area measurements through a series of semithin sections. The specialized appositions constituting the vomeronasal nerve synapses were identified in electron micrographs of the glomerular layer by the characteristic electron dense matrix, vesicles, shape and arrangement of the presynaptic elements, and the pale, vesicle-containing dendritic postsynaptic elements.The volume of the glomerular layer showed an overall increase from 66 x 10³ cu. μm at 1 month of age to 82 x 10⁶ cu. μm at 12 months (although there was almost equally large intergroup variation probably not associated with age). The number of synapses per unit area was between 0.114 and 0.131 per sq. μm and unrelated to age. The average length of the synaptic appositions increased from0.32 ± 0.01 μm at 1 month to0.36 ± 0.02 μm at 12 months (the major increase occurring between 1 and 4 months). The calculated total numbers of synapses in the glomerular layer were between 20 and 21 millions at 1, 4 and 8 months and showed a statistically insignificant increase to slightly over 23 millions at 12 months. Since there are about 140 thousand vomeronasal axons, each axon makes on the average about 150 synapses.New vomeronasal neurosensory cells are continually being formed during life and the new cells develop axons which grow to the bulb. However, both the overall number of cells in the epithelium and the total number of synapses in the bulb are constant after about 8 months of age. This could be achieved without synaptic turnover if the newly formed cells die before making synapses. Alternatively, it could be achieved if the entire population of neurosensory cells and their synapses are undergoing continual replacement.     

Ultrastructural localization of LH-RH-immunoreactive synapses in the hamster accessory olfactory bulb

Electron microscopic immunocytochemistry was used to localize luteinizing hormone-releasing (LH-RH) immunoreactivity within the male golden hamster accessory olfactory bulb. Two LH-RH-immunoreactive fiber populations were identified in the accessory olfactory bulb. A superficial system of immunoreactive axons was localized to the vomeronasal nerve and glomerular layers, and a periventricular system appeared in granule cell and periventricular layers. LH-RH-immunoreactive varicosities were observed to contain large reactive vesicles (80-120 nm) as well as a variable degree of cytoplasmic reaction product. Additionally, small vesicles with unreactive lumens and mitochondria were often present. Intravaricose segments of immunoreactive fibers invariably displayed fewer reactive vesicles than did varicosities. Within both glomerular and periventricular layers, some LH-RH-immunoreactive varicosities were observed to form asymmetric contacts characterized by prominent postjunctional densities. In the glomerular layer, these junctions could be identified as synaptic by several features. The presence of LH-RH-immunoreactivity in presynaptic elements supports a neuromodulatory role for LH-RH. As the accessory olfactory system is critically involved in the initiation of mating behavior of the male golden hamster, LH-RH-immunoreactive synapses in the accessory olfactory bulb may function to regulate reproductive behavior.     

Neuropeptide- and neurotransmitter-related immunoreactivities in the developing rat olfactory bulb

The development of neuropeptide and neurotransmitter-related immunoreactivities in the rat olfactory bulb were investigated immunohistochemically by using antisera raised against substance P (SP), cholecystokinin-8 (CCK), neurotensin (NT), leucine-enkephalin or methionine-enkephalin-Arg6-Gly7-Leu8 (ENK), somatostatin (SOM), neuropeptide Y (NPY) and tyrosine hydroxylase (TH). Results obtained for the adult olfactory bulb confirmed previous observations, except for SP-like immunoreactive (SP-IR) granule cells in the main olfactory bulb (MOB) and NT-IR neurons around the modified glomerular complex (MGC) (Teicher et al., Brain Res. 194:530-535, 1980). SP-, CCK- and NT-IR neurons were observed in the MOB of the rat fetus. SP-IR neurons also appeared in the accessory olfactory bulb (AOB). Among them, NT-IR neurons in the MOB and SP-IR neurons in the AOB were observed on embryonic day 16. SP- and CCK-IR neurons in the MOB appeared on embryonic day 18. Most of these neurons were presumed to be projecting neurons. SOM-, NPY-, ENK- and TH-IR neurons appeared in the newborn rats. The number and intensity of immunostaining of these neurons continued to increase with age, producing the adult pattern, except for NT-IR neurons in the MGC and SP-IR neurons in the mitral cell layer of the AOB, which were more numerous and intensely stained in young animals.     

Accessory olfactory bulb function is modulated by input from the main olfactory epithelium

Although it is now established that sensory neurons in both the main olfactory epithelium and the vomeronasal organ may be activated by both general and pheromonal odorants, it remains unclear what initiates sampling by the vomeronasal organ. Anterograde transport of wheat germ agglutinin–horseradish peroxidase was used to determine that adequate intranasal syringing with zinc sulfate interrupted all inputs to the main olfactory bulb but left intact those to the accessory olfactory bulb. Adult male treated mice were frankly anosmic when tested with pheromonal and non‐pheromonal odors and failed to engage in aggressive behavior. Treated juvenile females failed to show puberty acceleration subsequent to exposure to bedding from adult males. Activation of the immediate early gene c‐Fos and electrovomeronasogram recording confirmed the integrity of the vomeronasal system in zinc sulfate‐treated mice. These results support the hypothesis that odor detection by the main olfactory epithelium is required to initiate sampling by the vomeronasal system.     

Convergence of olfactory and vomeronasal projections in the rat basal telencephalon

Olfactory and vomeronasal projections have been traditionally viewed as terminating in contiguous non-overlapping areas of the basal telencephalon. Original reports, however, described areas such as the anterior medial amygdala where both chemosensory afferents appeared to overlap. We addressed this issue by injecting dextran amines in the main or accessory olfactory bulbs of rats and the results were analyzed with light and electron microscopes. Simultaneous injections of different fluorescent dextran amines in the main and accessory olfactory bulbs were performed and the results were analyzed using confocal microscopy. Similar experiments with dextran amines in the olfactory bulbs plus FluoroGold in the bed nucleus of the stria terminalis indicate that neurons projecting through the stria terminalis could be integrating olfactory and vomeronasal inputs. Retrograde tracing experiments using FluoroGold or dextran amines confirm that areas of the rostral basal telencephalon receive inputs from both the main and accessory olfactory bulbs. While both inputs clearly converge in areas classically considered olfactory-recipient (nucleus of the lateral olfactory tract, anterior cortical amygdaloid nucleus, and cortex-amygdala transition zone) or vomeronasal-recipient (ventral anterior amygdala, bed nucleus of the accessory olfactory tract, and anteroventral medial amygdaloid nucleus), segregation is virtually complete at posterior levels such as the posteromedial and posterolateral cortical amygdalae. This provides evidence that areas so far considered receiving a single chemosensory modality are likely sites for convergent direct olfactory and vomeronasal inputs. Therefore, areas of the basal telencephalon should be reclassified as olfactory, vomeronasal, or mixed chemosensory structures, which could facilitate understanding of olfactory-vomeronasal interactions in functional studies.     

Activation of accessory olfactory bulb neurons during copulatory behavior after deprivation of vomeronasal inputs in male rats

We examined whether the vomeronasal organ (VNO) is the sole receptor for pheromonal cues for male sexual behavior. Males carrying surgical removal of the VNO (VNOx) mated with stimulus females as sham-operated males, with a comparable number of mounts but a prolonged latency for ejaculation. In sham-operated males, mating increased cFos immunoreactivity in the granule and mitral cell layers of the accessory olfactory bulb and in the medial amygdala. VNOx diminished baseline as well as mating-induced cFos in the granule cell layer and in the medial amygdala; VNOx had no effect on either basal or induced cFos immunoreactivity in the mitral cell layer. Thus, during mating encounter, cFos expression in the mitral cell layer did not depend on VNO inputs. The medial amygdala may be modulated by impulses other than of mitral cell origin.     

Light microscope differentiation of two populations of rat olfactory bulb granule cells

Examination of glycolmethacrylate embedded olfactory bulbs of normal rats revealed that the granule cells of the accessory olfactory bulb were dissimilar from the majority of main olfactory bulb granule cells. Cells from these structures can be discriminated on the basis of spherical form. nuclear diameter, nucleoplasm staining, basal dendritic arborizations, and susceptibility to loss after neonatal X-irradiation. Based on their staining qualities, we have called these cells light and dark granule cells. In the normal rat the dark granules make up about 85% of granule cells in the main olfactory bulb and is the cell type preferentially killed by neonatal X-irradiation. Timing of postnatal X-irradiation and consequent differential loss of the dark type of main olfactory bulb granule cell suggests that the light type is largely prenatally formed.     

Light microscopic differentiation of two populations of rat olfactory bulb granule cells

Examination of glycolmethacrylate embedded olfactory bulbs of normal rats revealed that the granule cells of the accessory olfactory bulb were dissimilar from the majority of main olfactory bulb granule cells. Cells from these structures can be discriminated on the basis of spherical form, nuclear diameter, nucleoplasm staining, basal dendritic arborizations, and susceptibility to loss after neonatal X-irradiation. Based on their staining qualities, we have called these cells light and dark granule cells. In the normal rat the dark granules make up about 85% of granule cells in the main olfactory bulb and is the cell type preferentially killed by neonatal X-irradiation. Timing of postnatal X-irradiation and consequent differential loss of the dark type of main olfactory bulb granule cell suggests that the light type is largely prenatally formed.     

Efferents and centrifugal afferents of the main and accessory olfactory bulbs in the hamster

The efferents and centrifugal afferents of the hamster olfactory bulbs were studied using orthograde and retrograde tracing techniques. Following injections of tritiated amino acids which were restricted to the main olfactory bulb (MOB), autoradiographic grains were observed ipsilaterally over layer IA of the entire anterior olfactory nucleus (AON), the ventral portion of the hippocampal rudiment (HR), the entire prepyriform cortex and olfactory tubercle, the anterior and posterolateral cortical amygdaloid nuclei and the lateral entorhinal cortex. An ipsilateral projection to the nucleus of the lateral olfactory tract (nLOT) was also indicated. No subcortical or contralateral projections were observed. Amino acid injections into the accessory olfactory bulb (AOB) revealed ipsilateral projections to the superficial plexiform layer of the medial and posteromedial cortical amygdaloid nuclei and to the bed nucleus of the accessory olfactory tract (nAOT) and the bed nucleus of the stria terminalis (nST). Following injections of HRP which were restricted to the MOB, contralateral HRP-positive neurons were found predominantly in pars externa and to a lesser extent in the other subdivisions of the AON. Centrifugal projections to the MOB were identified ipsilaterally from the entire AON, the ventral portion of the HR, the anterior portion of the prepyriform cortex, and the nLOT. No labelled neurons were found in the olfactory tubercle, the anterior and posterolateral cortical amygdaloid nuclei or the entorhinal cortex. Centrifugal projections to the MOB were also identified from subcortical structures of the ipsilateral basal forebrain and from midline structures of the midbrain. Labelling occurred in the fusiform neurons of the diagonal band near the medial base of the forebrain at the level of caudal olfactory tubercle. Heavy labelling was seen in a distinct group of large, predominantly multipolar neurons (magnocellular preoptic area) that continued from the level of caudal olfactory tubercle to the level of the nLOT. This band of HRP-positive neurons could be followed more caudally to a position dorsal and medial to the nLOT near the lateral margin of the lateral anterior hypothalamic area. The midbrain projections to the MOB originated in the dorsal and median raphe nuclei. After injections of HRP into the AOB, centrifugal projections were identified from the nAOT and the posteromedial cortical amygdaloid nucleus. In addition, isolated neurons were labelled in the medial cortical amygdaloid nucleus but no labelled neurons were found in the nST. These results support the notion of two anatomically distinct olfactory systems and demonstrate two previously unreported pathways through which the limbic system may modulate sensory processing in the olfactory bulb.     

Sexual differences in the development of accessory olfactory bulbs in the rat

The aim of this investigation was 1) to compare the development of AOB in male and female rats before birth, early after birth, and at later stages of sexual immaturity and maturity 2) to examine the effects of early and delayed castration after birth on AOB development in the male rat. A significant increase of the surface area of AOB was observed in both sexes from birth until postnatal day 7, but AOB was found to be larger in males than in females. From the end of the first postnatal week AOB stopped growing until day 40 in both males and females. After this time AOB resumed its development until day 60 in males, while no changes occurred in females over the same period. Castration of males at birth or six hours later impaired development of AOB. Castration of males on postnatal days 20 or 30 also impaired AOB development until day 60. The results strongly suggest that the development of AOB in the male rat is dependent on the two well known "perinatal" and "adult" phases of the endocrine activity of the testes.     

Noradrenaline-induced enhancement of oscillatory local field potentials in the mouse accessory olfactory bulb does not depend on disinhibition of mitral cells

The olfactory bulb differs from other brain regions by its use of bidirectional synaptic transmission at dendrodendritic reciprocal synapses. These reciprocal synapses provide tight coupling of inhibitory feedback from granule cell interneurons to mitral cell projection neurons in the accessory olfactory bulb (AOB), at the first stage of vomeronasal processing. It has been proposed that both the mGluR2 agonist DCG-IV and noradrenaline promote mate recognition memory formation by reducing GABAergic feedback on mitral cells. The resultant mitral cell disinhibition is thought to induce a long-lasting enhancement in the gain of inhibitory feedback from granule to mitral cells, which selectively gates the transmission of the learned chemosensory information. However, we found that local infusions of both noradrenaline and DCG-IV failed to disinhibit AOB neural activity in urethane-anaesthetised mice. DCG-IV infusion had similar effects to the GABA(A) agonist isoguvacine, suggesting that it increased GABAergic inhibition in the AOB rather than reducing it. Noradrenaline infusion into the AOB also failed to disinhibit mitral cells in awake mice despite inducing long-term increases in power of AOB local field potentials, similar to those observed following memory formation. These results suggest that mitral cell disinhibition is not essential for the neural changes in the AOB that underlie mate recognition memory formation in mice.     

Localized changes in olfactory bulb morphology associated with early olfactory learning

Young rats exposed to an odor while receiving reinforcing stimulation come to approach that odor upon subsequent presentation. In addition, such pups have increased 14C-2-deoxyglucose (2DG) uptake within focal areas of the glomerular layer in response to that odor, compared to control animals experiencing the odor for the first time. In this study, the morphology of the glomerular areas underlying these 2DG foci was examined to determine whether early olfactory learning imposed local structural changes that could produce the enhanced 2DG uptake. Alternate sections either were processed with a silver and a Nissl stain to label both cell bodies and their processes or were histochemically treated for the mitochondrial enzymes cytochrome oxidase (CO) or succinic dehydrogenase (SDH) to define the glomerular core of the bulb; 2DG autoradiographs were aligned with adjacent stained sections, and regions underlying the high 2DG uptake foci were examined. In odor-familiar animals, large glomerular clusters that protruded into the external plexiform layer or the olfactory nerve layer were associated with the focal areas of increased 2DG uptake. Morphometric analysis of these regions revealed that the glomerular layer underlying the foci of high 2DG uptake was 30% wider in odor-familiar animals than comparable areas in odor-unfamiliar animals; the cross-sectional areas of individual glomeruli were 21% larger in odor-familiar animals. The foci of enhanced 2DG uptake therefore appear to be associated with groups of enlarged glomeruli. These data demonstrate that early olfactory learning influences the morphology of the olfactory bulb.     

Topographical and laminar localization of 2-deoxyglucose uptake in rat olfactory bulb induced by electrical stimulation of olfactory nerves

Experiments were carried out to examine the topographical projection of the olfactory nerves to the olfactory bulb in the rat, using the Sokoloff [14C]2-deoxyglucose (2-DG) technique. Electrical stimulation of a medially located bundle of olfactory nerves produced a discrete zone of 2-DG uptake at the rostral pole of the bulb. Increasing stimulus strength yielded a slightly larger focus at this site. In contrast, electrical stimulation of laterally situated bundles of olfactory nerves resulted in a broad zone of activity extending along the lateral wall of the bulb, and increasing stimulus intensity produced a more extensive area of uptake. Laminar analyses provided information on the relation between activity in the glomerular layer, where the olfactory nerves terminate, and activity in deeper layers. The results support previous studies of the topographical projections of the olfactory nerves to the olfactory bulb. They also support the hypothesis that odor-induced 2-DG uptake in the olfactory bulb represents activation of groups of receptors in the olfactory epithelium whose axons terminate in activated glomerular regions in the olfactory bulb.