A sex-specific feedback projection from aromatase-expressing neurons in the medial amygdala to the accessory olfactory bulb

The accessory olfactory bulb (AOB) plays a critical role in classifying pheromonal signals. Here we identify two previously undescribed sources of aromatase signaling in the AOB: (1) a population of aromatase-expressing neurons in the AOB itself; (2) a tract of aromatase-expressing axons which originate in the ventral medial amygdala (MEA) and terminate in the AOB. Using a retrograde tracer in conjunction with a transgenic strategy to label aromatase-expressing neurons throughout the brain, we found that a single contiguous population of neurons in the ventral MEA provides the only significant feedback by aromatase-expressing neurons to the AOB. This population expresses the estrogen receptor alpha (ERα) and displayed anatomical sex differences in the number of neurons (higher in male mice) and the size of cell bodies (larger in females). Given the previously established relationship between aromatase expression, estrogen signaling, and the function of sexually dimorphic circuits, we suggest that this feedback population is well-positioned to provide neuroendocrine feedback to modulate sensory processing of social stimuli in the AOB.     

Adjacent laminar terminations of two centrifugal afferent pathways to the accessory olfactory bulb in the mouse

Anterograde and retrograde axonal tracing methods have been combined with transection of the stria terminalis to investigate the centrifugal afferent connections of the accessory olfactory bulb in the mouse.Injection of tritiated proline into the postero-medial cortical amygdaloid nucleus (C₃) gives rise to anterograde autoradiographic labelling of a pathway terminating in the internal granular layer of the accessory olfactory bulb (AOB). Transection of the ipsilateral stria terminalis completely abolishes labelling of this pathway. Injections further rostral, in the bed nucleus of the accessory olfactory tract (bnAOT) and medial amygdaloid nucleus (M), give rise to labelling of a second ipsilateral afferent pathway to the AOB which terminates in the internal plexiform layer (IPL) and is unaffected by strial transection.Injections of wheat germ lectin-HRP conjugate into the AOB confirm that it receives afferents from the ipsilateral bnAOT, M and C₃, and from a few cells in the contralateral C₃. Transection of the ipsilateral stria terminalis prevents retrograde labelling of any cells in the ipsilateral C₃, but does not affect labelling of cells in M or bnAOT (or contralateral C₃). The conjugate is also transported anterogradely in this system, labelling the efferent projections of the AOB to bnAOT, M and C₃.It is concluded that the AOB receives at least two sets of ipsilateral afferents: one set from C₃, via the stria terminalis, terminating in the internal granular layer, and a second set from M and/or bnAOT terminating in the IPL and probably running in the accessory olfactory tract.     

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.     

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.     

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.     

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.     

Second-order input to the medial amygdala from olfactory sensory neurons expressing the transduction channel TRPM5

Recent anatomical tracing experiments in rodents have established that a subset of mitral cells in the main olfactory bulb (MOB) projects directly to the medial amygdala (MeA), traditionally considered a target of the accessory olfactory bulb. Neurons that project from the MOB to the MeA also show activation in response to conspecific (opposite sex) volatile urine exposure, establishing a direct role of the MOB in semiochemical processing. In addition, olfactory sensory neurons (OSNs) that express the transient receptor potential M5 (TRPM5) channel innervate a subset of glomeruli that respond to putative semiochemical stimuli. In this study, we examined whether the subset of glomeruli targeted by TRPM5-expressing OSNs is innervated by the population of mitral cells that projects to the MeA. We injected the retrograde tracer cholera toxin B (CTB) into the MeA of mice in which the TRPM5 promoter drives green fluorescent protein (GFP). We found overlapping clusters of CTB-labeled mitral cell dendritic branches (CTB(+) ) in TRPM5-GFP(+) glomeruli at significantly greater frequency than expected by chance. Despite the significant degree of colocalization, some amygdalopetal mitral cells extended dendrites to non-TRPM5-GFP glomeruli and vice versa, suggesting that, although significant overlapping glomerular innervation is observed between these two features, it is not absolute.     

Differential projections of the main and accessory olfactory bulb in the frog.

The central projections of the main olfactory bulb and the accessory olfactory bulb of the adult leopard frog (Rana pipiens) were reexamined, by using a horseradish peroxidase anterograde tracing method that fills axons with a continuous deposit of reaction product. The fine morphology preserved by this method allowed the terminal fields of the projection tracts to be delineated reliably, and for the first time. Herrick's amygdala has been newly subdivided into cortical and medial nuclei on the basis of cytoarchitecture, dendritic morphology, and the differential projections of the main and accessory olfactory tracts. The main olfactory bulb projects through the medial and lateral olfactory tracts to the postolfactory eminence, the rostral end of the medial cortex, the rostral end of the medial septal nucleus, the cortical amygdaloid nucleus, the nucleus of the hemispheric sulcus, and both the dorsal and ventral divisions of the lateral cortex, including its retrobulbar fringe. The lateral olfactory tract overlaps the dorsal edge of the striatal plate along the ventral border of the lateral cortex, but it is not certain whether any striatal cells are postsynaptic to the tract fibers. The lateral cortex is the largest of these territories, and receives the terminals of the main olfactory projection throughout its extent. It extends from the olfactory bulb to the posterior pole, and from the striatum to the summit of the hemisphere, where it borders the dorsal cortex. The medial and lateral olfactory tracts combine in the region of the amygdala to form a part of the stria medullaris thalami. These fibers cross in the habenular commissure and terminate in the contralateral cortical amygdaloid nucleus and periamygdaloid part of the lateral cortex. Cells projecting to the main olfactory bulb are found in the diagonal band and adjacent cell groups, but there is no evidence of an interbulbar projection arising from either the olfactory bulb proper or a putative anterior olfactory nucleus. The accessory olfactory bulb projects through the accessory olfactory tract to the medial and cortical amygdaloid nuclei. A fascicle of the tract crosses in the anterior commissure to terminate in the contralateral amygdala. While the main and accessory olfactory projections may converge in the cortical amygdaloid nucleus, the medial amygdaloid nucleus is connected exclusively with the accessory olfactory bulb.     

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.     

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.     

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.     

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.     

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.     

The main and the accessory olfactory systems interact in the control of mate recognition and sexual behavior

In the field of sensory perception, one noticeable fact regarding olfactory perception is the existence of several olfactory subsystems involved in the detection and processing of olfactory information. Indeed, the vomeronasal or accessory olfactory system is usually conceived as being involved in the processing of pheromones as it is closely connected to the hypothalamus, thereby controlling reproductive function. By contrast, the main olfactory system is considered as a general analyzer of volatile chemosignals, used in the context of social communication, for the identification of the status of conspecifics. The respective roles played by the main and the accessory olfactory systems in the control of mate recognition and sexual behavior are at present still controversial. We summarize in this review recent results showing that both the main and accessory olfactory systems are able to process partially overlapping sets of sexual chemosignals and that both systems support complimentary aspects in mate recognition and in the control of sexual behavior.     

Changes in granule cells of the ferret olfactory bulb associated with imprinting on prey odours

The maturation of the granule cells of the ferret olfactory bulb around the time of odour imprinting has been examined. Rapid Golgi impregnation studies revealed a temporal overshoot in the development of the spines on the external and internal dendrites of the granule cells. In contrast, the number of somatic spines decreased continuously. Electron microscopical examinations of the synaptic contacts in the external plexiform layer revealed that the time course of synapse and reciprocal synapse formation was similar to that of the formation of the spines on the external dendrites. The results were taken as evidence that both the Golgi and the electron microscopical investigations described the same developmental process of postnatal synaptic rearrangement.     

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.     

Odor vapor pressure and quality modulate local field potential oscillatory patterns in the olfactory bulb of the anesthetized rat

A central question in chemical senses is the way that odorant molecules are represented in the brain. To date, many studies, when taken together, suggest that structural features of the molecules are represented through a spatio-temporal pattern of activation in the olfactory bulb (OB), in both glomerular and mitral cell layers. Mitral/tufted cells interact with a large population of inhibitory interneurons resulting in a temporal patterning of bulbar local field potential (LFP) activity. We investigated the possibility that molecular features could determine the temporal pattern of LFP oscillatory activity in the OB. For this purpose, we recorded the LFPs in the OB of urethane-anesthetized, freely breathing rats in response to series of aliphatic odorants varying subtly in carbon-chain length or functional group. In concordance with our previous reports, we found that odors evoked oscillatory activity in the LFP signal in both the beta and gamma frequency bands. Analysis of LFP oscillations revealed that, although molecular features have almost no influence on the intrinsic characteristics of LFP oscillations, they influence the temporal patterning of bulbar oscillations. Alcohol family odors rarely evoke gamma oscillations, whereas ester family odors rather induce oscillatory patterns showing beta/gamma alternation. Moreover, for molecules with the same functional group, the probability of gamma occurrence is correlated to the vapor pressure of the odor. The significance of the relation between odorant features and oscillatory regimes along with their functional relevance are discussed.