Impairment of olfactory memory by local infusions of non-selective excitatory amino acid receptor antagonists into the accessory olfactory bulb

Female mice form a long-term olfactory memory to the pheromones of the male that mates with them. This memory is dependent on neural mechanisms within the accessory olfactory bulb. In this study we show that localized infusions of the excitatory amino acid receptor blocker, gamma-D-glutamylglycine, into the accessory olfactory bulb prevents memory formation. This is in marked contrast to the effects of infusions of the specific N-methyl-D-aspartate receptor antagonists, D-2-amino-5-phosphonovaleric acid and MK 801, which are without effect on memory formation. Excitatory amino acid receptor blockade by localized infusion of these drugs into the accessory olfactory bulb induced seizures. This paradoxical effect could only be due to disinhibition of granule cell GABAergic inhibitory feedback to the mitral cell. This was confirmed by the pregnancy blocking effect of these drugs, an event which also occurs with bicuculline infusions into the accessory olfactory bulb. These findings strongly implicate excitatory amino acid receptors in memory formation to the pheromones of the mating male and localize the mechanism to the reciprocal dendro-dendritic synapse between mitral and granule cells.     

The effect of microinfusions of drugs into the accessory olfactory bulb on the olfactory block to pregnancy

Female mice which have mated and are subsequently exposed to the odour (pheromones) of a strange male undergo hormonal changes resulting in a block to their pregnancy. The fact that the stud male's odours can also block pregnancies, that is other than his own, implies the formation of a memory or some form of recognition process by the female for this male's pheromones at the time of mating. The purpose of this study was to evaluate the effect of microinfusions of drugs which interfere with neural transmission, into the accessory olfactory bulbs. This was carried out immediately after mating over a 4-h period during which the "memory" to the stud male's pheromones is formed. Infusions of the alpha-blocker, phentolamine, blocked the formation of the olfactory memory, while the GABA receptor blocker, bicuculline, itself blocked pregnancy, but was without effect on memory formation. Protein synthesis inhibition or calpain inactivation in the accessory bulb was without effect on memory formation at any of the doses used. These studies demonstrate that GABAergic transmitter blockade in the accessory olfactory bulb at the time of mating can prevent subsequent blastocyst implantation some 3 days later, while alpha-noradrenergic blockade can prevent the formation of an olfactory memory to the stud male.     

The expression of the immediate-early genes c-fos, egr-1 and c-jun in the accessory olfactory bulb during the formation of an olfactory memory in mice.

Female mice form a memory for the pheromones of the male with which they mate. It has been proposed that the site of the synaptic changes underlying this memory is the accessory olfactory bulb, at the first level of the accessory olfactory system. In this study we have examined the expression of the immediate-early genes c-fos, c-jun and egr-1 in the mitral and granule cells of the accessory olfactory bulb immediately after mating, during the period of memory formation. Transient increases were seen in the number of granule cell nuclei expressing c-fos and the number of granule and mitral cell nuclei expressing egr-1, during the period of memory formation. No changes were observed in the expression of c-jun during this period. The increase in the number of cells expressing c-fos and egr-1 required the association of mating and pheromonal exposure, conditions also required for memory formation. Large increases in the number of mitral and granule cell nuclei expressing c-fos and egr-1 were also observed following the infusion of the drug bicuculline into the accessory olfactory bulb in the absence of mating. This procedure has previously been shown to result in the formation of a nonspecific memory for male pheromones. These results associate the expression of c-fos and egr-1 in the accessory olfactory bulb with the conditions required for the formation of an olfactory memory for male pheromones.     

Differential plasticity of microglial cells in the rostrocaudal neuraxis of the accessory olfactory bulb of female mice following mating and stud male exposure

The formation of an olfactory recognition memory by female mice for the stud male pheromones requires two fundamental conditions: incidence of mating and retention of the stud male with the female for a critical 6h interval following mating. This biologically critical recognition memory results from plasticity of reciprocal dendrodendritic synapses in the accessory olfactory bulb (AOB). In this study, a microglia marker antibody (ionized calcium-binding adaptor protein, Iba1) was used to determine how mating and stud pheromones affect microglia in the AOB rostrocaudal axis in female mice. The results showed that compared with estrus and mating only, mating and pheromone exposure significantly increased Iba1 immunoreactivity in the AOB evidenced by increased complexity of ramified microglial processes characteristic of resting microglial morphological phenotype, particularly in the rostral AOB. The density of Iba1 staining after mating and stud pheromone exposure was higher in the rostral - compared to caudal - AOB and was most prevalent in the external plexiform layer, the site of reciprocal mitral-granule dendrodendritic synapses. While cells with activated phenotype were observed in caudal AOB during estrus, mating/pheromone exposure appeared to induce a morphological transformation to the resting microglia phenotype. Since previous evidence implicate the rostral AOB in processing pheromonal signals and microglial cells monitor active synapses, these observations have important functional implications for a potential role for microglia in processing pheromonal signals in the AOB during the formation of olfactory memory.     

Hippocampal lesions are without effect on olfactory memory formation in the context of pregnancy block

Female mice which have mated and are subsequently exposed to male urine odours (pheromones), which differ from those of the male that mated (stud male), undergo hormonal changes resulting in a block to pregnancy. Since the stud male's odour blocks the pregnancy of females other than those he mated, this would suggest that a memory specific for this male's odour is established at the time of mating. Hippocampal lesions, when made prior to mating, did not disrupt memory formation to the odour of the stud male. This male's odour did not block pregnancy, while changing odour to that of the strange male did block pregnancy. In order to establish the functional effectiveness of these hippocampal lesions, olfactory discriminations were examined in a modified T-maze. The odour discrimination was urine-soiled bedding taken from a cage of different strain males versus same strain bedding. After 35 trials, neither control nor lesioned females were performing above chance level (50% correct responses), indicating the difficulty in cognitively learning this discrimination. These female mice were therefore trained to distinguish a novel odour (butyl acetate) from familiar strain urine-soiled bedding. Hippocampal-lesioned females were slower to acquire this discrimination and did not perform above chance level after 35 trials, in contrast to the 70% success of sham-lesioned females. Hence, hippocampal lesions, while producing a deficit in olfactory learning in the maze test, are without effect on the formation or retrieval of the specific olfactory memory formed for the stud male at mating, as revealed from pregnancy block tests.     

Hormonal enhancement of neurogenesis and its relationship to the duration of olfactory memory

On mating with a stud male, virgin female mice from an olfactory memory to this male which is essential to their reproductive success. Failure to form this memory results in the stud male being treated as strange, and hence, his pheromones block pregnancy. This study investigates the duration of the olfactory memory, and the factors which determine this. The results show that a single prolonged exposure to the male during mating forms a life-long olfactory memory trace unless pregnancy ensues. In the event of pregnancy the olfactory memory fades significantly faster, an effect which can be replicated by implants of oestradiol in non-pregnant females. Anatomical studies indicate that neurogenesis of the vomeronasal receptors is enhanced during pregnancy, an event which we interpret as being important for the duration of this olfactory memory.     

Properties of reciprocal synapses in the mouse accessory olfactory bulb.

Reciprocal dendrodendritic synapses between mitral and granule cells in the accessory olfactory bulb have been implicated in a specialized form of olfactory learning in mice, in which a female forms a memory to the pheromonal signal of the male that mates with her. Relatively little is known, however, about the mechanism of synaptic transmission at the reciprocal synapses. We analyzed synaptic currents generated in accessory olfactory bulb mitral cells in slice preparations with the patch-clamp technique in nystatin-perforated whole-cell configuration. A brief (5-20-ms) depolarizing voltage step from -70 to 0 mV applied to a single mitral cell evoked GABA(A) receptor-mediated inhibitory postsynaptic currents. The inhibitory postsynaptic currents persisted in the presence of tetrodotoxin, indicating that the inhibitory postsynaptic current in mitral cells can be elicited through purely dendritic interactions. The inhibitory postsynaptic currents were greatly enhanced by washout of extracellular Mg(2+). In Mg(2+)-free solution, the N-methyl-D-aspartate (NMDA) receptor antagonist D,L-2-amino-5-phosphonovaleric acid greatly reduced the inhibitory postsynaptic currents, whereas the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX) slightly reduced them.These data demonstrate that NMDA receptors play an important role in the generation of dendrodendritic inhibition in mitral cells of the mouse accessory olfactory bulb.     

Expression and regulation of the immediate-early gene product Arc in the accessory olfactory bulb after mating in male rat

Recent studies of the accessory olfactory bulb have shown that the expression of immediate-early genes, e.g., c-fos, c-jun and egr-1, can be used as a marker of neuronal activity in response to pheromonal cues. In this study, we analyzed the expression pattern, in response to mating, of the novel immediate-early gene product Arc (an activity-regulated cytoskeleton-associated protein). Arc is hypothesized to play a role in activity-dependent neuronal plasticity in the hippocampus. In a control group of male rats, only a small number of Arc-immunoreactive cells were observed in the accessory olfactory bulb. In a mating group, however, a marked increase in the number of Arc-immunoreactive cells was observed only in the granule cell layer of the accessory olfactory bulb. The increase in the number of Arc-immunoreactive cells after mating was similar to that observed for other immediate-early genes. However, for the mating group, the increase in Arc-positive cells was limited to the granule cell layer. Granule cells have been shown to exhibit a strong synaptic plasticity in response to pheromonal stimulation.From these findings we suggest that Arc plays an important role in neuronal plasticity in the accessory olfactory bulb.     

Oxytocin facilitates the induction of long-term potentiation in the accessory olfactory bulb

When female mice are mated, they form a memory to the pheromonal signal of their male partner. Several lines of evidence indicate that the neural changes underlying this memory occur in the accessory olfactory bulb (AOB) at the first stage of the vomeronasal system. The formation of this memory depends on the mating-induced release of noradrenaline in the AOB. In addition to noradrenaline, the neuropeptide oxytocin (OT) is also released within the central nervous system during mating. Because OT has been implicated in social memory and its receptors are expressed in the AOB, we hypothesized that OT might promote the strength of synaptic transmission from mitral to granule cells in the AOB. To test this hypothesis, we analyzed the lateral olfactory tract-evoked field potential that represents the granule cell response to mitral cell activation and its plasticity in parasagittal slices of the AOB. Of the 10-, 20-, 50-, and 100-Hz stimulations tested, the 100-Hz stimulation was optimal for inducing long-term potentiation (LTP). OT paired with 100-Hz stimulation that only produced short-term potentiation enhanced LTP induction in a dose-dependent manner. OT-paired LTP was blocked by both the selective OT antagonist desGly-NH₂,d(CH₂)₅[Tyr(Me)²,Thr⁴]-ornithine vasotocin and the N-methyl-d-aspartate (NMDA) receptor antagonist dl-2-amino-5-phosphonovaleric acid. These results indicate that OT can function as a gate to modulate the establishment of NMDA receptor-dependent LTP at the mitral-to-granule cell synapse in the AOB.     

Synaptology of the olfactory bulb of an elasmobranch fish, Sphyrna tiburo

The ultrastructure of the elasmobranch olfactory bulb was examined in order to determine the synaptology of the olfactory circuitry in the bonnethead shark, Sphyrna tiburo. The compartmentalization of the bulb, together with the lack of mitral cell basal dendrites, suggests a different way of performing lateral communication between mitral cells of the olfactory bulb. The results show that granule cells assume an important role by directly interlinking mitral cells. A corollary of this is the segregation of the input onto the mitral cell dendritic arborization: afferent fibers synapse onto the intraglomerular mitral terminals, whereas most local circuit interactions utilize extraglomerular synapses located on the shafts and the somas of the mitral dendrites. Therefore, the elasmobranch synaptic pattern is different from that of higher vertebrates; This might represent the use of a different neural route to achieve the same processing task.     

GABAergic mechanisms are involved in the control of tuberoinfundibular arcuate neurons by the accessory olfactory bulb

In this study we examined electrophysiologically the involvement of the intrinsic GABAergic system of the accessory olfactory bulb (AOB) in controlling the activity of tuberoinfundibular (TI) arcuate neurons in anaesthetized female mice. Local infusions of the γ-aminobutyric acid-A (GABA_A) receptor antagonist, bicuculline into the AOB enhanced the spontaneous firing activity of TI arcuate neurons with excitatory inputs from the AOB. This finding reveals a neural mechanism responsible for the pregnancy blocking effect of this drug in freely behaving female mice and, taken together with the cytoarchitecture of the AOB, suggests that the reciprocal dendrodendritic interaction between mitral cells and GABAergic granule cells in the AOB is critical to control of AOB output to TI arcuate neurons as part of the final common pathway of the accessory olfactory system.     

Synaptology of the olfactory bulb of an elasmobranch fish, Sphyrna tiburo

The ultrastructure of the elasmobranch olfactory bulb was examined in order to determine the synaptology of the olfactory circuitry in the bonnethead shark, Sphyrna tiburo. The compartmentalization of the bulb, together with the lack of mitral cell basal dendrites, suggests a different way of performing lateral communication between mitral cells of the olfactory bulb. The results show that granule cells assume an important role by directly interlinking mitral cells. A corollary of this is the segregation of the input onto the mitral cell dendritic arborization: afferent fibers synapse onto the intraglomerular mitral terminals, whereas most local circuit interactions utilize extraglomerular synapses located on the shafts and the somas of the mitral dendrites. Therefore, the elasmobranch synaptic pattern is different from that of higher vertebrates; This might represent the use of a different neural route to achieve the same processing task.     

Patterns of expression of the immediate-early gene egr-1 in the accessory olfactory bulb of female mice exposed to pheromonal constituents of male urine

Male mice excrete large quantities of major urinary proteins that have been proposed to have an important pheromonal role either alone or by way of their bound ligands. We have found that these major urinary proteins are not only likely to mediate the pregnancy blocking effects of male urine, but that they also convey the strain recognition signal of the male pheromone. Recent molecular biological investigations have characterized two classes of pheromonal receptor in the vomeronasal organ that appear to project separately to anterior and posterior regions of the accessory olfactory bulb. However, it is not known whether these separate pathways handle fundamentally different types of pheromonal information. We have attempted to investigate this question using the expression of the immediate-early gene egr-1 as a marker for activity of neurons in the accessory olfactory bulb of female mice in response to putative pheromonal constituents. Exposure to 2,3 dihydro-exo-brevicomin and 2-sec-butyl-4,5-dihydro-thiazole, the main ligands bound to the major urinary proteins, elicited expression of egr-1 in clusters of presumed mitral neurons at the medial and lateral margins of the posterior accessory olfactory bulb. Whole male urine and a preparation of major urinary proteins that had been stripped of their ligands induced egr-1 expression in mitral cells of the anterior half of the accessory olfactory bulb in addition to the posterior clusters.

This would suggest that the anterior and posterior halves of the accessory olfactory bulb are processing different aspects of the male pheromone signal with the anterior region, which responds preferentially to major urinary proteins, being principally concerned with the strain recognition component.     

Analysis of synaptic events in the mouse accessory olfactory bulb with current source-density techniques.

The accessory olfactory bulb of the mouse was studied by current source-density analysis of field potentials to determine the laminar and temporal distribution of synaptic currents evoked by electrical stimulation of the vomeronasal organ. The one-dimensional current source-density analysis revealed two major spatially and temporally distinct inward membrane currents (sinks): one in the glomerular layer and the other in the external plexiform layer. The glomerular layer sink preceded the external plexiform layer sink by a mean of 5.5 ms. Local infusions of the broad-spectrum excitatory amino acid antagonist, kynurenate, into the accessory olfactory bulb blocked the external plexiform layer sink without an obvious effect on the glomerular layer sink. The selective non-N-methyl-D-aspartate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione produced a dose-dependent blockade of the external plexiform layer sink, whereas the selective N-methyl-D-aspartate receptor antagonist D-2-amino-5-phosphonovalerate was without effect. These results, taken together with the cytoarchitecture of the accessory olfactory bulb, suggest that the glomerular layer sink results mainly from synaptic excitation evoked in the glomerular dendritic branches of mitral cells by the vomeronasal afferent fibres and the external plexiform layer sink mainly from non-N-methyl-D-aspartate receptor-mediated synaptic excitation in the peripheral processes of granule cells via the mitral to granule cell dendrodendritic synapse.     

Induction of pseudopregnancy in pregnancy-blocked mice by re-exposure to stud males

Re-exposure to stud males beginning on day 4 post coitum induced pseudopregnancy in a large proportion of female mice whose pregnancies were terminated by a single injection of 0.5 mg of bromocriptine on day 1 post coitum. By contrast, exposure to alien or strange males was ineffective in inducing pseudopregnancy in pregnancy-blocked females. The results suggest that the newly inseminated female retains the memory of the stud male for some days after mating. Activation of the 'memory' by pheromones of the stud males exerts a luteotrophic effect in pregnancy-blocked females resulting in the incidence of pseudopregnancy. This may account for the inability of the stud male to block pregnancy in his coital partner and also for the inability of alien males to block implantation in newly inseminated females housed with stud males.     

Functional synapse formation between rat olfactory receptor neurons and olfactory bulb neurons in vitro

To explore the mechanism of the synapse formation between olfactory receptor neurons and neurons in the olfactory bulb, blocks of olfactory epithelium and slices of olfactory bulb were cocultured by a modified slice culture method. After 4 days in culture, neuron specific fibers from the olfactory epithelium block extending to the olfactory bulb slice were observed. After approximately 20 days in culture, application of forskolin, which induces excitatory responses in olfactory receptor neurons, to the epithelium induced inward current responses in olfactory bulb neurons under the voltage clamp conditions, indicating that functional synapses between olfactory receptor neurons and olfactory bulb neurons had been established in vitro conditions.     

Role of the brain and accessory olfactory system in the block to pregnancy in mice

Selective lesions have been made to the receptors in the main and accessory olfactory systems. In the absence of the accessory receptors, female mice are not able to show a neuroendocrine response to male pheromones but are able to detect male odours which induce the response. In the absence of main olfactory receptors, such discrimination of male odours is not possible, but the neuroendocrine response resulting in a block to pregnancy is maintained.These experiments suggest that cognitive aspects of olfaction are not essential for pregnancy block to occur and that in mice the dual olfactory systems are functionally as well as anatomically distinct. Moreover, strain recognition can occur at the level of the accessory olfactory system without the female being able to display a behavioural awareness of this ‘recognition’.     

Effect of bilateral accessory olfactory bulb lesions on volatile urinary odor discrimination and investigation as well as mating behavior in male mice

Previous research raises the possibility that urinary volatiles from estrous female mice activate mitral cells in the accessory olfactory bulb (AOB) of male mice following detection via the main olfactory epithelium as opposed to the vomeronasal organ. We asked whether bilateral lesions of the AOB would disrupt the ability of male mice to discriminate between urinary volatiles from mice of different sexes or endocrine states, or affect their interest in investigating these odors when they were presented sequentially in home-cage habituation/dishabituation tests. Males with either partial or complete bilateral lesions of the AOB resembled sham-operated control males in their ability to discriminate between ovariectomized and estrous female urinary volatiles as well as between male and estrous female urinary volatiles. However, males with either complete or partial AOB lesions spent significantly less time than sham-operated control males investigating urinary volatiles from estrous females, especially during tests when the alternative stimulus presented was male urine. Placement of AOB lesions failed to disrupt males' mating performance. Our results suggest that the incentive value of opposite-sex (female) volatile urinary odors which are initially detected by the main olfactory system is enhanced when they are further processed by the male's AOB.     

Functional synapse formation between cultured rat accessory olfactory bulb neurons and vomeronasal pockets

To investigate the interaction between vomeronasal receptor neurons and accessory olfactory bulb neurons during pheromonal signal processing and specific synapse formation, partially dissociated rat vomeronasal receptor neurons were co-cultured with accessory olfactory bulb neurons. Between 7 and 14 days in co-culture, a few bundles of fibers from a spherical structure, termed the vomeronasal pocket, of cultured vomeronasal receptor neurons extended to the accessory olfactory bulb neurons. An optical recording of the intracellular Ca(2+) concentration was used to monitor the synaptic activation of cultured accessory olfactory bulb neurons. Electrical stimulation of the vomeronasal pocket between 7 and 14 days in co-culture had no effects on most of the cultured neurons tested, although it occasionally evoked weak responses in a small number of neurons. In contrast, vomeronasal pocket stimulation after 21 days in co-culture evoked clear calcium transients in a substantial number of cultured accessory olfactory bulb neurons. These responses of accessory olfactory bulb neurons were reversibly suppressed by the application of 6-cyano-7-nitroquinoxaline-2,3-dione; the calcium transients disappeared in most of the neurons and were diminished in the others. The application of d-2-amino-5-phosphonopentanoic acid partially affected the calcium transients, but blocked spontaneous calcium increases, which were observed repeatedly in accessory olfactory bulb-alone cultures. The application of both 6-cyano-7-nitroquinoxaline-2,3-dione and d-2-amino-5-phosphonopentanoic acid completely blocked the evoked calcium transients. These results suggest that functional glutamatergic synapses between vomeronasal receptor neurons and accessory olfactory bulb neurons were formed at around 21 days in co-culture.     

Axons from the olfactory bulb transplanted into the cerebellum form synapses with dendrites in the granular layer, as demonstrated by mouse allelic form of Thy-1 and electron microscopy.

Transplant-to-host axon projection and synapse formation from the olfactory bulb (OB) transplant to the host cerebellum were studied using the mouse allelic form of Thy-1 (AKR strain of Thy-1.1 was used as host and BALB/c strain of Thy-1.2 as graft). Thy-1.2 immunohistochemical and ultrastructural examination showed that numerous OB axons elongate into the cerebellar granular layer and form asymmetrical synapses there with dendrites of host origin (perhaps of the host granule neuron). Factors which support this mismatched synapse formation are discussed.