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.     

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.     

Excitatory influence of the accessory olfactory bulb on tuberoinfundibular arcuate neurons of female mice and its modulation by oestrogen

The role of the accessory olfactory bulb in conveying pheromonal information to tuberoinfundibular arcuate neurons was examined electrophysiologically in chloral hydrate-anaesthetized, oestrogen (0.5 micrograms in silastic capsules)-treated and untreated ovariectomized Balb/c female mice. Electrical stimulation of the accessory olfactory bulb orthodromically excited part of tuberoinfundibular neurons which were antidromically stimulated from the median eminence and histologically verified as being located within the arcuate nucleus. No inhibitions followed accessory bulb stimulation. The excitatory response to accessory bulb stimulation was reversibly blocked by the local anaesthetic lignocaine infused into the amygdala. The percentage of tuberoinfundibular arcuate neurons responding to accessory bulb stimulation was significantly higher in oestrogen-treated than in untreated animals. There was no difference between the two groups for the antidromic activation threshold, spontaneous firing rate, absolute refractory period or frequency of successful antidromic propagation into the soma of tuberoinfundibular arcuate neurons. In oestrogen-treated preparations, tuberoinfundibular arcuate neurons responsive and unresponsive to accessory bulb stimulation could be distinguished by the frequency of successful antidromic propagation into the soma. These studies demonstrate that olfactory relay neurons in the accessory olfactory bulb act to enhance the activity of a subpopulation of tuberoinfundibular arcuate neurons via the amygdala and that this neural transmission is modulated by oestrogen.     

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.     

Neuropeptide Y modulates excitatory synaptic transmission in the olfactory bulb

Although the olfactory bulb contains one of the highest concentrations of neuropeptide Y in the CNS, its function in the bulb remains unclear. In this study, we used whole-cell electrophysiological, molecular, and primary culture techniques to investigate neuropeptide Y gene expression and neuromodulatory actions of neuropeptide Y on rat olfactory bulb neurons. Northern analysis showed that neuropeptide Y mRNA increases with animal age or time in culture, in a parallel manner. In electrophysiology experiments, agonists that activate neuropeptide Y receptors (whole neuropeptide Y) and the Y2 receptor subtype (neuropeptide Y 13-36) reduced spontaneous excitatory activity in bulb interneurons. In investigating potential presynaptic effects, both agonists reduced the amplitude of calcium channel currents in the presynaptic (mitral/tufted) cell. Also consistent with a presynaptic effect, both agonists reduced the frequency but not the amplitude of miniature excitatory postsynaptic currents (or "minis") in interneurons. In examining potential postsynaptic effects, both agonists slightly increased membrane resistance but had no effect on currents evoked by glutamate. Together, these data suggest that neuropeptide Y inhibits excitatory neurotransmission between olfactory bulb neurons via a presynaptic effect on transmitter (glutamate) release.     

Functional properties of dopaminergic neurones in the mouse olfactory bulb

The olfactory bulb of mammals contains a large population of dopaminergic interneurones within the glomerular layer. Dopamine has been shown both in vivo and in vitro to modulate several aspects of olfactory information processing, but the functional properties of dopaminergic neurones have never been described due to the inability to recognize these cells in living preparations. To overcome this difficulty, we used a transgenic mouse strain harbouring an eGFP (enhanced green fluorescent protein) reporter construct under the promoter of tyrosine hydroxylase, the rate-limiting enzyme for cathecolamine synthesis. As a result, we were able to identify dopaminergic neurones (TH-GFP cells) in living preparations and, for the first time, we could study the functional properties of such neurones in the olfactory bulb, in both slices and dissociated cells. The most prominent feature of these cells was the autorhythmicity. In these cells we identified five main voltage-dependent conductances: the two having largest amplitude were a fast transient Na ⁺ current and a delayed rectifier K ⁺ current. In addition, we observed three smaller inward currents, sustained by Na ⁺ ions (persistent type) and by Ca^{2 +} ions (LVA and HVA). Using pharmacological tools and ion substitution methods we showed that the pacemaking process is supported by the interplay of the persistent Na ⁺ current and of a T-type Ca^{2 +} current. We carried out a complete kinetical analysis of the five conductances present in these cells, and developed a Hodgkin-Huxley model of TH-GFP cells, capable of reproducing accurately the properties of living cells, including autorhytmicity, and allowing a precise understanding of the process.     

Responses of olfactory bulb neurones to the dipeptide carnosine

Summary Carnosine has been applied by microiontophoresis to identified neurones in the olfactory bulb of the rat from solutions of different pH. Although mainly without effect when compared with conventional excitatory and inhibitory amino acids, the dipeptide tended to be depressive when ejected as a cation and excitant when ejected as an anion. The results obtained are not in favour of this substance being an excitatory transmitter in the primary olfactory pathway.     

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.     

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.     

An experimental study of the projection of the amygdala to the accessory olfactory bulb and its relationship to the concept of a dual olfactory system

Summary The present anatomical findings point to the existence of a separate subdivision of the olfactory system whose connections are quite different from the principal part. The main olfactory bulb has olfactory afferents from the receptors of the general olfactory mucosa, while the accessory bulb has afferents from receptors in the vomeronasal organ. The main bulb projects to the olfactory tubercle and pyriform cortex, while the accessory bulb projects to the amygdala. In turn these areas are further related with the medial forebrain bundle in the case of the pyriform cortex and olfactory tubercle, and with the medial preoptic area and medial hypothalamus in the case of the amygdala. The main and accessory olfactory bulbs are further distinguished by their centrifugal connections, the main bulb receiving fibres from the olfactory tubercle passing through the lateral olfactory tract, and the accessory olfactory bulb receiving fibres from the amygdala through the stria terminalis. The centrifugals to the accessory olfactory bulb resemble those to the main bulb in that both appear to terminate upon granule cells, although further projections to the external plexiform layer or to the periglomerular region have not been demonstrated for the accessory bulb. By virtue of its neural connections the accessory olfactory system is ideally placed to mediate the effects of olfactory stimuli on reproduction.     

Oxytocin enhances presynaptic and postsynaptic glutamatergic transmission between rat olfactory bulb neurones in culture

Although oxytocin (OT) within the olfactory bulb has been implicated in maternal behaviour and olfactory recognition, the cellular mechanisms of action remain to be clarified. We examined the effects of OT on glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs) in cultured granule cells with the use of whole-cell patch-clamp recordings. OT reversibly increased both the frequency and amplitude of sEPSCs. The effects of OT on sEPSCs were blocked by the selective OT receptor antagonist desGly-NH(2)(9),d (CH(2))(5)-[Thy(Me)(2),Thr(4)]-ornithine vasotocin. OT had no detectable effect, however, on high voltage-activated Ca2+ currents in mitral/tufted cells, suggesting that OT acts presynaptically on step(s) in the release process downstream from calcium influx. OT augmented the membrane current in granule cells evoked by exogenous application of glutamate, indicating a postsynaptic site of action. These results indicate that OT facilitates sEPSCs in granule cells by both pre- and postsynaptic mechanisms.     

Influence of amygdala stimulation on the activity of identified tuberoinfundibular neurones in the rat hypothalamus.

1. Extracellular action potentials were recorded from 1246 neurones in the mediobasal hypothalamus of pentobarbitone or urethane anaesthetized male rats. Antidromic invasion from the surface of the median eminence identified 165 cells, located in the arcuate and ventromedial nuclei and the periventricular area, as tuberoinfundibular neurones. The majority (65%) of these cells displayed no spontaneous activity. 2. Latencies for antidromic invasion from median eminence ranged from 0-5 to 14-0 msec (mean 4-3 +/- 2-9 msec, S.D.). Conduction velocities for axons of tuberoinfundibular neurones were under 1-0 m/sec, and were slowest (under 0-2 m/sec) for those tuberoinfundibular neurones located in the arcuate nucleus. 3. Single 1 HZ stimulation of amygdala evoked short latency (mean 18-8 +/- 7-0 msec; n = 30) excitation of tuberoinfundibular neurones in the ventromedial nucleus. Stria terminalis stimulation evoked similar responses at a shorter latency (mean 10-2 +/- 3-5 msec; n = 12) from other ventromedial tuberoinfundibular neurones. Three of these neurones were also excited by amygdala stimulation at comparably longer latencies. In spontaneously active tuberoinfundibular cells, the initial excitation was followed by a decrease in excitability lasting 70-150 msec. Tuberoinfundibular neurones soldom followed orthodromic activation at frequencies beyond 30 HZ. 4. An initial decrease in activity at latencies of 18-40 msec (mean 29-2 +/- 10-2 msec) characterized the amygdala evoked responses from nine tuberoinfundibular neurones. A similar response from one other tuberoinfundibular neurone followed stria terminalis stimulation at a latency of 11 msec. Most of these tuberoinfundibular neurones were located in the dorsal part of the ventromedial nucleus. 5. Two ventromedial tuberoinfundibular neurones also displayed antidromic invasion from the amygdala; interaction studies suggested an axon collateral pathway that originated close to the origin of the axon. 6. Tuberoinfundibular neurones unresponsive to amygdala stimulation were usually located in the arcuate nucleus or periventricular area. 7. These results provide electrophysiological evidence for a direct influence of the amygdala on the activity of tuberoinfundibular neurones in the ventromedial hypothalamic nucleus. There are also data to indicate that some ventromedial tuberoinfundibular neurones have axon collaterals that return to the amygdala. These reciprocal connexions between the amygdala and ventromedial tuberoinfundibular neurones may indicate neural circuits important for extrahypothalamic modulation of adenohypophyseal secretion.     

Olfactory nerves and their excitatory action in the olfactory bulb

Summary Extracellular field potentials were recorded from the rabbit olfactory bulb following stimulation of the surface and the lateral olfactory tract. Experiments were also performed on olfactory nerve rootlets and a conduction velocity of .34 m/sec and an absolute refractory period of 2.7 msec were obtained. Stimulation of the surface of the bulb produced in a nearby surface recording electrode an initial sharp triphasic potential followed by two slow negative waves and a later positive wave. It was concluded that the initial triphasic potential represents the compound action potential of the olfactory nerves. The first slow negative wave is due to the postsynaptic excitation of the dendrites of secondary neurons by the olfactory nerves. The second negative wave is due to the synaptic depolarization of the granule cell processes by the dendrites of secondary neurons. The later positive wave represents the inhibitory postsynaptic potentials generated in the relay neurons by the granule cell processes.     

Dopamine depresses synaptic inputs into the olfactory bulb.

Both observations in humans with disorders of dopaminergic transmission and molecular studies point to an important role for dopamine in olfaction. In this study we found that dopamine receptor activation in the olfactory bulb causes a significant depression of synaptic transmission at the first relay between olfactory receptor neurons and mitral cells. This depression was found to be caused by activation of the D2 subtype of dopamine receptor and was reversible by a specific D2 receptor antagonist. A change in paired-pulse modulation during the depression suggests a presynaptic locus of action. The depression was found to occur independent of synaptic activity. These results provide the first evidence for dopaminergic control of inputs to the main olfactory bulb. The magnitude and locus of dopamine's modulatory capabilities in the bulb suggest important roles for dopamine in odorant processing.     

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.     

Response properties of neurones in the rat olfactory bulb to various parameters of odour stimulation

Carbon fibre filled microelectrodes were used for extracellular recording of spikes discharges from 345 neurones of the lateral aspect of the left olfactory bulb of the rat under pentobarbitone anaesthesia. The identity of the neurones was determined by antidromic activation of the cell axons by electrical stimuli applied to the lateral olfactory tract (LOT) using collision blocking and twin stimuli. The bulbar units were classified as mitral (269) or tufted (16) cells; units in a third class were not identified (60). Application of stimuli (21) was made over a period of 15 s while slowly increasing the odorant concentration monitored by a flame ionization device (FID). The stimulating efficiency reached a maximum at a nasal flow of about 8 cm3 s-1. The response type excitation was seen in 12.0% and inhibition in 11.8% of the 7245 odour presentations. Each response type was subdivided into four classes according to the reactions to ramp stimulation. In the majority of cases the spike discharge remained constant or followed the odour concentration for the entire stimulation period. In 18% of the cases, the response adapted quickly towards the spontaneous activity level.     

Responses of olfactory bulb neurones to odour stimulation of small nasal areas in the salamander

1. Previous experiments have suggested that one way odours may be discriminated is by different spatial patterns of response at both the olfactory bulb and receptor level. The present experiments were designed to test to what extent the position of an odour on the receptor mucosa can influence the activity of olfactory bulb neurones.2. To deliver odours to small areas on the nasal receptor sheet a new method for local application of odour was developed. The flow rate, concentration, and time course of the odour were controlled using the olfactometer described in the preceding paper.3. In thirty olfactory bulb units in the salamander it was found that if the response of a unit to odour delivered to the entire exposed receptor epithelium were suppression (type S), then the unit tended to be suppressed when odour was delivered to a number of localized epithelial regions. If the response were excitation (type E) to stimulation of the entire epithelium, then stimulation to only one or two localized regions would elicit the maximum response.4. Different epithelial regions had the ability to cause excitation in the same bulbar unit depending on the odour being used. Two odours, camphor and amyl acetate, elicited maximum excitation when they were presented to different mucosal areas. The areas at which presentation of these odours gave excitation were surprisingly consistent from unit to unit and animal to animal.5. The data presented here suggest the presence of restricted excitatory receptive fields for some olfactory bulb neurones for a particular odour.6. The presence of spatial response patterns using odour delivery to small nasal receptor regions and thus the presence of receptive fields is discussed with reference to bulbar neuronal circuitry.     

General organization of the perinatal and adult accessory olfactory bulb in mice

The vomeronasal system is currently a topical issue since the dual functional specificity, vomeronasal system-pheromones, has recently been questioned. Irrespective of the tools used to put such specificity in doubt, the diversity of the anatomy of the system itself in the animal kingdom is probably of more importance than has previously been considered. It has to be pointed out that a true vomeronasal system is integrated by the vomeronasal organ, the accessory olfactory bulb, and the so-called vomeronasal amygdala. Therefore, it seems reasonable to establish the corresponding differences between a well-developed vomeronasal system and other areas of the nasal cavity in which putative olfactory receptors, perhaps present in other kinds of mammals, may be able to detect pheromones and to process them. In consequence, a solid pattern for one such system in one particular species needs to be chosen. Here we report on an analysis of the general morphological characteristics of the accessory olfactory bulb in mice, a species commonly used in the study of the vomeronasal system, during growth and in adults. Our results indicate that the critical period for the formation of this structure comprises the stages between the first and the fifth day after birth, when the stratification of the bulb, the peculiarities of each type of cell, and the final building of glomeruli are completed. In addition, our data suggest that the conventional plexiform layers of the main olfactory bulb are not present in the accessory bulb.     

Electrophysiological studies of the tongue and accessory olfactory bulb in garter snakes

Tongue flicking in snakes transports environmental chemicals to the vomeronasal (Jacobson's) organ (VNO) which acts as a chemoreceptor. This paper provides the first electrophysiological evidence that the vomeronasal/accessory-olfactory system is activated following tongue flick. In acute experiments with partially anesthetized snakes, single units recorded in the accessory olfactory bulb (AOB) failed to show changes in firing rate following tongue flicks but changes in AOB activity did occur when cotton swabs soaked in prey extract were pressed to the roof of the mouth (where the VNO ducts open). In chronic experiments, multiunit recordings from electrodes implanted in the AOB of freely moving animals did reveal changes in neural activity following tongue flicks. EMG recordings from the hyoglossus (tongue retractor) muscle (and in one animal from the genioglossus [tongue extender] as well) were used to indicate precisely the time of tongue flicking. The techniques used in these experiments are detailed.