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Various neural systems cooperate in feeding behaviour, and olfaction plays crucial roles in detecting and evaluating food objects. While odour-mediated feeding behaviour is highly adaptive and influenced by metabolic state, hedonic cues and learning processes, the underlying mechanism is not well understood. Feeding behaviour is regulated by orexigenic and anorexigenic neuromodulatory molecules. However, knowledge of their roles especially in higher olfactory areas is limited. Given the potentiation of feeding behaviour in hunger state, we systemically examined the expression of feeding-related neuromodulatory molecules in food-restricted mice through quantitative PCR, in the olfactory bulb (OB), olfactory tubercle (OT), and remaining olfactory cortical area (OC). The OT was further divided into attraction-related anteromedial, aversion-related lateral and remaining central regions. Examination of 23 molecules including neuropeptides, opioids, cannabinoids, and their receptors as well as signalling molecules showed that they had different expression patterns, with many showing elevated expression in the OT, especially in the anteromedial and central OT. Further, in mice trained with odour-food association, the expression was significantly altered and the increase or decrease of a given molecule varied among areas. These results suggest that different olfactory areas are regulated separately by feeding-related molecules, which contributes to the adaptive regulation of feeding behaviour.

The formation of mate recognition memory in mice is associated with neural changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cell (MC) projection neurons and GABAergic granule cell (GC) interneurons in the accessory olfactory bulb (AOB). Although noradrenaline (NA) plays a critical role in the formation of the memory, the mechanism by which it exerts this effect remains unclear. Here we used extracellular field potential and whole-cell patch-clamp recordings to assess the actions of bath-applied NA (10 µM) on the glutamatergic transmission and its plasticity at the MC-to-GC synapse in the AOB. Stimulation (400 stimuli) of MC axons at 10 Hz but not at 100 Hz effectively induced N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP), which exhibited reversibility. NA paired with subthreshold 10-Hz stimulation (200 stimuli) facilitated the induction of NMDA receptor-dependent LTP via the activation of α2-adrenergic receptors (ARs). We next examined how NA, acting at α2-ARs, facilitates LTP induction. In terms of acute actions, NA suppressed GC excitatory postsynaptic current (EPSC) responses to single pulse stimulation of MC axons by reducing glutamate release from MCs via G-protein coupled inhibition of calcium channels. Consequently, NA reduced recurrent inhibition of MCs, resulting in the enhancement of evoked EPSCs and spike fidelity in GCs during the 10-Hz stimulation used to induce LTP. These results suggest that NA, acting at α2-ARs, facilitates the induction of NMDA receptor-dependent LTP at the MC-to-GC synapse by shifting its threshold through disinhibition of MCs.

Background. We have observed changes in body reactions during cooking, which is one of the treatment modalities used in occupational therapy. The perception of food-related odors during cooking may have behavioral effects on human activities through the activation of appetitive motivation. Objectives. We investigated whether odor components contained in seasonings could facilitate the human motor system and the specificity of this effect. Methods. The subjects were 72 healthy adults, randomly assigned to a water exposure group, a phenylethyl alcohol (PEA, pleasant rose-like odor) exposure group, and a Japanese soy sauce (Koikuchi Shoyu) exposure group (n=24 each). The subjects’ olfactory sense was stimulated by their sniffing of three different test tubes containing 5 ml of water, PEA, or Japanese soy sauce for 20 sec while they were seated. The modified Functional Reach Test (mFRT), which mimics a functional activity that is required in daily living and assesses a reliable measure of sitting balance, was performed prior to and immediately after the sniffing. Results. Sniffing the soy sauce increased the subjects’ mFRT scores. This facilitation effect was odorant-specific and was absent when the subjects were presented with water or PEA. Conclusions. Cooking interventions are aimed at improving tool-handling skills such as using knives and chopsticks. The results indicate that treatment interventions using odors of seasonings would be effective for improving subjects’ physical functions.

Olfaction plays an important role in social recognition in most mammals. Central arginine vasopressin (AVP) plays a role in this olfaction-based recognition. The high level of expression of AVP receptors in the accessory olfactory bulb (AOB) at the first relay of the vomeronasal system highlights the importance of AVP signaling at this stage. We therefore analyzed the effects of AVP on the synaptic plasticity of glutamatergic transmission from mitral cells to granule cells in AOB slices from male mice. To monitor the strength of the glutamatergic transmission, we measured the maximal initial slope of the lateral olfactory tract-evoked field potential, which represents the granule cell response to mitral cell activation. AVP paired with 100-Hz stimulation that only produced short-term potentiation enhanced the induction of long-term potentiation (LTP) in a dose-dependent manner. AVP-paired LTP was blocked by the selective AVP receptor 1a (AVPR1a) antagonist, d(CH2)5[Tyr(Me)2]AVP (Manning compound), but not by the AVPR1b antagonist SSR149415, and it was mimicked by the selective AVPR1a agonist [Phe2, Ile3, Orn8]-vasopressin. We further examined the effect of AVP on the reciprocal transmission between mitral and granule cells by stimulating a mitral cell and recording the evoked inhibitory postsynaptic currents (IPSCs) from the same cell using conventional whole-cell patch-clamp techniques. AVP reduced the reciprocal IPSCs triggered by endogenous glutamate release from the excited mitral cell. These results suggest that AVP promotes the induction of LTP at the mitral-to-granule cell synapse via the activation of AVPR1a through an as-yet-to-be-determined mechanism in the AOB of male mice.

The formation of mate recognition memory in mice is associated with neural changes at the reciprocal dendrodendritic synapses between glutamatergic mitral cell (MC) projection neurons and GABAergic granule cell (GC) interneurons in the accessory olfactory bulb (AOB). Although noradrenaline (NA) plays a critical role in the formation of the memory, the mechanism by which it exerts this effect remains unclear. Here we used extracellular field potential and whole-cell patch-clamp recordings to assess the actions of bath-applied NA (10 µM) on the glutamatergic transmission and its plasticity at the MC-to-GC synapse in the AOB. Stimulation (400 stimuli) of MC axons at 10 Hz but not at 100 Hz effectively induced N-methyl-d-aspartate (NMDA) receptor-dependent long-term potentiation (LTP), which exhibited reversibility. NA paired with subthreshold 10-Hz stimulation (200 stimuli) facilitated the induction of NMDA receptor-dependent LTP via the activation of α2-adrenergic receptors (ARs). We next examined how NA, acting at α2-ARs, facilitates LTP induction. In terms of acute actions, NA suppressed GC excitatory postsynaptic current (EPSC) responses to single pulse stimulation of MC axons by reducing glutamate release from MCs via G-protein coupled inhibition of calcium channels. Consequently, NA reduced recurrent inhibition of MCs, resulting in the enhancement of evoked EPSCs and spike fidelity in GCs during the 10-Hz stimulation used to induce LTP. These results suggest that NA, acting at α2-ARs, facilitates the induction of NMDA receptor-dependent LTP at the MC-to-GC synapse by shifting its threshold through disinhibition of MCs.

Epigenetic mechanisms play an important role in memory formation and synaptic plasticity. Specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. In keeping with the classical conditioning paradigm, young rats have been shown to exhibit aversion to an odor stimulus initially presented during foot shock. We previously showed that synaptic plasticity at the dendrodendritic synapses between mitral and granule cells in the olfactory bulb (OB) underlies this aversive olfactory learning. However, the epigenetic mechanisms involved are not well characterized. Therefore, we examined whether intrabulbar infusion of trichostatin A (TSA), a histone deacetylase inhibitor, facilitates olfactory learning in young rats. TSA infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner and prolonged the learned aversion. Western blot and immunohistochemical analyses showed that the level of histone H4 acetylation significantly increased until 4 h after odor-shock training in both mitral and granule cells in the OB, whereas histone H3 acetylation returned to the control level at 2 h after the training. We also obtained evidence that TSA infusion elevated acetylation of histone H4 or H3. Furthermore, in vitro electrophysiological analysis using slices of the OB revealed that application of TSA significantly enhanced the long-term potentiation induced in synaptic transmission from mitral to granule cells at dendrodendritic synapses. Taken together, these results provide evidence that histone H4 and H3 acetylation in the OB is an epigenetic mechanism associated with aversive olfactory learning in young rats.

Tunicamycin (TM) induces endoplasmic reticulum (ER) stress and inhibits N-glycosylation in cells. ER stress is associated with neuronal death in neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease, and most patients complain of the impairment of olfactory recognition. Here we examined the effects of TM on aversive olfactory learning and the underlying synaptic plasticity in the main olfactory bulb (MOB). Behavioral experiments demonstrated that the intrabulbar infusion of TM disabled aversive olfactory learning without affecting short-term memory. Histological analyses revealed that TM infusion upregulated C/EBP homologous protein (CHOP), a marker of ER stress, in the mitral and granule cell layers of MOB. Electrophysiological data indicated that TM inhibited tetanus-induced long-term potentiation (LTP) at the dendrodendritic excitatory synapse from mitral to granule cells. A low dose of TM (250nM) abolished the late phase of LTP, and a high dose (1μM) inhibited the early and late phases of LTP. Further, high-dose, but not low-dose, TM reduced the paired-pulse facilitation ratio, suggesting that the inhibitory effects of TM on LTP are partially mediated through the presynaptic machinery. Thus, our results support the hypothesis that TM-induced ER stress impairs olfactory learning by inhibiting synaptic plasticity via presynaptic and postsynaptic mechanisms in MOB.

The throughput of information from the accessory olfactory bulb (AOB) to downstream structures is controlled by reciprocal dendrodendritic inhibition of mitral cells by granule cells. Given the high expression levels of mGluR2, a metabotropic glutamate receptor, in the AOB and the fact that the activation of mGluR2 permits the formation of a specific olfactory memory, we reasoned that mGluR2 might play an important role in regulating dendrodendritic inhibition. To test this hypothesis, we examined the effects of pharmacological and genetic manipulations of mGluR2 on synaptic responses measured from mitral or granule cells in slice preparations from 23- to 36-day-old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from -70 to 0 mV or a threshold current stimulus adjusted to elicit action potential(s) was applied to a mitral cell using either a nystatin-perforated or conventional whole-cell configuration. We found that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG-IV [(2S,1'R,2'R,3'R)-2-(2,3-dicarboxycyclopropyl)glycine], suppressed, whereas the mGluR2/mGluR3 antagonist LY341495 [(αS)-α-amino-α-[(1S,2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid] enhanced dendrodendritic inhibition. Genetic ablation of mGluR2 markedly impaired the effects of DCG-IV and LY341495 on dendrodendritic inhibition. DCG-IV reduced both the frequency and the amplitude of spontaneous miniature excitatory postsynaptic currents recorded from granule cells. Additionally, DCG-IV inhibited high-voltage-activated calcium currents in both mitral and granule cells. These results suggest that mGluR2 reduces dendrodendritic inhibition by inhibiting synaptic transmission between mitral cells and granule cells in the AOB.

Propylene glycol (PG) is commonly used as a solvent for odorous chemicals employed in studies of the olfactory system because PG has been considered to be odorless for humans and other animals. However, if laboratory rats can detect the vapor of PG and if exposure to this influences behaviors, such effects might confound data obtained from experiments exposing conscious rats to odorants dissolved in PG. Therefore, we examined this issue using differences in the acoustic startle reflex (ASR) as an index. We also conducted a habituation/dishabituation test to assess the ability of rats to detect the vapor of PG. In addition, we observed Ca(2+) responses of vomeronasal neurons (VNs) in rats exposed to PG using the confocal Ca(2+)-imaging approach. Pure PG vapor significantly enhanced the ASR at a dose of 1 x 10(-4) M, which was much lower than the dose for efficiently detecting. In Ca(2+) imaging, VNs were activated by PG at a dose of 1 x 10(-4) M or lower. These results suggest that PG vapor acts as an aversive stimulus to rats at very low doses, even lower than those required for its detection, indicating that we should consider such effect of PG when it is employed as a solvent for odorants in studies using conscious rats. In addition, our study suggests that some non-pheromonal volatile odorants might affect animal behaviors via the vomeronasal system.

When female mice are mated, they form a memory to the pheromonal signal of their male partner. The neural changes underlying this memory occur in the accessory olfactory bulb, depend upon vaginocervical stimulation at mating and involve changes at the reciprocal synapses between mitral and granule cells. However, the action of vaginocervical stimulation on the reciprocal interactions between mitral and granule cells remains to be elucidated. We have examined the effects of vaginocervical stimulation on paired-pulse depression of amygdala-evoked field potentials recorded in the external plexiform layer of the accessory olfactory bulb (AOB) and the single-unit activity of mitral cells antidromically stimulated from the amygdala in urethane-anaesthetized female mice. Artificial vaginocervical stimulation reduced paired-pulse depression (considered to be due to feedback inhibition of the mitral cell dendrites from the granule cells via reciprocal dendrodendritic synapses) recorded in the AOB external plexiform layer. As would be expected from this result, vaginocervical stimulation also enhanced the spontaneous activity of a proportion of the mitral cells tested. These results suggest that vaginocervical stimulation reduces dendrodendritic feedback inhibition to mitral cells and enhances their activity.

Turtle vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the whole-cell and cell-attached configurations. The mean resting potential was -48, and the response to an injected current step consisted of either a single spike or a train of spikes. An injected current of 3–30 pA was required to depolarize the neuron to spike threshold near -50 mV. Voltage-clamped vomeronasal receptor neurons displayed transient inward currents followed by sustained outward currents in response to depolarizing voltage steps. In cell-attached recordings, 10 microM forskolin added to the bath caused a transient increase of spike rate. Intracellular application of cAMP evoked ann inward current in a dose-dependent manner from the neurons voltage clamped at -70 mV; 0.1 mM cAMP was sufficient to elicit an inward current in the neurons. The magnitude of the response to cAMP reached a plateau at 1 mM with an average peak amplitude of 176 +/- 34 pA. Intracellular application of 1 mM cGMP also evoked an inward current with an average peak amplitude of 227 +/- 61 pA. The reversal potentials of the induced components were estimated to be 10 +/- 7 mV for cAMP and -4 +/- 16 mV for cGMP. The reversal potential of the cAMP- induced current in external Cl(-)-free solution was similar to that in normal Ringer's solution, suggesting that Cl- current is not significantly involved in the current. The present results represent the first evidence of cyclic nucleotide-activated conductance in the vomeronasal receptor membranes.

To elucidate the signal transduction mechanisms in the turtle vomeronasal receptor neurons, the effects of forskolin, changes in mucosal Ca 2+ concentrations and ruthenium red on the responses of the accessory olfactory bulb to general odorants were examined. Forskolin elicited a large response, suggesting that there are CAMP-gated channels in the vomeronasal neurons. On the other hand, the dependence of the responses to general odorants on Ca 2+ concentrations was different from that of the response to forskolin. A large response to an odorant (n-amyl acetate) appeared after the CAMP-mediated pathway was fully desensitized by application of 50 μM forskolin. These results suggest that the CAMP-mediated pathway does not contribute significantly to generation of the response to general odorants. A concentration of 50 μM ruthenium red significantly reduced the responses to n-amyl acetate alone and after 50 μM forskolin desensitization, suggesting that the inositol triphosphate-mediated pathway contributes partly to generation of the responses to general odorants in the vomeronasal neurons. Chem Senses 21 : 763-771, 1996.

The turtle olfactory epithelium was treated with suspensions of various lipids and their effects on the olfactory responses were examined by measuring the olfactory bulbar responses. The phosphatidylserine (PS)-treatment greatly lowered the threshold for n-valeric acid and enhanced its responses at all concentrations examined. The responses to isovaleric acid and n-butyric acid were also greatly enhanced by the PS-treatment. The responses to ten other odorants examined were a little enhanced or unchanged by the PS-treatment. The enhanced responses to the fatty acids returned to the original level about 10 h after the treatment. It was confirmed that PS was incorporated into olfactory epithelium by incubating the epithelium with PS-suspension containing [14C]PS. The treatment of the epithelium with phosphatidic acid or cardiolipin unchanged or suppressed the responses to odorants including the fatty acids. The present results suggest that lipids as well as proteins in the receptor membranes play an important role in odor reception.

It is generally believed that odorants bind to receptor proteins and activate cAMP-dependent channels located in olfactory cilia. In the present paper, we discuss whether or not this mechanism holds for in vivo transduction in the olfactory and vomeronasal organs of a turtle: (1) Elimination of salts from the olfactory epithelium did not affect the responses to odorants; changes in concentration of NaCl or CaCl2 did not affect the olfactory responses. (2) The sensitivity of the vomeronasal system to various general odorants was essentially similar to that of the olfactory system, although the vomeronasal cells have no cilia. Elimination of salts from the surface of the vomeronasal organ, similar to the olfactory system, did not affect responses to odorants. (3) Liposomes having certain lipid compositions responded to odorants with a sensitivity comparable to that in the olfactory system. The liposomes containing phosphatidylserine (PS) exhibited strong responses, especially to fatty acids. (4) Application of PS-containing liposomes to the olfactory epithelium greatly enhanced the responses to fatty acids. The results suggest that the cation channels located at olfactory cilia may not contribute to in vivo olfactory transduction. The results also suggest that lipids in olfactory receptor membranes are important in odor reception.

Aversive olfactory learning was established in young rats after odor exposure paired with foot shock through a classical conditioning paradigm. Using behavioral pharmacology and Western blotting, we previously reported that plasticity in the main olfactory bulb (MOB) underlies aversive olfactory learning. Since long-term potentiation (LTP) observed in the hippocampus is believed to be a cellular substrate for aspects of memory, we attempted to induce LTP in the MOB. Using brain slices containing the MOB, we found that five tetani of the lateral olfactory tract evoked LTP that was blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist AP5. Although three tetani induced no significant changes in control slices, with noradrenaline (NA) application they produced clear LTP (NA-mediated LTP), which was not dependent on NMDA receptors. NA's facilitating effect on LTP induction was blocked by the beta-adrenoceptor antagonist timolol but not by the alpha-adrenoceptor antagonist phentolamine, and was mimicked by the beta-adrenoceptor agonist isoproterenol. The l-type calcium channel blocker nifedipine completely blocked LTP as well as NA-mediated LTP. In addition, we found that aversive olfactory learning was impaired by beta-adrenoceptor antagonist, timolol but not by alpha-adrenoceptor antagonist, phentolamine, and only odor training established olfactory learning by isoproterenol infusion. Moreover, we found that nifedipine but not AP5 prevented olfactory learning formation. These common properties provided evidence for neural correlates between NA-mediated LTP aversive olfactory learning in young rats.

No systematic electrophysiological study on differences in odor intensity and quality between optical isomers has been carried out. In the present study, we measured the turtle olfactory bulbar responses to six pairs of highly pure optical isomers and compared the differences in odor intensity and quality between the optical isomers. The results obtained indicated that with all odorants tested, there was no difference in odor threshold and intensity in the whole concentration range examined between optical isomers. The difference in odor quality of optical isomers was evaluated by a quantitative cross-adaptation method in which odorant concentration was varied. The degree of cross adaptation between optical isomers greatly varied with species of odorants. The rank order of the magnitude of the differences in odor quality between optical isomers was carvone greater than beta-citronellol greater than menthol greater than hydroxycitronellal greater than citronellal greater than limonene.

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.

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.

Oxytocin (OT) modulation of synaptic transmission between olfactory bulb neurones has been implicated in the induction of maternal behaviour, but the mechanism of action is unknown. We examined the action of OT on gamma-aminobutyric acid(A) (GABA(A)) receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) in cultured mitral/tufted (M/T) cells with the use of whole-cell patch-clamp recordings. OT reversibly reduced the frequency of sIPSCs without affecting the amplitudes. The effect of OT on sIPSCs was mimicked by the OT receptor agonist [Thr(4), Gly(7)]-OT in a reversible manner and blocked by the OT receptor antagonist desGly-NH(2)(9), d(CH(2))(5)-[Tyr(Me)(2), Thr(4)]-ornithine-vasotocin. OT has no effect, however, on the membrane currents evoked by exogenous application of GABA. These results demonstrate that OT depresses GABA(A) receptor-mediated sIPSCs in M/T cells by a presynaptic mechanism.

Snake vomeronasal receptor neurons in slice preparations were studied using the patch-clamp technique in the conventional and nystatin-perforated whole-cell configurations. The mean resting potential was approximately -70 mV; the average input resistance was 3 GOmega. Neurons required current injection of only 1-10 pA to display a variety of spiking patterns. Intracellular dialysis of 100 microM inositol 1,4,5-trisphosphate (IP(3)) evoked an inward current in 38% of neurons, with an average peak amplitude of 16.4 +/- 2.8 pA at a holding potential of -70mV. Application of 100 microM 3-deoxy-3-fluoro-D-myo-inositol 1,4,5-trisphosphate (F-IP(3)), a derivative of IP(3), also evoked an inward current in 4/8 (50%) neurons (32.6 +/- 58 pA at -70 mV, n = 4). The reversal potentials of the induced components were estimated to be -14 +/- 5 mV for IP(3) and -17 +/- 3 mV for F-IP(3). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP(3)-evoked inward current to 1.6 +/- 1.1 pA at -70 mV (n = 6). With Cs(+)-containing internal solution, neither the Ca(2+)-ATPase inhibitor thapsigargin (1-50 microM) nor the Ca(2+)-ionophore ionomycin (10 microM) evoked a significant current response, suggesting that IP(3) can elicit current response in the neurons without mediation by intracellular Ca(2+) stores. Intracellular application of 1 mM cAMP evoked no detectable current response. Extracellular application of chemoattractant for snakes evoked a very large inward current. The reversal potential of the chemoattractant-induced current was similar to that of the IP(3)-induced current. The present results suggest that IP(3) may act as a second messenger in the transduction of chemoattractants in the garter snake vomeronasal organ.

Electrovomeronasogram (EVG) recordings were made from adult garter snakes, Thamnophis sirtalis. Stimulation of vomeronasal epithelium with a stimulus prepared from prey, earthworm electric shock secretion (ESS), evoked EVG response in a dose-dependent manner. The magnitude of the EVG response to ESS was remarkably larger than n-amyl acetate and glutamate, which elicited insignificant responses, supporting the idea that the vomeronasal system is differentially sensitive to liquid delivery of biologically significant chemical stimuli. Fourteen days following vomeronasal axotomy, the magnitudes of the EVG responses of animals which received bilateral axotomy without cauterization or with cauterization was -0.19+/-0.07 mV or -0.05+/-0.02 mV respectively, compared with the normal EVG response of -0.41+/-0.10 mV. The epithelia of animals which received bilateral axotomy without cauterization exhibited remarkable degeneration of the bipolar neurons. Maximal depletion of bipolar neurons occurred in the epithelia denervated with cauterization, though the difference between cell densities in vomeronasal neuron layers in these epithelia was not statistically significant. The present results clearly indicate that the fewer neurons the epithelium contains, the smaller EVG response it generates, suggesting that the receptor neurons are the primary origin of EVG responses.

Recording of stimulant-induced waves (bulbar responses) from the main olfactory bulb is a useful tool for measuring quantitative stable olfactory responses. There is a good relationship between the olfactory bulbar response, olfactory nerve response and electroolfactogram (EOG), suggesting that the bulbar response reflects events in receptor cells. The modern whole-cell recording technique offers direct information on olfactory transduction in single cells, but it requires long experimental periods and many animals. On the other hand, analysis of bulbar responses provides useful information and requires the use of few animals. For example, we found that cAMP-increasing and IP3-increasing odorants were not distinctly received by the turtle olfactory organ by measuring olfactory bulbar responses and analyzed with a multidimensional scaling from about 60 animals. However, to record similar odor responses from isolated turtle olfactory neurons, at least 200 animals would be necessary. Bulbar responses are recorded with electrodes implanted into or located on the main olfactory bulb. When electrodes are located on the olfactory bulb surface, it is possible to record stable responses over a period of 3 days. These methods were applied successfully to the accessory olfactory bulb. In this paper, we describe the protocols used for recording of the stimulant-induced waves from the main and accessory olfactory bulb.

Inositol 1,4,5-trisphosphate (IP3) was injected into turtle vomeronasal receptor neurons in the slice preparation under a whole-cell patch clamp, and the evoked current was measured. Application of 0.1 mM IP3 evoked a prolonged, inward current (52 of 98 neurons) with an average peak amplitude of 89.9 +/- 10.9 pA. The reversal potential of the response induced by IP3 was estimated to be -32.3 +/- 1.5 mV (6 neurons). Bathing the neurons in 10 microM ruthenium red solution greatly reduced the IP3 evoked inward current to 18.0 +/- 4.6 pA (5 neurons). This is the first study to demonstrate that the membranes of the turtle vomeronasal neurons carry IP3-activated conductance.

It is generally believed that an olfactory response is induced by binding of an odorant to a specific receptor protein in olfactory receptor membranes (Buck and Axel, 1991). On the other hand, it has been shown that nonolfactory systems such as the turtle trigeminal nerve (Tucker, 1963), the Helix ganglion, the fly taste nerve, the frog taste cell, and the neuroblastoma cell respond to various odorants (Kashiwayanagi et al., 1984; 1985). These nonolfactory systems do not seem to provide specific receptor proteins for odorants.

It is generally considered that odorants bind to specific receptor proteins (Buck and Axel, 1991) and activate cAMP-sensitive channels located in olfactory cilia (Pace et al., 1985; Nakamura and Gold, 1987). The present paper discusses whether or not the above mechanism holds in the in vivo olfactory system.