Liposomes having high sensitivity to odorants.

The conditions to increase the sensitivities of liposomes to odorants were examined. The results obtained are as follows. (1) The minimum concentration of amyl acetate to induce the membrane potential changes (threshold) in phosphatidylcholine (PC) liposomes was about 10(-4) M and addition of 10 or 20% phosphatidylserine (PS) lowered the threshold to about 10(-9) M, which was lower than the thresholds for amyl acetate in the turtle and frog olfactory systems. (2) Similar to amyl acetate, addition of PS to PC greatly lowered the threshold for beta-ionone. On the other hand, addition of PS to PC in certain ratio increased the threshold for citral, suggesting that addition of PS to PC does not always increase the responses to all odorants. (3) The membrane fluidity change of the liposomes in response to odorants occurred at similar concentration region where the membrane potential changes occurred. The presence of CaCl2 in external solution much greatly increased both the magnitude of the membrane potential changes and the membrane fluidity changes of the PC-PS liposomes in response to amyl acetate than the presence of NaCl and MgCl2. These results suggest that the membrane fluidity change is related to generation of the membrane potential change. (4) It was estimated that adsorption of less than a few molecules of amyl acetate on single liposome elicits detectable changes in the membrane potential and the membrane fluidity.

Contribution of electrostatic and hydrophobic interactions of bitter substances with taste receptor membranes to generation of receptor potentials.

The effects of changed ionic environments on the frog taste nerve responses to the bitter substances were examined. The responses to quinine and strychnine carrying a positive charge were suppressed by an increase in ionic strength of stimulating solutions. It was concluded that electrostatic interaction of these positive bitter substances with the receptor membranes greatly contributes to the adsorption of the substances on the membranes and that this interaction was suppressed by an increase in ionic strength. The responses to neutral bitter substances (caffeine and theophylline) were unchanged by an increase in salt concentration. The zeta potential of the mouse neuroblastoma (N-18 clone), which was depolarized by various bitter substances similarly to a taste cell, was measured in the presence of the bitter substances. The zeta potential was a little changed by quinine and practically unchanged by strychnine, caffeine and theophylline. The membrane fluidity of the N-18 cell monitored with 2-(9-anthroyloxy)stearic acid was changed in response to the bitter substances, while the fluidity monitored with 12-(9-anthroyloxy)stearic acid or 1,6-diphenyl-1,3,5-hexatriene was unchanged. This suggested that the bitter substances are adsorbed on the hydrophobic region near the surface and induce a conformational change at the region. The depolarization by the bitter substances seems to stem from changes in the "boundary potential" at the region near the surface within the membrane interior.

Responses of Xenopus laevis water nose to water-soluble and volatile odorants.

Using the whole-cell mode of the patch-clamp technique, we recorded action potentials, voltage-activated cationic currents, and inward currents in response to water-soluble and volatile odorants from receptor neurons in the lateral diverticulum (water nose) of the olfactory sensory epithelium of Xenopus laevis. The resting membrane potential was -46.5 +/- 1.2 mV (mean +/- SEM, n = 68), and a current injection of 1-3 pA induced overshooting action potentials. Under voltage-clamp conditions, a voltage-dependent Na+ inward current, a sustained outward K+ current, and a Ca2+-activated K+ current were identified. Application of an amino acid cocktail induced inward currents in 32 of 238 olfactory neurons in the lateral diverticulum under voltage-clamp conditions. Application of volatile odorant cocktails also induced current responses in 23 of 238 olfactory neurons. These results suggest that the olfactory neurons respond to both water-soluble and volatile odorants. The application of alanine or arginine induced inward currents in a dose-dependent manner. More than 50% of the single olfactory neurons responded to multiple types of amino acids, including acidic, neutral, and basic amino acids applied at 100 microM or 1 mM. These results suggest that olfactory neurons in the lateral diverticulum have receptors for amino acids and volatile odorants.

Cell suspensions from porcine olfactory mucosa. Changes in membrane potential and membrane fluidity in response to various odorants.

A suspension of olfactory epithelial cells was prepared from porcine olfactory mucosa and the physiological functions of the suspension were examined. The membrane potential of the cell suspension, which was monitored by measuring the fluorescence changes of rhodamine 6G, was depolarized by an increase in the K+ concentration in the external medium. Various odorants depolarized the cell suspension in a dose-dependent fashion. The magnitude of depolarization by odorants was either unchanged or slightly increased by a reduction of the concentration of Na+, Ca2+, and Cl- in the external medium, which suggests that changes in the permeabilities of specific ions are not involved in depolarization by odorants. The application of various odorants to the cell suspension induced changes in the membrane fluidity at different sites of the membrane that were monitored with various fluorescent dyes [8-anilino-1-naphthalene sulfonate, n-(9-anthroyloxy) stearic acids, 12-(9-anthroyloxy) oleic acid, and (1,6-diphenyl-1,3,5-hexatriene)], which suggests that the odorants having different odors are adsorbed on different sites in the membrane. On the basis of these results, a possible mechanism of odor discrimination is discussed.

Taste transduction mechanism: similar effects of various modifications of gustatory receptors on neural responses to chemical and electrical stimulation in the frog.

Responses in the frog glossopharyngeal nerve induced by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCl2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCl at equal current intensities, as chemical stimulation with MgCl2 produced much larger responses than stimulation with NaCl at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-1-naphthalenesulfonate, NiCl2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCl2 resulted in a large response, whereas application of 1 M CaCl2 to the tongue adapted to 50 mM CaCl2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCl2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdCl2 and verapamil) to the perfusing solution of the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCl) and to anodal current with 10 mM NaCl. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.

A large contribution of a cyclic AMP-independent pathway to turtle olfactory transduction.

Although multiple pathways are involved in the olfactory transduction mechanism, cAMP-dependent pathway has been considered to contribute mainly to the transduction. We examined the degree of contribution of cAMP-independent pathway to the turtle olfactory response by recording inward currents from isolated cells, nerve impulses from cilia and olfactory bulbar responses. The results obtained by the three recordings were essentially consistent with each other, but detail studies were carried out by recording the bulbar response to obtain quantitative data. Application of an odorant cocktail to the isolated olfactory neuron after injection of 1 mM cAMP from the patch pipette elicited a large inward current. Mean amplitude of inward currents evoked by the cocktail with 1 mM cAMP in the patch pipette was similar to that without cAMP in the pipette. Application of the cocktail after the response to 50 microM forskolin was adapted also induced a large inward current. Application of the odorant cocktail to the olfactory epithelium, after the response to 50 microM forskolin was adapted, brought about an appreciable increase in the impulse frequency. The bulbar response to forskolin alone reached a saturation level around 10 microM. After the response to 50 microM forskolin was adapted, 11 species of odorants were applied to the olfactory epithelium. The magnitudes of responses to the odorants after forskolin were 45-80% of those of the control responses. There was no essential difference in the degree of the suppression by forskolin between cAMP- and IP3-producing odorants classified in the rat, suggesting that certain part of the forskolin-suppressive component was brought about by nonspecific action of forskolin. Application of a membrane permeant cAMP analogue, cpt-cAMP elicited a large response, and 0.1 mM citralva after 3 mM cpt-cAMP elicited 51% of the control response which was close to the response to citralva after 50 microM forskolin. A membrane permeant cGMP analogue, db-cGMP elicited a small response and the response to 0.1 mM citralva was unaffected by db-cGMP. It was concluded that cAMP-independent (probably IP3-independent) pathway greatly contributes to the turtle olfactory transduction.

Introductory remarks on umami taste.

Psychophysical and electrophysiological studies indicated that the umami substances have no enhancing activity on other primary tastes. Experiments using amiloride clearly show that the umami component of canine chorda tympani nerve response to umami substances is independent of the salt component. Single fiber analysis of the responses of the mouse glossopharyngeal nerve and the monkey primary taste cortex neuron show that the responses to umami substances are independent of other primary tastes. A large synergism between monosodium glutamate (MSG) and disodium 5'-inosinate (IMP) or disodium 5'-guanylate (GMP) is observed in dogs and is explained in terms of allosteric effect. The order of intensity of umami taste induced by a mixture of 0.5 mM GMP and 1.5 mM of various agonists for the glutamate receptors was glutamate > ibotenate > DL(+)-2-amino-4-phosphonobutyric acid (DL-AP4)-(+)-1- aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD). Kainate, N-methyl-D-aspartate (NMDA) and (RS)--amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), which are agonists for ionotropic receptors, have no umami taste. It was concluded that the umami receptor is not identical to any of known glutamate receptors, and there seems to be a unique receptor for umami.

Cluster of proliferating cells in rat vomeronasal sensory epithelium.

We investigated the properties of small cells in the vomeronasal sensory epithelium of adult rats. The sensory neurons in the sensory epithelium were stained by antibodies to G(i2alpha) and G(oalpha) in their cell bodies and dendrites, while the small cells, which formed a cluster in the epithelium, were not stained at all. Voltage-activated inward currents were not detected by patch-clamp recordings, but outward currents were induced by the application of voltage step pulses. These results suggest that the small cells are different from the vomeronasal sensory neurons. Bromodeoxyuridine (BrdU) labeling indicated that dividing cells existed in the cluster of small cells.

Forskolin enhanced off-response in the turtle olfactory system.

Olfactory responses appear not only at the onset of odor stimulation, but also at the termination of the stimulation. It is widely considered that the cAMP-pathway is involved in the generation of odor responses. We examined how cAMP affects the generation of off-responses. To explore the role of the cAMP-pathway, odorants were applied to the turtle olfactory epithelium after forskolin at high concentrations which saturated the olfactory response to forskolin. Various odorants induced off-responses after 50 microM forskolin, indicating that off-responses are not induced via the cAMP-dependent pathway. However, the magnitude of the off-response after forskolin varied from 100 to 1400% of those of off-responses in its absence, indicating that forskolin greatly enhanced the off-responses to some odorants. The off-response after 0.1 mM citralva was also enhanced by 3 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (cpt-cAMP). These results suggested that cAMP modulates off-responses in the turtle olfactory system.

Odor discrimination of 'cAMP-' and 'IP3-increasing' odorants at high temperature and at high NaCl concentration in turtle olfactory system.

The ability of the turtle olfactory system to discriminate between various cAMP- and IP3-increasing odorants at high temperature and at high NaCl concentration in the olfactory bulb was examined by the cross-adaptation technique. The degrees of discrimination in high [Na+] solution were similar to those in normal Ringer's solution, suggesting that selectivities of receptors coupled with cAMP- and IP3-dependent pathways are similar to those coupled with both cAMP- and IP3-independent pathways. The mean values of the degree of discrimination among the IP3-increasing odorants were higher than those among the cAMP-increasing odorants at high temperature and at high [Na+] concentration. The degrees of discrimination among the IP3-increasing odorants at 40 degrees C were greater than those at 25 degrees C, while those among the cAMP-increasing odorants at 40 degrees C were similar to those at 25 degrees C, suggesting that the features of the receptors of cAMP-increasing odorants are different from those which respond to IP3-increasing odorants.

Role of basal ganglia-brainstem pathways in the control of motor behaviors.

Here we review a role of a basal ganglia-brainstem (BG-BS) system throughout the mesopontine tegmentum in the control of various types of behavioral expression. First the basal ganglia-brainstem system may contribute to an automatic control of movements, such as rhythmic limb movements and adjustment of postural muscle tone during locomotion, which occurs in conjunction with voluntary control processes. Second, the basal ganglia-brainstem system can be involved in the regulation of awake-sleep states. We further propose the possibility that the basal ganglia-brainstem system is responsible for the integration of volitionally-guided and emotionally-triggered expression of motor behaviors. It can be proposed that dysfunction of the basal ganglia-brainstem system together with that of cortico-basal ganglia loop underlies the pathogenesis of behavioral disturbances expressed in basal ganglia dysfunction.

Evidence for non-receptor odor discrimination using neuroblastoma cells as a model for olfactory cells.

The mouse neuroblastoma cell (N-18 clone), which is independent of an olfactory cell, was depolarized by 20 odorants examined, suggesting that specific proteins are not required for reception of odorants. The mechanism of non-receptor-mediated odor discrimination was examined using the N-18 cell. Changes in the membrane fluidity of the cell induced by adsorption of odorants were measured with various fluorescence probes, which monitor the fluidity at the different depth and in the different phase of the membrane. The profiles of the membrane fluidity changes monitored with these dyes were different from one species of odorants to another, suggesting that odorants having different odors are adsorbed at different sites in the membranes. The alteration of the lipid composition of the cell membrane brought about by exogenous application of stearic acid and cholesterol led to modification of the responses (magnitude of depolarization) to various odorants. The extent and direction (increase or decrease) of changes in the responses greatly varied among species of odorants. The following mechanism on odor discrimination was proposed. A membrane composition of each olfactory cell is postulated to be different from cell to cell. Different combinations of lipids and proteins in the membranes provide different adsorption sites for odorants. Relative amounts of the membrane potential changes in many olfactory cells in response to an odorant are characteristic of the species of the odorant. The response profiles at the cell level determine the quality of the odor.

Neuroblastoma cell as model for olfactory cell: mechanism of depolarization in response to various odorants.

The mouse neuroblastoma cell (N-18 clone) was used as a model for an olfactory cell. The N-18 cell was found to be depolarized reversibly by various species of odorants. The minimum concentrations of odorants which induced depolarization (threshold concentration) varied greatly with the species of odorants. There was a good correlation between the order of the threshold concentrations for various odorants in the N-18 cell and that in the frog olfactory responses. Replacement of Na+ and Cl- with impermeable ions or reduction of calcium concentration from 1.8 mM to 0.1 mM had practically no effect on the magnitude of the depolarization response to odorants. The input membrane resistance was little changed during the depolarization induced by various odorants. No reversal potential was observed when the cell was depolarized by n-amyl acetate or vanillin. It is suggested that the depolarization of N-18 cell by odorants is induced by changes in the phase-boundary potential at the outer surface of the cell.

Enhancement of the turtle olfactory responses to fatty acids by treatment of olfactory epithelium with phosphatidylserine.

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.

Existence of multiple receptors in single neurons: responses of single bullfrog olfactory neurons to many cAMP-dependent and independent odorants.

The responses of single bullfrog olfactory neurons to various odorants were measured with the whole-cell patch clamp which offers direct information on cellular events and with the ciliary recording technique to obtain stable quantitative data from many neurons. A large portion of single olfactory neurons (about 64% and 79% in the whole-cell recording and in the ciliary recording, respectively) responded to many odorants with quite diverse molecular structures, including both odorants previously indicated to be cAMP-dependent (increasing) and independent odorants. One odorant elicited a response in many cells; e.g. hedione and citralva elicited the response in 100% and 92% of total neurons examined with the ciliary recording technique. To confirm that a single neuron carries different receptors or transduction pathways, the cross-adaptation technique was applied to single neurons. Application of hedione to a single neuron after desensitization of the current in response to lyral or citralva induced an inward current with a similar magnitude to that applied alone. It was suggested that most single olfactory neurons carry multiple receptors and at least dual transduction pathways.

Neuroblastoma cell as a model for a taste cell: mechanism of depolarization in response to various bitter substances.

The mouse neuroblastoma cell (N-18 clone) was used as a model for a taste cell. The N-18 cell was found to be reversibly depolarized by various bitter substances. The minimum concentrations of bitter substances which induced depolarization (threshold concentration) varied greatly with the type of the substance. There was a good correlation between the threshold concentrations for various bitter substances in the N-18 cell and those in the human taste responses. The input membrane resistance was little changed during the depolarization induced by the bitter substances. Replacement of Na+ and Cl- with impermeable ions had practically no effect on the depolarization response to the bitter substances and reduction of calcium concentration from 1.8 to 0.2 mM led to a slight increase in the responses. It was suggested that the depolarization of the N-18 cell by bitter substances mainly stems from changes in the phase-boundary potential at the outer surface of the cell.

Water response of frog olfactory system is induced by a decrease in osmotic pressure.

The frog olfactory response to deionized water (water response) was recorded from the olfactory bulb. The water response was suppressed by both electrolytes and non-electrolytes as a function of osmolarity, while the water response in taste cells was not suppressed by non-electrolytes. It was concluded that a decrease in osmotic pressure induced by application of deionized water is the origin of the water response in the frog olfactory system.

Mechanism of the water response in frog gustation: possible significance of surface potential.

The frog taste response to deionized water (water response) after adaptation of the tongue to salts was recorded from the glossopharyngeal nerve under various conditions. It was found that the frog water response exhibits different behavior from the carp water response examined in a previous paper13. (a) The frog water response did not decline during stimulation and lasted for at least 3 min, while the carp water response declined within 10 s after stimulation to a spontaneous level. (b) The frog water response was practically independent of species and concentrations of salts in adapting solutions when the tongue was adapted to salts of monovalent cations, while the carp water response was highly dependent of salt concentration in adapting solution. (c) The water response was increased with an increase of CaCl2 concentration in adapting solution, while it was decreased with an increase of MgCl2 concentration. (d) The water response was suppressed by the presence of electrolytes in stimulating solution: the data obtained with different species of salts were described by a single curve as a function of the ionic strength. (e) The mechanism of the frog water response together with the carp water response was explained in terms of the surface potential.

Increases in Fos-immunoreactivity after exposure to a combination of two male urinary components in the accessory olfactory bulb of the female rat.

Exposure to either the dialyzed urine preparation (<500 Da) or the remaining substances (>500 Da) did not induce expression of Fos-immunoreactive cells in the mitral/tufted cell layer of the accessory olfactory bulb (AOB), whereas exposure to a mixture of these preparation did induce expression. These results suggest that a combination of low and high molecular weight substances is necessary for the increases in Fos-immunoreactivity in the AOB.

Inositol-1,4,5-trisphosphate accumulation induced by urinary pheromones in female rat vomeronasal epithelium.

The mechanisms involved in pheromone-induced responses in the vomeronasal neurons, especially in mammals, are still unclear. In the present study, we examined the effects of rat urine samples containing various types of pheromones regulating gonadal functions on the accumulation of cAMP and inositol 1,4,5-trisphosphate (IP3) in a vomeronasal membrane preparation from the female Wistar rat. Stimulation of the preparation with forskolin induced cAMP accumulation, but stimulation with urine samples excreted from the male Wistar rat, the female Wistar rat, and the male Donryu rat did not change cAMP levels. These results were consistent with the electrophysiological results showing that dialysis of a high concentration of cAMP into the vomeronasal neuron does not induce currents. Stimulation with the three urine samples induced the accumulation of IP3 in the membrane preparation. These results are consistent with previous electrophysiological results [K. Inamura, M. Kashiwayanagi, K. Kurihara, Inositol-1,4,5-trisphosphate induces responses in receptor neurons in rat vomeronasal sensory slices, Chem. Senses 22 (1997) 93-103; K. Inamura, M. Kashiwayanagi, K. Kurihara, Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons, Neurosci. Lett. 233 (1997) 129-132]. After the treatment with Pertussis toxin (PTX), the male Wistar urine did not induce IP3 accumulation significantly. Application of the male Wistar urine decreased ADP-ribosylation of Gi with PTX, while that of the male Donryu urine decreased ADP-ribosylation of Go. Thus, the present results support a mechanism by which the responses of the rat vomeronasal neurons to urinary pheromones are mediated by IP3, Gi and/or Go.