Makoto Kashiwayanagi

Laminar distribution of pheromone‐receptive neurons in rat vomeronasal epithelium

1 Responses of vomeronasal sensory neurons to urine excreted from rats, mice and hamsters were studied by the on‐cell patch clamp method in slices of sensory epithelium from female Wistar rats. 2 The urine excreted from male and female Wistar rats, male Donryu rats and male C57BL/6 mice induced relatively large responses, while urine from male Sprague‐Dawley rats and male Syrian hamsters induced small responses. 3 Of the 62 neurons responding to urine, 57 responded to only one of the urine preparations. 4 The sensory neurons that responded to the male Wistar urine were localized in the apical position of the epithelium where one type of GTP‐binding protein, Gi2α, is selectively expressed. The neurons in the basal position of the epithelium, which express Goα, responded to urine from the other animals. 5 This study demonstrates that sensory neurons responsive to different urinary pheromones are localized in a segregated layer in the rat vomeronasal sensory epithelium.

Regionalization of Fos immunostaining in rat accessory olfactory bulb when the vomeronasal organ was exposed to urine

The distribution of Fos‐immunoreactive (Fos‐ir) cells in the accessory olfactory bulb (AOB) of rats following vomeronasal organ exposure to urine was studied. Following exposure to male and female Wistar rat urine, Fos‐ir cells were found in the mitral/tufted cell layer, granule cell layer and periglomerular cell layer of the AOB of female Wistar rat, with the highest number in the granule cell layer. Exposure to water or removal of the vomeronasal organ suppressed the expression of Fos‐ir cells. These results suggest that female Wistar rats specifically detect urinary substances derived from male or female Wistar rats via the vomeronasal organ. Exposure of the vomeronasal organ of female Wistar rats to male Wistar urine induced the appearance of many more Fos‐ir cells in all layers of the AOB than exposure to female Wistar urine. As for the mitral/tufted cell layer, the density of Fos‐ir cells in the rostral portion (Gi2α‐positive) of all regions of the AOB was about twice as high as that in the caudal portion when male urine was given. The distribution pattern of Fos‐ir cells in response to female urine was not identical to that in response to male urine. That is, the density of Fos‐ir cells in the caudal portion was slightly larger than that in the rostral portion in the lateral region, while in other regions the density in the rostral portion was higher than that in the caudal portion. It is likely that information from different pheromones is transmitted to the higher brain regions through the different regions of the AOB.

Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons

The mammalian vomeronasal system is involved in the effects of urinary chemicals on gonadal functions and sexual behaviors. For example, exposure to urine affects the timing of oestrous cycles in rats. Rat vomeronasal sensory neurons in slice preparation were studied under on-cell patch clamp conditions. We found that urine excreted from male Wistar rats increased impulse frequency in vomeronasal sensory neurons of female Wistar rats. The urinary responses were blocked by an inositol-1,4,5-trisphosphate (IP3)-channel inhibitor (10 microM ruthenium red) or phospholipase C inhibitors (10 microM U-73122 and 1 mM neomycin), suggesting that pheromone-like substances in the urine induce the response in the rat vomeronasal sensory neurons via the IP3-dependent transduction pathway.

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.

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.

Intracellular injection of inositol 1,4,5-trisphosphate increases a conductance in membranes of turtle vomeronasal receptor neurons in the slice preparation

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.

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 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.

Inward current responses to urinary substances in rat vomeronasal sensory neurons

No study has yet demonstrated an inward current in response to pheromonal substances in vomeronasal sensory neurons. Using female rat vomeronasal sensory neurons, we here successfully recorded inward currents in response to urine from various sources. Of the neurons that responded to urine, 77% responded to only one type of urine. Male Wistar urine induced responses preferentially in the apical layer of the sensory epithelium, whilst male Donryu and female Wistar urine induced responses mainly in the basal layer of the epithelium. The amplitude of inward currents induced by application of male Wistar urine was voltage‐dependent with average amplitude of −47.1 ± 6.2 pA at −74 mV. The average reversal potential for male Wistar urine was −9.3 ± 6.1 mV, which was not apparently different from the reversal potentials for urine from different species. It is likely that the urine‐induced inward currents in response to different types of urine are mediated via a similar channel. The simultaneous removal of Na+ and Ca2+ from extracellular solution eliminated the response. The magnitude of the urine‐induced inward current in Cl–‐free external solution was similar to that in normal solution, suggesting that the urine‐induced current is cation selective. Removal of external Ca2+ enhanced the amplitude of the urine‐induced current and prolonged the response. Application of the constant‐field equation indicated a very high permeability coefficient for Ca2+. This study first demonstrated that substances contained in urine elicited inward currents, which induce an excitatory response in vomeronasal sensory neurons, through cation‐selective channels.

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.