- Email: [email protected]
Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons
Neuroscience Letters 233 (1997) 129–132
Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons Kouhei Inamura, Makoto Kashiwayanagi*, Kenzo Kurihara Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan Received 21 August 1997; received in revised form 25 August 1997; accepted 25 August 1997
Abstract 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 mM ruthenium red) or phospholipase C inhibitors (10 mM 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. 1997 Elsevier Science Ireland Ltd. Keywords: Inositol-1,4,5-trisphosphate; Inhibitors; Rat; Vomeronasal sensory neuron; Urine; Pheromone
The vomeronasal organ existing in many vertebrates for receiving pheromones relates social and sexual behavior [3,13,19,20]. Regulation of gonadal functions by urine was well established in rodent vomeronasal organ; male urinary factors elicited reflex ovulation in the absence of coitus and mounting [7], reduced oestrous cycle of the female rats from 5 to 4 days [1] and excreted the pubertydelay chemosignal [9]. Urinary compounds of low volatility stimulate the vomeronasal system and provide information that is normally not provided by gustation or olfaction [20]. In the garter snake, ES20, a chemoattractant extracted from its prey [6], induced inositol 1,4,5-trisphosphate (IP3) accumulation in the vomeronasal epithelium [11]. Dialysis of IP3 into the turtle and rat vomeronasal receptor neurons induced inward currents [5,17]. These results suggest that pheromonal information is mediated via the IP3dependent pathway in the vomeronasal receptor neurons. In the present study, we studied effects of phospholipase C inhibitors and an IP3-channel inhibitor on responses to urine in the rat vomeronasal receptor neuron to obtain information about the transduction mechanism of the mamma* Corresponding author. Tel.: +81 11 7063916; fax: +81 11 7064991; e-mail: [email protected]
lian vomeronasal receptor neurons under on-cell patch clamp conditions. The results obtained suggested that the urinary responses are mediated via the IP3-mediated transduction pathway in rat vomeronasal receptor neurons. Urine collected from male Wistar rats were treated with charcoal to reduce cAMP concentration. Concentration of cAMP in the charcoal-treated urine was less than 0.5 nM. Concentration of cAMP in the urine preparation was measured by the sensitive radioimmunoassay procedure as described by Honma et al. [4]. The urine preparation was further treated with anion and cation exchanger to reduce ionic concentration. Concentration of urine in the preparation was thus about 1/4 of that of original urine. The final concentrations of Na+, K+, Ca2+ and Mg2+ in urine stimulating solutions were 155–159.3 mM, 0.2–5.1 mM, 2–2.05 mM and 0–0.2 mM, respectively. A control salt solution containing 160 mM Na+, 5.7 mM K+ and 2.1 mM Ca2+ did not induce any response in the vomeronasal sensory neuron (data not shown). Slice preparations were prepared from female Wistar rats as descried previously [18]. Vomeronasal epithelium was quickly removed from the decapitated rats. The epithelia were cut into slices about 120 mm thick with a vibrating slicer (DTK-1000, D.T.K., Kyoto, Japan) in tyrode solution
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940 (97 )0 0655- 1
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at 0°C and stored at 4°C. Epithelial slices were fixed on the glass at the bottom of a recording chamber, permitting access to cells on the surface of the slice by the patch pipette. The preparations were viewed under an upright microscope (model OPTIPHOT, Nikon, Tokyo, Japan) using a 40 × water immersion lens. Membrane currents (holding potential, −70 mV) were recorded in the on-cell configuration using an EPC-7 patch clamp amplifier (List, Darmstadt, Germany) and stored on video cassette via a digital audio processor. All recordings were performed at room temperature. Analysis was carried out on a personal computer using pCLAMP and AXOSCOPE softwares (Axon Instruments, Foster City, USA). Extracellular tyrode solution consisted of (in mM): 145 NaCl, 2.5 KCl, 2 CaCl2, 10 glucose, 10 HEPES-NaOH (pH 7.4). Patch pipettes were usually filled with a normal internal solution (in mM): 140 KCl, 2.5 MgCl2, 0.5 ATP, 0.5 EGTA, 10 HEPES-KOH (pH 7.2). Neomycin, ruthenium red, 1-[6-[[17b-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), 1-[6-[[17b3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343) and charcoal (Norit SX plus) were obtained from Wako (Tokyo, Japan). Ion exchanger (AG 501-X8 Resin) was obtained from Bio-Rad (CA, USA). All chemicals used were of the highest grade available. The effects of urine on the female rat vomeronasal sensory neurons were examined using the on-cell patch clamp mode. Urine was treated with charcoal and ion exchangers to reduce the concentration of cAMP and ions in the urine.
Fig. 1. An increase in impulse frequency of a female Wistar vomeronasal sensory neuron recorded with the cell-attached mode in response to the male Wistar urine in (a) normal tyrode solution without blockers, (b) 10 mM U-73122 and (c) 10 mM U-73343. (d) The response of the cell to sustained current injection with 10 mM U-73122. Data were obtained from the same neurons. Bars at the bottom of the traces indicate periods of stimulation. Large electrical noise shown in the traces was induced by electrical actual valves at the ‘on’ time of stimulation.
Fig. 2. An increase in impulse frequency of a female Wistar vomeronasal sensory neuron recorded with the cell-attached mode in response to the male Wistar urine in (a) normal tyrode solution without blockers and (b) 1 mM neomycin. (c) The response of the cell to sustained current injection with 1 mM neomycin. Data were obtained from the same neurons. Bars at the bottom of the traces indicate periods of stimulation. Large electrical noise shown in the traces was induced by electrical actual valves at the ‘on’ time of stimulation.
Introduction of the urine preparation thus obtained from the male Wistar rat increased impulse frequency in the sensory neuron (Fig. 1a). An increase in impulse frequency in response to the male Wistar urine varied from 0.3 to 14 Hz. In the previous paper, we showed that dialysis of IP3 into the sensory neurons of the female Wistar rat induced inward currents [5]. To explore whether urinary responses are mediated via the IP3-dependent pathway, U-73122, an inhibitor of phospholipase C [15], was introduced. As shown in Fig. 1b, 10 mM U-73122 completely inhibited the responses to urine in all three cells that were examined. Stimulation with depolarizing current pulses injected from the patch pipette (Fig. 1d) or high K+-solution (data not shown) induced a train of impulses, suggesting that depolarization generates action potentials even in the presence of U-73122. In the presence of 10 mM U-73343, which has a similar molecular structure to U-73122 but does not inhibit phospholipase C [15], the urine preparation appreciably increased impulse frequency, although the magnitude of the responses was decreased (Fig. 1c). Similar results were observed with the three cells that were examined. Neomycin, a compound known to interact with polyphosphoinositide [14], was also shown to inhibit phospholipase C [2]. Likewise, application of 1 mM neomycin completely blocked urinary responses in all three cells (Fig. 2b). A train of impulses was induced by application of depolarizing current pulses (Fig. 2c) or high K+-solution (data not shown), suggesting that neomycin does not block the generation of action potentials non-specifically. These results suggest that urinary responses are mediated via phospholipase C although effects of U-73122 and neomycin on phospholipase C-independent-pathways cannot be completely precluded. Previous results showed that ruthenium red (10 mM)
K. Inamura et al. / Neuroscience Letters 233 (1997) 129–132
Fig. 3. An increase in impulse frequency of a female Wistar vomeronasal sensory neuron recorded with the cell-attached mode in response to the male Wistar urine in (a) normal tyrode solution without blockers and (b) 10 mM ruthenium red. (c) The response of the cell to sustained current injection with 10 mM ruthenium red. Data were obtained from the same neurons. Bars at the bottom of the traces indicate periods of stimulation. Large electrical noise shown in the traces was induced by electrical actual valves at the ‘on’ time of stimulation.
blocked inward currents in turtle [17] and rat [5] vomeronasal sensory neurons induced by dialysis of IP3 from the patch pipette. In the presence of 10 mM ruthenium red, the male Wistar urine did not increase the impulse frequency (Fig. 3b). Similar inhibition was observed with all three cells. During the treatment with ruthenium red, action potentials were recorded from the same neurons in response to currents injected from the patch pipette (Fig. 3c), suggesting that ruthenium red did not inhibit the generation of action potentials in response to urine by blocking voltagedependent ion channels. The blockage of urinary responses by the inhibitors for phospholipase C and the inhibitor for IP3-activated channel suggests that an increase in the impulse frequency in response to the male Wistar urine was mediated via the IP3-dependent pathway. The functional adenylyl cyclase was shown biochemically in the snake and turtle vomeronasal sensory epithelia [11,12]. In the turtle, dialysis of cyclic nucleotides into the vomeronasal sensory neurons induced inward currents [18], and application of forskolin to the vomeronasal sensory epithelium induced accessory olfactory bulbar responses [16], both indicating that excitable responses via the cAMP-dependent channels are transmitted to the accessory olfactory bulb. Similarly, in the garter snake, a chemoattractant (ES20) derived from worm induced changes in cAMP concentrations in the vomeronasal epithelium [11]. It is therefore possible that the cAMP-dependent pathway is at least partly involved in the transduction process in reptile vomeronasal sensory neurons. It has been, however, found that even high concentrations such as 0.5 mM and 1 mM of cAMP did not induce any response in the mouse [10] and rat vomeronasal sensory neurons (Inamura et al., unpublished data), respectively. These results suggest that cAMP is not a
131
second messenger in the pheromone reception for mammalian vomeronasal organs. On the other hand, this study, together with previous studies, supports the conclusion that the vomeronasal sensory responses in both reptiles and mammals are IP3-dependent. In the previous study, we have shown that dialysis of IP3 induced inward currents in the turtle vomeronasal sensory neurons [17], while Luo et al. [11] report that ES20 induced IP3 accumulation in the garter snake vomeronasal epithelium, suggesting that IP3 is a second messenger in reptile vomeronasal sensory neurons. Similarly, dialysis of IP3 into the rat vomeronasal sensory neurons was found to induce inward currents in more than 50% of cells examined [13], while this study showed that increases in impulse frequency of the sensory neurons in response to the male urine were blocked by ruthenium red, U-73122 and neomycin. These results indicate that the response of the rat vomeronasal sensory neurons to urine is generated via the IP3dependent pathway. In a separate study, urine excreted from male and female Wistar rat was applied to the female Wistar vomeronasal sensory neurons to explore specificity of the urinary responses (Inamura et al., unpublished data). The female Wistar urine as well as the male urine induced responses in the neurons, but the number of cells which responded to the female urine was smaller than that which responded to the male one. In addition, the neurons which responded to the male urine never responded to the female urine. These results indicate that there are neurons which selectively respond only to the male urine. Recently, aphrodisin, a pheromone excreted from the female hamster, was reported to induce IP3 accumulation in the preparation of the male hamster vomeronasal sensory epithelium [8]. These results together with those from this study strongly suggest that mammalian vomeronasal sensory neurons transduce pheromonal information into electrical signals via the IP3mediated pathway. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan and by a grant from the Human Frontier Science Program. [1] Chateau, D., Roos, J., Plas-Roser, S., Roos, M. and Aron, C., Hormonal mechanisms involved in the control of oestrous cycle duration by the odour of urine in the rat, Acta Endocrinol., 82 (1976) 426– 435. [2] Cockcroft, S., Howell, T. and Gomperts, B., Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphoinositide phosphodiesterase and exocytosis, J. Cell Biol., 105 (1987) 2745–2750. [3] Halpern, M., The organization and function of the vomeronasal system, Annu. Rev. Neurosci., 10 (1987) 325–362. [4] Honma, M., Satoh, T., Takezawa, J. and Ui, M., An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small volume sample from blood and tissue, Biochem. Med., 18 (1977) 257–273.
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[5] Inamura, K., Kashiwayanagi, M. and Kurihara, K., Inositol-1,4,5trisphosphate induces responses in receptor neurons in rat vomeronasal sensory slices, Chem. Senses, 22 (1997) 93–104. [6] Jiang, X.C., Inouchi, J., Wang, D. and Halpern, M., Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes, J. Biol. Chem., 265 (1990) 8736–8744. [7] Johns, M.A., Feder, H.H., Komisaruk, B.R. and Mayer, A.D., Urineinduced reflex ovulation in anovulatory rats may be a vomeronasal effect, Nature, 272 (1978) 446–448. [8] Kroner, C., Breer, H., Singer, A.G. and O’Connell, R.J., Pheromoneinduced second messenger signaling in the hamster vomeronasal organ, NeuroReport, 7 (1996) 2989–2992. [9] Lepri, J.J., Wysocki, C.J. and Vandenbergh, J.G., Mouse vomeronasal organ: effects on chemosignal production and maternal behavior, Physiol. Behav., 35 (1985) 809–814. [10] Liman, E.R. and Corey, D.P., Electrophysiological characterization of chemosensory neurons from the mouse vomeronasal organ, J. Neurosci., 16 (1996) 4625–4637. [11] Luo, Y., Lu, S., Chen, P., Wang, D. and Halpern, M., Identification of chemoattractant receptors and G-proteins in the vomeronasal system of garter snakes, J. Biol. Chem., 269 (1994) 16867–16877. [12] Okamoto, K., Tokumitsu, Y. and Kashiwayanagi, M., Adenylyl cyclase activity in turtle vomeronasal and olfactory epithelium, Biochem. Biophys. Res. Commun., 220 (1996) 98–101.
[13] Powers, J.B. and Winans, S.S., Vomeronasal organ: critical role in mediating sexual behavior of the male hamster, Science, 187 (1975) 961–963. [14] Schacht, J., Purification of polyphosphoinositides by chromatography on immobilized neomycin, J. Lipid Res., 19 (1978) 1063–1067. [15] Smith, R., Sam, J., Gordon, L., Bala, G. and Bleasdale, J., Receptorcoupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness, J. Pharmacol. Exp. Ther., 253 (1990) 688–697. [16] Taniguchi, M., Kanaki, K. and Kashiwayanagi, M., Difference in behavior between responses to forskolin and general odorants in turtle vomeronasal organ, Chem. Senses, 21 (1996) 763–771. [17] Taniguchi, M., Kashiwayanagi, M. and Kurihara, K., Intracellular injection of inositol 1,4,5-trisphosphate increases a conductance in membranes of turtle vomeronasal receptor neurons in the slice preparation, Neurosci. Lett., 188 (1995) 5–8. [18] Taniguchi, M., Kashiwayanagi, M. and Kurihara, K., Intracellular dialysis of cyclic nucleotides induces inward currents in turtle vomeronasal receptor neurons, J. Neurosci., 16 (1996) 1239–1246. [19] Wysocki, C. J., and Meredith, M., The vomeronasal system. In T.E. Finger and W.L. Silver (Eds.), Neurobiology of Taste and Smell, Wiley, New York, 1987, pp. 125–150. [20] Wysocki, C.J., Wellington, J.L. and Beauchamp, G.K., Access of urinary non-volatiles to the mammalian vomeronasal organ, Science, 207 (1980) 781–783.
Blockage of urinary responses by inhibitors for IP3-mediated pathway in rat vomeronasal sensory neurons Kouhei Inamura, Makoto Kashiwayanagi*, Kenzo Kurihara Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan Received 21 August 1997; received in revised form 25 August 1997; accepted 25 August 1997
Abstract 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 mM ruthenium red) or phospholipase C inhibitors (10 mM 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. 1997 Elsevier Science Ireland Ltd. Keywords: Inositol-1,4,5-trisphosphate; Inhibitors; Rat; Vomeronasal sensory neuron; Urine; Pheromone
The vomeronasal organ existing in many vertebrates for receiving pheromones relates social and sexual behavior [3,13,19,20]. Regulation of gonadal functions by urine was well established in rodent vomeronasal organ; male urinary factors elicited reflex ovulation in the absence of coitus and mounting [7], reduced oestrous cycle of the female rats from 5 to 4 days [1] and excreted the pubertydelay chemosignal [9]. Urinary compounds of low volatility stimulate the vomeronasal system and provide information that is normally not provided by gustation or olfaction [20]. In the garter snake, ES20, a chemoattractant extracted from its prey [6], induced inositol 1,4,5-trisphosphate (IP3) accumulation in the vomeronasal epithelium [11]. Dialysis of IP3 into the turtle and rat vomeronasal receptor neurons induced inward currents [5,17]. These results suggest that pheromonal information is mediated via the IP3dependent pathway in the vomeronasal receptor neurons. In the present study, we studied effects of phospholipase C inhibitors and an IP3-channel inhibitor on responses to urine in the rat vomeronasal receptor neuron to obtain information about the transduction mechanism of the mamma* Corresponding author. Tel.: +81 11 7063916; fax: +81 11 7064991; e-mail: [email protected]
lian vomeronasal receptor neurons under on-cell patch clamp conditions. The results obtained suggested that the urinary responses are mediated via the IP3-mediated transduction pathway in rat vomeronasal receptor neurons. Urine collected from male Wistar rats were treated with charcoal to reduce cAMP concentration. Concentration of cAMP in the charcoal-treated urine was less than 0.5 nM. Concentration of cAMP in the urine preparation was measured by the sensitive radioimmunoassay procedure as described by Honma et al. [4]. The urine preparation was further treated with anion and cation exchanger to reduce ionic concentration. Concentration of urine in the preparation was thus about 1/4 of that of original urine. The final concentrations of Na+, K+, Ca2+ and Mg2+ in urine stimulating solutions were 155–159.3 mM, 0.2–5.1 mM, 2–2.05 mM and 0–0.2 mM, respectively. A control salt solution containing 160 mM Na+, 5.7 mM K+ and 2.1 mM Ca2+ did not induce any response in the vomeronasal sensory neuron (data not shown). Slice preparations were prepared from female Wistar rats as descried previously [18]. Vomeronasal epithelium was quickly removed from the decapitated rats. The epithelia were cut into slices about 120 mm thick with a vibrating slicer (DTK-1000, D.T.K., Kyoto, Japan) in tyrode solution
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at 0°C and stored at 4°C. Epithelial slices were fixed on the glass at the bottom of a recording chamber, permitting access to cells on the surface of the slice by the patch pipette. The preparations were viewed under an upright microscope (model OPTIPHOT, Nikon, Tokyo, Japan) using a 40 × water immersion lens. Membrane currents (holding potential, −70 mV) were recorded in the on-cell configuration using an EPC-7 patch clamp amplifier (List, Darmstadt, Germany) and stored on video cassette via a digital audio processor. All recordings were performed at room temperature. Analysis was carried out on a personal computer using pCLAMP and AXOSCOPE softwares (Axon Instruments, Foster City, USA). Extracellular tyrode solution consisted of (in mM): 145 NaCl, 2.5 KCl, 2 CaCl2, 10 glucose, 10 HEPES-NaOH (pH 7.4). Patch pipettes were usually filled with a normal internal solution (in mM): 140 KCl, 2.5 MgCl2, 0.5 ATP, 0.5 EGTA, 10 HEPES-KOH (pH 7.2). Neomycin, ruthenium red, 1-[6-[[17b-3-methoxyestra-1,3,5(10)-trien-17-yl] amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), 1-[6-[[17b3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidine-dione (U-73343) and charcoal (Norit SX plus) were obtained from Wako (Tokyo, Japan). Ion exchanger (AG 501-X8 Resin) was obtained from Bio-Rad (CA, USA). All chemicals used were of the highest grade available. The effects of urine on the female rat vomeronasal sensory neurons were examined using the on-cell patch clamp mode. Urine was treated with charcoal and ion exchangers to reduce the concentration of cAMP and ions in the urine.
Fig. 1. An increase in impulse frequency of a female Wistar vomeronasal sensory neuron recorded with the cell-attached mode in response to the male Wistar urine in (a) normal tyrode solution without blockers, (b) 10 mM U-73122 and (c) 10 mM U-73343. (d) The response of the cell to sustained current injection with 10 mM U-73122. Data were obtained from the same neurons. Bars at the bottom of the traces indicate periods of stimulation. Large electrical noise shown in the traces was induced by electrical actual valves at the ‘on’ time of stimulation.
Fig. 2. An increase in impulse frequency of a female Wistar vomeronasal sensory neuron recorded with the cell-attached mode in response to the male Wistar urine in (a) normal tyrode solution without blockers and (b) 1 mM neomycin. (c) The response of the cell to sustained current injection with 1 mM neomycin. Data were obtained from the same neurons. Bars at the bottom of the traces indicate periods of stimulation. Large electrical noise shown in the traces was induced by electrical actual valves at the ‘on’ time of stimulation.
Introduction of the urine preparation thus obtained from the male Wistar rat increased impulse frequency in the sensory neuron (Fig. 1a). An increase in impulse frequency in response to the male Wistar urine varied from 0.3 to 14 Hz. In the previous paper, we showed that dialysis of IP3 into the sensory neurons of the female Wistar rat induced inward currents [5]. To explore whether urinary responses are mediated via the IP3-dependent pathway, U-73122, an inhibitor of phospholipase C [15], was introduced. As shown in Fig. 1b, 10 mM U-73122 completely inhibited the responses to urine in all three cells that were examined. Stimulation with depolarizing current pulses injected from the patch pipette (Fig. 1d) or high K+-solution (data not shown) induced a train of impulses, suggesting that depolarization generates action potentials even in the presence of U-73122. In the presence of 10 mM U-73343, which has a similar molecular structure to U-73122 but does not inhibit phospholipase C [15], the urine preparation appreciably increased impulse frequency, although the magnitude of the responses was decreased (Fig. 1c). Similar results were observed with the three cells that were examined. Neomycin, a compound known to interact with polyphosphoinositide [14], was also shown to inhibit phospholipase C [2]. Likewise, application of 1 mM neomycin completely blocked urinary responses in all three cells (Fig. 2b). A train of impulses was induced by application of depolarizing current pulses (Fig. 2c) or high K+-solution (data not shown), suggesting that neomycin does not block the generation of action potentials non-specifically. These results suggest that urinary responses are mediated via phospholipase C although effects of U-73122 and neomycin on phospholipase C-independent-pathways cannot be completely precluded. Previous results showed that ruthenium red (10 mM)
K. Inamura et al. / Neuroscience Letters 233 (1997) 129–132
Fig. 3. An increase in impulse frequency of a female Wistar vomeronasal sensory neuron recorded with the cell-attached mode in response to the male Wistar urine in (a) normal tyrode solution without blockers and (b) 10 mM ruthenium red. (c) The response of the cell to sustained current injection with 10 mM ruthenium red. Data were obtained from the same neurons. Bars at the bottom of the traces indicate periods of stimulation. Large electrical noise shown in the traces was induced by electrical actual valves at the ‘on’ time of stimulation.
blocked inward currents in turtle [17] and rat [5] vomeronasal sensory neurons induced by dialysis of IP3 from the patch pipette. In the presence of 10 mM ruthenium red, the male Wistar urine did not increase the impulse frequency (Fig. 3b). Similar inhibition was observed with all three cells. During the treatment with ruthenium red, action potentials were recorded from the same neurons in response to currents injected from the patch pipette (Fig. 3c), suggesting that ruthenium red did not inhibit the generation of action potentials in response to urine by blocking voltagedependent ion channels. The blockage of urinary responses by the inhibitors for phospholipase C and the inhibitor for IP3-activated channel suggests that an increase in the impulse frequency in response to the male Wistar urine was mediated via the IP3-dependent pathway. The functional adenylyl cyclase was shown biochemically in the snake and turtle vomeronasal sensory epithelia [11,12]. In the turtle, dialysis of cyclic nucleotides into the vomeronasal sensory neurons induced inward currents [18], and application of forskolin to the vomeronasal sensory epithelium induced accessory olfactory bulbar responses [16], both indicating that excitable responses via the cAMP-dependent channels are transmitted to the accessory olfactory bulb. Similarly, in the garter snake, a chemoattractant (ES20) derived from worm induced changes in cAMP concentrations in the vomeronasal epithelium [11]. It is therefore possible that the cAMP-dependent pathway is at least partly involved in the transduction process in reptile vomeronasal sensory neurons. It has been, however, found that even high concentrations such as 0.5 mM and 1 mM of cAMP did not induce any response in the mouse [10] and rat vomeronasal sensory neurons (Inamura et al., unpublished data), respectively. These results suggest that cAMP is not a
131
second messenger in the pheromone reception for mammalian vomeronasal organs. On the other hand, this study, together with previous studies, supports the conclusion that the vomeronasal sensory responses in both reptiles and mammals are IP3-dependent. In the previous study, we have shown that dialysis of IP3 induced inward currents in the turtle vomeronasal sensory neurons [17], while Luo et al. [11] report that ES20 induced IP3 accumulation in the garter snake vomeronasal epithelium, suggesting that IP3 is a second messenger in reptile vomeronasal sensory neurons. Similarly, dialysis of IP3 into the rat vomeronasal sensory neurons was found to induce inward currents in more than 50% of cells examined [13], while this study showed that increases in impulse frequency of the sensory neurons in response to the male urine were blocked by ruthenium red, U-73122 and neomycin. These results indicate that the response of the rat vomeronasal sensory neurons to urine is generated via the IP3dependent pathway. In a separate study, urine excreted from male and female Wistar rat was applied to the female Wistar vomeronasal sensory neurons to explore specificity of the urinary responses (Inamura et al., unpublished data). The female Wistar urine as well as the male urine induced responses in the neurons, but the number of cells which responded to the female urine was smaller than that which responded to the male one. In addition, the neurons which responded to the male urine never responded to the female urine. These results indicate that there are neurons which selectively respond only to the male urine. Recently, aphrodisin, a pheromone excreted from the female hamster, was reported to induce IP3 accumulation in the preparation of the male hamster vomeronasal sensory epithelium [8]. These results together with those from this study strongly suggest that mammalian vomeronasal sensory neurons transduce pheromonal information into electrical signals via the IP3mediated pathway. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan and by a grant from the Human Frontier Science Program. [1] Chateau, D., Roos, J., Plas-Roser, S., Roos, M. and Aron, C., Hormonal mechanisms involved in the control of oestrous cycle duration by the odour of urine in the rat, Acta Endocrinol., 82 (1976) 426– 435. [2] Cockcroft, S., Howell, T. and Gomperts, B., Two G-proteins act in series to control stimulus-secretion coupling in mast cells: use of neomycin to distinguish between G-proteins controlling polyphosphoinositide phosphoinositide phosphodiesterase and exocytosis, J. Cell Biol., 105 (1987) 2745–2750. [3] Halpern, M., The organization and function of the vomeronasal system, Annu. Rev. Neurosci., 10 (1987) 325–362. [4] Honma, M., Satoh, T., Takezawa, J. and Ui, M., An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small volume sample from blood and tissue, Biochem. Med., 18 (1977) 257–273.
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