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The vomeronasal organ VNO is important for activating accessory olfactory pathways that are involved in sexually dimorphic mating behavior. The VNO of male garter snakes is critically important for detection of, and response to, female sex pheromones.
In the present study, under voltage-clamp conditions, male snake VNO neurons were stimulated with female sexual attractiveness pheromone. The amplitude of the inward current was dose dependent, and the relationship could be fitted by the Hill equation. Under current-clamp conditions, application of pheromone produced membrane depolarizing responses and increases in firing frequency. These suggest that the female pheromone directly affects male snake VNO neurons and in opening of ion channels, thereby converting the pheromone al to an electrical al.
The response to female pheromone is sexually dimorphic, that is, the pheromone does not evoke responses in VNO neurons of female snakes. An associated finding of the present study is that the female sex pheromone, which is insoluble in aqueous solutions, became soluble in the presence of Harderian gland homogenate.
The vomeronasal organ VNO of vertebrates is a chemoreceptive organ that has been implicated in the detection of, and response to, sex pheromones Halpern, ; Wysocki and Meredith, ; Meredith, ; Keverne, ; Johnston, ; McClintock, ; Halpern and Martinez-Marcos, One of the few vertebrate pheromones that has been isolated, purified, and characterized is the sex pheromone of the red-sided garter snake Thamnophis sirtalis parietalis Mason et al.
This pheromone, a mixture of 13 long-chain C 29 —C 37 saturated and monounsaturated methyl ketones, is expressed on the dorsal surface of adult female snakes during the mating season. When males encounter a female expressing the pheromone, they exhibit stereotyped courtship behaviors including chin rubbing, rapid tongue-flicks, and caudocephalic body undulations Noble, ; F.
Blanchard and F. Blanchard, ; Aleksiuk and Gregory, ; Crews, ; Kubie et al. Male garter snakes deprived of a functional vomeronasal VN system are unable to detect or respond appropriately to this pheromone Noble, ; Kubie et al. Although behavioral studies have established the critical involvement of the VN system in detection of this pheromone, the transduction mechanism by which the pheromone activates snake sensory neurons has yet to be elucidated. The present study was deed to examine the effects of the purified female snake sex pheromone on the membrane potential and firing properties of VNO sensory neurons.
We used VNO neurons from male and female red-sided garter snakes, testing them under whole-cell voltage- and current-clamp protocols to identify neural responses to the purified pheromone. Prior to conducting the electrophysiological study, we tested the effect of the female sex pheromone on the generation of IP 3 in male garter snake VN sensory epithelium homogenates. IP 3 is a known second messenger in the snake VN al transduction pathway for prey chemicals Luo et al.
The female sex pheromone is insoluble in aqueous solutions. Since solubility in the aqueous medium filling the VNO is required for odorant access to the VN sensory epithelium and the major source of fluid in the VNO is derived from the Harderian gland Rehorek, ; Rehorek et al. Therefore, this paper describes the effects of female snake sex pheromone, solubilized in Harderian gland homogenate, on the membrane potential and firing properties of VN sensory neurons of male garter snakes.
Thirty-six male and four female red-sided garter snakes T. Mason during the early portion of the mating season early May and transported to Brooklyn, NY, for the electrophysiological experiment. An additional 10 male snakes from the same source were used in a preliminary study to determine whether the pheromone would have the effect of increasing the production of IP 3. The animals were maintained in a cool environment to prolong their sexually active period.
Prior to electrophysiological experimentation, all males were tested for courtship behavior with females to ensure that they were still in mating condition. A total 10 male animals 20 VN organs were used for preparing VN homogenate. For each reaction, the amount of homogenate used was based on protein concentration only. Experimental animals received an overdose of Brevital sodium 0. The VN organs were isolated immediately after the snakes were killed.
The sensory epithelium of each organ was carefully dissected on ice. VN sensory epithelial homogenate 12 mg of protein was incubated with snake pheromone 5 ml in Harderian homogenate mg protein in a reaction solution 50 ml of 25 mM Tris acetate, pH 7. Harderian homogenate alone served as the control.
The supernatants were transferred into new vials. To each vial, 16 ml of 1. Three separate experiments were performed, each in duplicate. Adult, sexually attractive female red-sided garter snakes of varying size snout-vent length range: The animals were killed with an overdose of Brevital sodium. The resulting residues were resuspended in fresh hexane 1—2 ml and sealed in 9-ml glass vials with polyethylene-lined caps for storage.
To isolate the methyl ketones composing the sexual attractiveness pheromone, we fractionated the skin lipid extracts using column chromatography Mason et al. The resulting methyl ketone residues were weighed on a digital scale Mettler AT and resuspended in 1 ml fresh hexane. Each female yielded an average of 1. This pooled solution was subsequently used in the experiments as the stock pheromone solution. Harderian glands were isolated after the snakes were killed with an overdose of Brevital sodium and homogenized in binding buffer with a 5-ml Dounce glass homogenizer.
Pheromone, collected from female garter snake skin lipids, was dissolved in hexane. Pheromone of 2. This mixture was vortexed and exposed to helium gas stream briefly before adding required aliquot to the reaction vial. Electric shock—induced earthworm secretion ESS was prepared as described elsewhere Jiang et al. In response to this shock regime, earthworms secreted a mucus-like fluid that drained into a collection beaker.
This secretion, known to contain chemoattractants for garter snakes Halpern et al. Slices of VNO were prepared from garter snakes as described ly Taniguchi et al. Briefly, the animals were immobilized by cooling on ice for 30—40 min. The slice was placed in a glass-bottomed chamber and fixed in place with a grid of parallel nylon thre supported by a U-shaped silver wire weight. During the experiment, the slice in the recording chamber was perfused constantly with Ringer's solution at a rate of 1—1.
Data were acquired through a DigiData A interface onto a personal computer using pClamp software 9. The al was low-pass filtered at 2 kHz and sampled at 5 kHz. Off-line analysis was performed using Clampfit 9. We estimated the magnitude of inward currents from just before the response to the peak. Liquid junction potentials were measured with a microelectrode containing 3 M KCl Neher, All data in this report have been corrected for junction potentials.
Statistical comparisons were determined using Student's t -test. Curve fitting was performed using Igor Pro 4. Using sky blue dye, we determined that fluid from the micropipette tip reached the epithelial surface in less than 1 s. As expected, due to the high molecular weight and the aliphatic chain length C 29 —C 37we found that the nonpolar female sex pheromone was not soluble in aqueous solvent, even with small amounts less than 0. We tried to partition the nonpolar pheromone into more polar organic solvents by using a mixture of pheromone in hexane and ethanol, acetone, or dimethylsulfoxide, but none of these mixtures were successful in dissolving the pheromone in aqueous reaction buffer.
The amount of detergent required to solubilize the pheromone would have been deleterious to the integrity of the VN sensory epithelium and would have rendered the tissue unusable. As indicated above, since Harderian gland secretions fill the VNO, and it is generally understood that secretions in the oral cavity coat the snake's tongue, it was reasonable to suppose that Harderian gland secretions might normally act to facilitate transfer of female sex pheromone to VNO receptor cells under normal conditions.
We found that Harderian gland homogenate added to female pheromone with a small amount of detergent did, indeed, solubilize the pheromone. Since NP has been widely utilized in other studies e. We initially demonstrated that garter snake sexual attractiveness pheromone activated neurons in the VN sensory epithelium by incubating the pheromone and Harderian gland homogenate with VN sensory epithelium homogenate and assaying for IP 3 production. The effect of garter snake sexual attractiveness pheromone on the yield of IP 3 in VN sensory epithelial homogenate of male garter snake.
VN homogenate 12 mg of protein was incubated with snake pheromone in Harderian homogenate in a reaction solution 50 ml of 25 mM Tris acetate, pH 7. Harderian homogenate was used as a control. The data are means of three sets of separate experiments and each treatment with duplicate samples.
Whole-cell currents were recorded from receptor neurons of male VNOs. Nine cells exhibited spontaneous spike discharges in the absence of external stimulation. The resting membrane potential of these spontaneously active cells was measured when the cells were, on occasion, silent and was recorded at To exclude the effect of Harderian gland on pheromone-induced current, we applied Harderian gland homogenate before application of female pheromone.
Increasing the amount of pheromone caused an increase in the observed currents Figure 1A. The pheromone-induced current was activated with a latency of 5. The dose-response pheromone-induced inward currents were fit to the Hill equation, giving a Hill coefficient of 3. Female sexual attractiveness pheromone evoked inward currents in male VNO neurons. A Representative currents induced by various concentrations of pheromone. The bar at the top indicates the timing of application.
Pheromone-induced current was not observed with buffer solution or Harderian gland homogenate without pheromone.
B Dose-response curve for pheromone induced inward currents. Under current-clamp conditions, of the 20 cells tested, four cells responded to pheromone stimulation with membrane depolarization. The membrane depolarization in response to pheromone was ificantly different from that observed to control Pheromone alone did not ificantly increase action potential firing. During injection of a 2-pA current step to the same cell, pheromone application increased the of action potentials from 2.
Action potential changes before, during, and after application of pheromone are illustrated in Figure 2. Under current-clamp condition, application of pheromone produced a depolarizing membrane potential leading to increased of action potentials in response to a current pulse of 2 pA. In four of 21 cells recorded from 12 male snakes, the time course of the rising phase of the responses at different voltages did not change. In Figure 3a typical reversal potential is illustrated.
The linear I—V plot indicates that the conductance was voltage dependent. B Current—voltage relationships of pheromone induced currents plotted from A at peak current. We examined changes in membrane conductance during application of female snake sex pheromone under voltage-clamp conditions by applying a series of 10 mV depolarizing voltage pulses 10 ms, 0. In four of 15 cells, pheromone application increased the amplitude of voltage step—induced response from The remaining 11 cells did not respond to pheromone and did not change membrane conductance.
A typical response is illustrated in Figure 4. Membrane conductance was increased by the application of pheromone. Under voltage-clamp conditions, a series of mV depolarizing voltage pulses 0. Finally, we examined whether female pheromone also had an effect on female VNO neurons. Female pheromone did not evoke inward currents in female of VN organ neurons. None of the female VN organ sensory neurons responded to the pheromone, whereas three of 11 responded to ESS with induced inward current.
In the present work, we describe for the first time the physiological responses in snake VN neurons to purified female sexual attractiveness pheromone. Stimulation with female pheromone produced inward currents in patch-clamped neurons of male snake VNO slices in a dose-dependent manner. This inward current was accompanied by an increase in membrane conductance. These suggest that the effect of female pheromone on male VNO neurons is to open ion channels causing membrane depolarization and initiation of action potentials.
Inward currents in VN sensory neurons in response to urine have been reported in female rats Inamura and Kashiwayanagi, However, the response latency observed in the present study was ificantly longer than that observed in rat VNO neurons Inamura and Kashiwayanagi, but similar to that reported for snake VNO neurons 2—3 s, Taniguchi et al.
Dissociated mouse receptor cells from female mouse VNOs respond to dehydro-exo-brevicomin DHBa pheromone present in male mouse urine, with outward current at negative holding potentials. In current-clamp mode, DHB causes a hyperpolarization of the neurons Moss et al. Note that this finding contrasts with the studies referenced above and by Leinders-Zufall et al.
Female snake pheromone-induced current was inward at negative holding potentials and outward at positive holding potentials. These indicate that the pheromone-induced membrane response is mediated via IP 3. However, in addition, diacyglycerol DAG may also be a critical member of the al transduction pathway for VNO neurons response to female pheromone. The response of VNO neurons to chemoattractants and pheromones is known to involve the phospholipase C second messenger aling cascade resulting in an increase in intracellular IP 3 and DAG Luo et al.Sex women with snake online
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