Patch-clamp analysis of voltage-activated and chemically activated currents in the vomeronasal organ of Sternotherus odoratus (stinkpot/musk turtle)
1 The Florida State University, Program in Neuroscience and Molecular Biophysics, Biomedical Research Facility, Tallahassee, FL 32306, USA and
2 Yale University School of Medicine, Department of Physiology, New Haven, CT 06520, USA
*e-mail: dfadool{at}bio.fsu.edu
Accepted 9 October 2001
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Summary |
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Key words: vomeronasal, turtle, pheromone, Sternotherus odoratus, olfaction.
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Introduction |
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We hypothesized that Sternotherus odoratus, the common musk or stinkpot turtle, would afford an electrophysiological advantage for the study of functional transduction in the vertebrate vomeronasal organ. Turtles are historically well-suited as robust physiological preparations because of their high tolerance of room temperature and oxygen-reduced recording environments compared with mammalian preparations. This particular semi-aquatic species has a proportionately larger vomeronasal organ (VNO) compared with the main olfactory epithelium (MOE); the former is separate and easily accessible (Murphy et al., 2001). Sternotherus odoratus also has a well-characterized reproductive physiology and pattern of mating behavior (Carpenter and Ferguson, 1984
). The animal secretes a musk produced from specialized glands that it uses in both courtship and predator avoidance (Eisner et al., 1977
).
In previous work, we described the developmental and gender-dependent expression of GTP-binding proteins (Gi and Gß) and the transient receptor potential channel (trp2) in the musk turtle vomeronasal epithelium (Murphy et al., 2001
). The presence of these putative signal-transduction proteins supports their involvement in encoding chemical information, but the functional role of these proteins remains incompletely determined. Ultimately, the initiation and characterization of the electrical event elicited via this transduction cascade shapes the information processed in the VNO. Here, we report a study of the vertebrate VNO in which multiple natural chemicals were presented to single isolated neurons to elicit currents recorded in the whole-cell patch-clamp configuration. Important biophysical properties of these neurons, including a narrow breadth of responsiveness to a chemical and a degree of sexual dimorphism in electrical properties, are described.
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Materials and methods |
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Turtle capture and care
Sternotherus odoratus (stinkpot or common musk turtle) were captured at Auburn University arboretum (Auburn, AL, USA), Lake Jackson (Tallahassee, FL, USA) or Indian Pines (Opelika, AL, USA) [mean male plastron length was 56.5±1.9 mm (N=10) while that of the female was 60.4±0.8 mm (N=22); mean male mass was 88.6±7.6 g (N=10) compared with 98.0±3.9 g (N=22) for females; means ± S.E.M.] from April to September, the approximate peak reproductive season for this species (Carpenter and Ferguson, 1984). Turtles were captured either by hand or using raw chicken-baited hoop nets 0.31 m in diameter. Turtles were housed for periods of 12 months in 38 l aquaria, maintained on a 15 h:9 h light:dark cycle and fed a standard diet of chicken, beef or catfish pellets. Southern populations of S. odoratus reach reproductive maturity at 4 years of age (Tinkle, 1961
). Dr Mary Mendonça, a reproductive herpetologist at Auburn University, assessed that all the animals in this study were adults by noting secondary sexual and other developmental characteristics such as an enlarged tail base, scale patches on the hind legs, unmarked patterning of the carapace, the absence of a vertebral keel and a carapace length greater than 60 mm (Ernst et al., 1994
). In addition, the majority of females at this age had developing eggs in the oviduct.
Collection of natural body fluids
Musk was harvested from the turtles by milking the posterior, reproductive musk glands. Approximately 12 µl of musk could be obtained from one animal. Urine was collected through a 27 gauge needle inserted directly into the bladder. The collected fluids were immediately placed on ice and then stored at 80°C until diluted in turtle Ringer for experimentation. Urine and musk samples used in this study were collected over the 19982000 summer reproductive months (AprilSeptember).
Vital dye labeling and cryosectioning
Turtles were immobilized and anesthetized using ice hypothermia for 30 min. Tetramethylrhodamine-conjugated dextran (Mr 10 000, neutral; Molecular Probes, Eugene, OR, USA) was introduced into the VNO orifice using a handmade syringe pump that delivered 7 µl to the cavity (Friedrich and Korsching, 1997; Wachowiak and Cohen, 1999
). The dextran dye was diluted in PBS plus 0.5 % Triton-X 100 to a final concentration ranging between 2 and 4 % and applied for 20 min. The animal then recovered at room temperature (23°C) for 30 min prior to reintroduction into the aquarium. After migration of the dye over a 2-week period, turtles were reanesthetized on ice and were given a lethal injection of sodium pentabarbitol (Butler Company, Columbus, OH, USA) before decapitation. The VNO and combined olfactory bulb (OB) and accessory olfactory bulb (AOB) were quickly dissected from the turtles using methods approved by the American Veterinary Association and NIH, as reviewed by the Animal Care and Use Committees of Florida State University and the Marine Biological Laboratories.
For cryosections, whole VNOs and combined OB/AOBs were fixed in 4 % paraformaldehyde in PBS for 3 h followed by overnight infiltration with 10 % sucrose in PBS and then 4 h with 30 % sucrose in PBS. Tissues were cut to a thickness of 912 µm on an Ames Microtome-Cryostat (model 4550; Tarrytown, NY, USA). Sections were transferred to 1 % gelatin-coated glass slides (Sigma) and stored at 20°C until use.
Histochemistry and photomicroscopy
Rhodamine-conjugated dextran-labeled cryosections were viewed at 10x and 40x magnification without any further treatment using a Nikon Microphot-FX (Melville, NY, USA) microscope equipped with epifluorescence and a Nikon FX35WA automatic photomicrographic system using Kodak Elite II 400 ASA film. Other cryosections were histochemically stained using standard protocols for Massons Trichrome staining as cited by Humason (1972). In both instances, 35 mm films were scanned with a Hewlett-Packard Photosmart Scanner (model 106-816) or a Nikon LS2000. Digital files were imported into Photoshop (Adobe 5.5) to facilitate multiple panel/figure output on a Fuji Pictographic 3000 printer.
Electrophysiology
Turtles were anesthetized on ice and then killed by administering a lethal injection of sodium pentabarbitol (Butler Company) followed by decapitation. Neurons from the VNO were isolated either by incubation in a low-Ca2+, hyperosmotic dissociation saline (DS) followed by a brief incubation in 0.5 µg ml1 DNase I solution or by incubation in Ca2+-free, cysteine-activated papain (50 units; Worthington Biochemicals, Lakewood, NJ, USA) in turtle Ringer. For either dissociation protocol, the VNO was cut into quarters and incubated in appropriate dissociation saline for 1518 min with gentle rotation at 30 revs min1 on an orbital shaker at room temperature. Tissue quarters were then removed from the incubation saline and placed into cold turtle Ringer. Tissue quarters could be held at 4°C for up to 8 h prior to physical dissociation. A VNO quarter was dissociated by trituration using a size-graded series of fire-polished siliconized Pasteur pipettes. The resulting single vomeronasal cells were plated onto 0.01 % poly-D-lysine-hydrobromide-coated (Mr 49 00053 000; Sigma) and 0.1 % laminin-coated (laminin-like engineered protein polymer, Sigma) Corning dishes (Fisher no. 25000) (Leinders-Zufall et al., 1997).
Isolated vomeronasal neurons were viewed at 40x magnification (Axiovert 135, Carl Zeiss, Thornwood, NY, USA) with Hoffman modulation contrast optics for patch-clamp recording (Hamill et al., 1981). Patch pipettes were fabricated from Jencons borosilicate glass (catalog number M15/10; Jencons Limited, Bedfordshire, UK), fire-polished to approximately 1 µm (bubble number 5.0) (Mittman et al., 1987
) and coated near the tip with beeswax to reduce the pipette capacitance. Pipette resistances were between 7 and 10 M
, which produced high-resistance seals (between 8 and 14 G
) by applying gentle suction to the lumen of the pipette upon contact with the cell. In all experiments, cells were voltage-clamped at a holding potential (Vh) of 60 mV unless specified otherwise. Each cell was photographed (Minolta X-700, Ramsey, NJ, USA) to allow for correlation of soma size, length of process and cell morphology with physiological properties.
Voltage- and chemically activated currents were recorded in the whole-cell configuration using an integrating patch-clamp amplifier (Axopatch 200B, Axon Instruments, Foster City, CA, USA). The analog output was filtered at 5 kHz and digitally sampled every 0.5-4 ms. Data acquisition and subsequent storage and analysis of the digitized recordings were carried out using pClamp8.0 software (Axon Instruments) in combination with the analysis packages Origin (MicroCal Software, Northampton, MA, USA) and Quattro Pro (Borland International, Jericho, NJ, USA). Data traces were subtracted linearly for leakage conductance. The inactivation of the outward macroscopic current was fitted to a single-exponential function by minimizing the sums of squares. Differences between sexes in a particular biophysical property were analyzed by Fmax-test and then by Students t-test. No tests with unequal sample sizes violated homogeneity of variance (i.e. none failed the Fmax-test), and statistical significance in all tests was defined at the 95 % confidence interval (Steel and Torrie, 1980).
Chemical stimulation
Chemicals were puffer-applied to the vomeronasal neurons for 700 ms from a seven-barrelled glass micropipette (1.2 mm outer diameter, catalog no. 17-12-M; Frederick Haer, Bowdoinham, ME, USA) coupled to a pressurized valve system (Picospritzer, General Valve, Fairfield, NJ, USA) (Fadool et al., 1991). In most trials, fluorescein, the position of which was varied from one experiment to another, was used as an indicator in one barrel of the pipette to position the tip of the pipette relative to the cell and to ensure that the delivered compound completely surrounded the cell and its associated processes. The magnitude of the response to 0.5 mol l1 KCl was found to be independent of which of the six barrels contained the depolarizing solution. Dilution of the chemical between the pipette and the cell surface, an average distance of two cell diameters, was estimated to be approximately 9 % on the basis of the calculated K+ permeability method of Firestein and Werblin (1989
).
Chemical concentrations are reported as the pipette concentration and are not corrected for this dilution. The pipette concentration of urine varied between 1:5 and 1:20 dilution from full strength and that for musk varied between 1:100 and 1:300. Catfish extract was prepared from a commercially available pellet (Florida Farm and Feed, Tallahassee, FL, USA). Pellets (2 g) were hydrated in 20 ml of high-quality water, ground by mortar and pestle, and then clarified by low-speed centrifugation for 5 min at room temperature. The extract was stored at 80°C and diluted 100- to 1000-fold for use. All dilutions were prepared daily in turtle Ringer, which served as the control vehicle in all conditions.
If a neuron responded to the control, it was assumed to be a mechanical stretch-activated response, and no further use was made of the cell. The peak magnitude of a response was measured as the difference in current from the baseline prior to presentation of the chemical to the peak outward- or inward-evoked current within 500 ms of valve activation of the picospritzer. Zero current (no response) was defined as no observable deflection or a deflection that was less than four times the total noise level (membrane plus equipment) under control baseline conditions.
The number of different compounds that stimulated a given cell (the response spectrum) was quantified using the breadth of responsiveness metric of Smith and Travers (1979). Here, the breadth of responsiveness (H) is defined as:
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where K is a proportionality constant, n is the number of chemicals tested, pi=|pA| is the absolute current (pA) elicited by the nth chemical and expressed as a proportion of the total current elicited by all chemicals. This equation is an application of the entropy equation as a measure of diversity in neural responsiveness. The pi values for each neuron are derived by converting the neural response profile for that neuron into a proportional profile in which the response to each chemical is expressed as a proportion of the total response to all five chemicals (Smith and Travers, 1979). The value of K=1.4306 was calculated so that H=1.0 when pi=1/n. Therefore, when there is a response to only one of the five chemicals, the breadth of responsiveness is minimal (H=0), and it is maximal (H=1.0) when there is an equal response to each of the five chemicals (no selectivity or diversity in response) (Smith and Travers, 1979
).
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Results |
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Several protocols for dissociation of neurons were explored to achieve acute isolation of single vomeronasal neurons and, ultimately, to preserve the proteins of the chemosensory receptors and related signal cascade. The least mechanical damage and tightest seals were achieved with a low-Ca2+, hyperosmotic dissociation saline (Dionne, 1992) or a weak and brief incubation in cysteine-activated papain (see Materials and methods). Both these methods yielded viable neurons with identical electrophysiological properties. Since the latter method was less time-consuming and more reliable, all neurons used to generate results for this report were subjected to this brief papain treatment for dissociation. Doubling the papain concentration, long orbital rotations, isolation with DNase I only, mechanical trituration without the use of a siliconized pipette surface or incubation with the bacterial enzyme subtilisin resulted in the loss of either voltage-activated Na+ currents or chemically activated currents or resulted in an inability to patch the VN membrane, even though each of these protocols has been used to isolate neurons in other sensory organs (Fadool et al., 1993
; Lumpkin and Hudspeth, 1995
; Trotier et al., 1998
).
Isolated VN neurons were viable for up to 8 h following initial dissection of the organ. The neurons had a morphology similar to that observed in other vertebrate species (Taniguchi et al., 1995; Trotier et al., 1998
), namely a distinct lengthy dendrite terminating in a tuft of microvilli (Fig. 1E,F). In preparations that were first dextran-labeled and then dissociated, the morphological cell type shown in Fig. 1E,F was consistently labeled. Cells of fusiform or spherical soma morphology, both of which lacked processes, were not studied. Cells of varying dendritic process length and soma diameter were deliberately sampled to ascertain whether a particular neuronal morphology was correlated to a higher degree of either voltage- or chemically activated sensitivity. The mean soma diameter of VN neurons harvested from female animals was significantly greater than that calculated for male animals (8.5±1.6 µm, N=68 for females; 7.4±1.5 µm, N=32 for males; Students t-test,
=0.05, means ± S.E.M.) (Fig. 2A,B). The mean process length for neurons from male animals was 45.9±32.7 µm (N=27) and that from female animals was 35.6±20.0 µm (N=60) (Fig. 2C,D), and these values were not significantly different (Students t-test,
=0.05). We could not determine whether the high variability in process length was real or was attributable to the dissociation process. The following measurements were made in both sexes and found to be gender-independent, with correlations coefficients ranging from r=0.27 to +0.26: dendritic length or soma diameter versus chemically activated current magnitude; dendritic length or soma diameter versus voltage-activated Na+ current magnitude; and dendritic length or soma diameter versus inactivation kinetics of K+ currents. Because no strong correlation (r>0.90) existed between cell shape and evoked-current properties, subsequent data were not grouped by morphology (i.e. dendritic process length or soma diameter) but were treated as a whole.
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The peak amplitude of currents of both polarities evoked from the five natural stimuli ranged from 92 to 180 pA (Fig. 6A), demonstrating a degree of variability. The mean magnitude of the inward current (13.2±3 pA, mean ± S.E.M., N=48) was significantly less than that of the outward current (36.5±18 pA, mean ± S.E.M., N=9) irrespective of the type of chemical stimulus (N=57 responses to chemicals, recorded from 33 neurons; t'-statistic, =0.05). In selecting a mate, Sternotherus odoratus has the capacity to orient towards the opposite sex by using water-borne chemical cues up to a 1.6 km radius from the site of release (Carpenter and Ferguson, 1984
). Urine could act as this chemical cue, and the pheromone constituents in mammalian urine are thought to be plural, with the ability to alter inter-male aggression and endocrine behavior patterns (Chateau et al., 1976
; Johns et al., 1978
; Menzies et al., 1992
). Our electrophysiological data indicate that both male and female urine are detected by VN neurons harvested from musk turtles of both sexes.
Female urine and male urine applied to neurons harvested from female animals evoked inward currents that were approximately 23 times larger than those elicited by the same compounds presented to neurons harvested from male animals. It is not clear whether large urine-evoked currents in females are important for the coding of this signal, but it is noteworthy that 90 % of the outward currents were elicited by urine and detected by female VN neurons. The peak inward current elicited by female urine or male urine in female VN neurons was, however, significantly smaller than that evoked by female musk or male musk (Table 2). Musk-evoked inward currents were three times the mean magnitude of urine-evoked or catfish-extract-evoked inward currents (Fig. 5) (Table 2). Neurons harvested from female animals exhibited musk-activated inward currents, while those harvested from male animals failed to respond to musk, whether it was female or male musk (Table 2).
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Discussion |
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Sternotherus odoratus initiates courtship by tactile contact between the nose of the male and the posterior musk gland and cloacal region of the female. If the male finds the female suitable for mating, through chemical cues in the musk or urine, he will bite her head and climb to position her under his plastron (ventral shell surface) for mating (Ernst et al., 1994). Although it is not clear whether these sampled odors are processed through the VNO or the MOE, our data demonstrate that both male and female musk evoke inward currents in neurons of the vomeronasal organ. The magnitude of musk-evoked inward currents is 35 times that evoked by urine, implying that this compound could provide a different chemical or reproductive cue encoded by strength of response. Unlike urine, musk elicits only inward current, not currents of either polarity. Interestingly, and uncommon in olfactory sensory neurons, the chemosensitivity of the VN neurons is also gender-dependent. The peak magnitude of urine-evoked currents from female VN neurons is three times that elicited in male VN neurons.
Most unexpected, and incongruent with the reproductive behavior of this species, is the fact that male VN neurons failed to respond to female or male musk. It is possible that the component(s) in the musk to be detected by the male VN neuron is not completely soluble in the manner in which we are applying the stimuli to the neuron. It is also conceivable that the component(s) in the musk is quite labile and that the more sensitive female VN neurons have the ability to retain electrical responses to this component(s) whereas male VN neurons do not. It cannot be ruled out that the failure of these male neurons to detect female or male musk was a result of the small number of male animals that we were able trap. Further electrophysiological studies are needed to increase the number of male VN neurons sampled. The present study was undertaken utilizing S. odoratus trapped during the peak breeding season only, and it will be interesting to test the chemosensitivity of non-breeding animals captured during the winter months and of animals captured prior to reproductive maturity (as juveniles). We have previously demonstrated that juvenile S. odoratus express a distinct G-protein that appears to be redistributed to the microvillar layer following maturation to adult animals (Murphy et al., 2001). It would be interesting to determine whether the components of the electrical transduction event, as assessed biophysically, were also expressed differently in juveniles.
The majority of chemically activated responses were inward currents of moderate amplitude (less than 25 pA), but a small proportion of the responses were outward currents (Fig. 6). It is noteworthy that no single responsive VN neuron was observed to produce chemosignal responses of both polarities, but our data cannot rule out this possibility. The data are not inconsistent with having two signal-transduction cascades operational in this population of VN neurons, each mediating either an inward or an outward current. This suggests that there could be two different functional neuronal types in the vomeronasal organ, each containing different signaling cascades, rather than a single neuron containing dual transduction cascades. Further experimentation will be required to clarify this.
Pharmacological and kinetic data suggest that the balance of the contribution of voltage-activated channels in VN neurons may be slightly different across species. In mammalian and amphibian VN neurons, there is a TTX-sensitive and a TTX-insensitive Na+ current (Trotier et al., 1993; Liman and Corey, 1996
; Inamura et al., 1997a
), whereas in S. odoratus the Na+ current is completely blocked by TTX and its magnitude is much smaller. In the frog VNO, the outward K+ current was only slightly deactivated after 250 ms (Trotier et al., 1993
). In the turtle (present report) (Taniguchi et al., 1996
) and in mammals (Liman and Corey, 1996
; Inamura et al., 1997b
), there are two kinetic components of K+ current inactivation, which is consistent either with two channel species or a single channel type with multiple kinetic rates of inactivation. Also similar in these two models is the relatively slow rate of activation of the K+ current; in the mouse, the time to half-activation is of the order of 10 ms and in the turtle it is 24 ms (depending upon sex), whereas mammalian olfactory sensory neurons are rapidly activated with a half-activation time of 1.3 ms (Liman and Corey, 1996
).
Earlier electrophysiological studies did not correlate voltage-activated properties with the sex of the animal, so it is not clear whether the dimorphic biophysical properties observed in the musk turtle reflect general principles of vertebrate VN neurons or are restricted to this species, which is clearly dimorphic in body size (Ernst et al., 1994). It is curious that, in the musk turtle, the transient component of the outward current makes a longer contribution to the K+ current in neurons from males than from females. The differential inactivation kinetics of the outward current would be consistent with the presence of two different channel species in the two sexes. Because only a fraction of the K+ current would be activated during the 13 ms required for an action potential to rise from 0 to +40 mV in the VNO (Liman and Corey, 1996
), male VN neurons would have the capacity to carry a larger fraction of the current during this period, which is critical for achieving threshold and interspike frequency. The additional difference in inward current between sexes would be consistent with a greater current density of Na+ channels in male compared with female neurons, with a concomitant enhanced electrical excitability of the male VN neuron.
Recent electrophysiological studies suggest that there may be differences in the properties of chemically activated currents of VN neurons amongst species (Inamura et al., 1999; Inamura and Kashiwayanagi, 2000
; Taniguchi et al., 2000
). Very different when comparing the chemically activated current in turtle neurons with that in rat and snake neurons are the polarity, the magnitude and the rate of activation and recovery of the response. The response polarity for turtle VN neurons to urine could be both inward or outward, whereas only inward currents have been reported in rat (Inamura and Kashiwayanagi, 2000
). The mean response magnitude for inward currents in the turtle was approximately one-third to half the magnitude of those reported for rat. By comparison, the mean value for snake chemically activated currents was 25 times larger (791 pA) than for either rat or turtle (Taniguchi et al., 2000
). The most striking difference in the three species is the kinetics of the response. Turtle VN neurons respond to a 700 ms chemical stimulation within several hundred milliseconds, and the response decays to baseline levels over 34 s (Fig. 5). In rat and snake, a 1520 s stimulation is required for full development of the response. The response then decays to baseline levels over 30240 s. In spite of differences in the chemical delivery systems and recording configurations in these studies, the response profiles and underlying activation mechanisms must have inherent differences. Both turtle and snake appear to use the VNO to detect compounds derived from prey items (catfish extract in the case of turtles and earthworm secretion in the case of snakes) (Luo et al., 1994
; Taniguchi et al., 2000
). These data suggest the possibility that, in some species, the VNO may subserve a function for prey identification and not function solely for reproductive and social chemical communication.
Several informative electrophysiological studies have been conducted in the turtle and, although none has explored chemically activated currents using whole-cell patch-clamp recording, they contribute to our knowledge of the biophysics of these neurons. It is important to note that the relative size and function of the two olfactory divisions [main olfactory epithelium (MOE) versus vomeronasal organ (VNO)] have been shown to be extremely habitat-dependent in turtles. Caution must be taken in interpreting the function or biophysical properties of these neurons without regard to species. The earliest electrophysiological studies on the VNO relied on extracellular recordings and were modeled on a terrestrial species, Terrapene carolina. In this species, it was found that general odorants caused an increase in action potential firing in both the VNO and MOE, but that several single compounds could be detected at concentrations two orders of magnitude lower by the MOE compared with the VNO (Graziadei and Tucker, 1970).
Most recent electrophysiological studies in turtle VNO were modeled on a semi-aquatic species, Geoclemys reevesii, and demonstrated that patch electrode dialysis of either cyclic AMP or inositol 1,4,5-trisphosphate (InsP3) evoked inward currents in these VN neurons (Taniguchi et al., 1995, 1996
). In G. reevesii, no chemical has been identified that activates either the cyclic AMP or the InsP3 cascade, so the biological roles of these second-messenger-gated ion channels have not been determined. Given the localization of Gi
and Gß G-proteins to the microvillar layer in S. odoratus (Murphy et al., 2001
), either or both of these messengers could mediate chemically activated currents in the musk turtle. A role for InsP3 signaling pathways in musk turtles is suggested by the presence of transient receptor potential channel expression (Murphy et al., 2001
) and type III InsP3 receptor expression (Fadool et al., 2001
) in western blot analyses of purified VNO membranes. We do not know how or whether these proteins are gated by natural chemicals, but our preliminary findings indicate that the type III InsP3 receptor and the trp2 are in a proteinprotein interacting complex (Fadool et al., 2001
).
Chemically activated currents form the basis of electrical information passed through the vomeronasal organ to encode reproductive, social, territorial and species-recognition behavior. Further electrophysiological analysis of the biophysical properties of single VN neurons will provide the mechanistic details of signal transduction in chemical communication in vertebrates.
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Acknowledgments |
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