1Center for Biomedical Engineering and 2Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555-0456
Submitted 22 January 2004 ; accepted in final form 16 June 2004
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ABSTRACT |
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snake; thermosensory; trigeminal; ion conductance
The pit organ shows structural specializations indicative of a high-resolution thermal detector. The temperature-sensing terminal nerve endings innervate an ultra-thin (<25 µm) pit membrane (29) that acts as the heat-sensing surface. This thin membrane and the associated terminal nerve mass are suspended in a facial cavity in front of an inner air space, giving the pit organ an extremely low thermal mass. The pit organ also has a rich capillary network that is thought to act as a rapid heat sink (12). This combination of low thermal mass and rapid heat dissipation contributes to the precise temporal and spatial resolution of thermal stimuli. Multiple- and single-unit neuronal activity recorded from pit organ afferents demonstrates that these receptors are sensitive to very small temperature changes (0.003°C) (3) and can transmit this thermal information over a broad range of temperatures (1537°C). This suggests that the pit organ must have highly sensitive neurons and ion channel mechanisms capable of receiving and transmitting thermal information and that these complement the anatomic specializations of the pit organ for heat reception. However, the cellular and molecular machinery responsible for converting temperature information into neuronal signals in the pit organ is completely unknown.
A generator potential for thermal stimuli at the pit organ has been identified using extracellular recordings (34), and this supports the hypothesis that the terminal nerve mass may express temperature-sensitive integral membrane proteins. This is also consistent with reports on mammalian thermoreceptors, especially thermal nociceptors, where the activation-temperature threshold, ion conductance, and even behavioral features (6, 7) have been correlated to the presence of a temperature-gated cation channel TRPV1. Thermal nociceptive neurons are fairly plentiful and thus benefited studies linking TRPV1 to thermal responses. The snake pit organ is a unique system for the study of these lower temperature-threshold thermoreceptors because it has the highest known density of warm receptors (4) and there are far fewer "warm" receptors in mammals (3, 15, 24). By analogy to TRPV1, ion conductances corresponding to warm receptors may be abundant in neurons that project to the pit organ.
Here, we demonstrate for the first time that dissociated neurons from the pit viper trigeminal ganglia (TG), which supplies the pit organ (4), show a heat-sensitive current with temperature-threshold and biophysical properties unlike those identified in mammalian neurons. Voltage-clamp recordings revealed an inward monovalent cation current (IT) that increased with heating and tracked temperature change. I
T was found in a large proportion of TG neurons that were isolated and had a threshold of
18°C.
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MATERIALS AND METHODS |
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Culture of TG. Cell bodies that have sensory terminal nerve specializations in the IR/heat-sensitive pit organ are found in the ganglia of ophthalmic, maxillary, and mandibular branches of the trigeminal nerve (4). Snakes were anesthetized with Isofluorane USP (Abbott Laboratories, North Chicago, IL) and then decapitated; the head was placed on ice to minimize cell death after decapitation. The TG was located visually, removed, and placed in ice-cold F-12:DMEM (Gibco BRL). Cells were dissociated from the ganglia by treatment with collagenase (1 mg/ml, 45 min, 28°C) and trypsin (2.5 mg/ml, 5 min, 22°C) followed by mechanical dissociation with plastic pipettes. The dissociated cell mixture was layered onto 25% percoll (Sigma, St. Louis, MO) and centrifuged for 10 min at 500 g to remove large cell bodies and debris (11). Cells were cultured in F-12:DMEM supplemented with 10% fetal bovine serum, 10 U/ml penicillin, 10 µg/ml streptomycin (Gibco BRL), and 2 mM glutamine (Gibco BRL) on poly-D-lysine-coated coverslips or in treated 12-well tissue culture plates (BD Falcon). Cells were maintained at 28°C in 7% CO2-93% air for 1696 h.
Electrophysiology.
Recordings from neurons were made in HEPES-buffered saline [HBS; in mM: 130 NaCl, 3.0 KCl, 2.0 CaCl2, 1.2 MgCl2, 10 HEPES (pH 7.3), 10 glucose]. Neurons were voltage-clamped using single-patch electrodes in the whole cell mode (14) and held at 65 mV using an Axon Instruments model 200A amplifier. Patch pipettes were made from 1.65-mm-outer diameter 7052 glass (Garner Glass) and pulled to a resistance of 25 M using a Sutter Instruments P87 puller. Patch electrode solutions contained 140 mM K-gluconate, 2 mM MgCl2, 2 mM BAPTA, 0.2 mM CaCl2, and 1 mM HEPES with pH 7.4. K-gluconate was substituted with CsCl for reversal potential (RP) experiments. Ion permeability ratios were calculated using modifications of the Goldman-Hodgkin-Katz equation (22). Voltage-clamp protocols were controlled using the pCLAMP (v. 6.2, Axon Instruments) suite of programs, and current traces were filtered using algorithms supplied in the software. Statistical analyses were preformed using StatView (ver. 5.0.1, SAS Institute, Cary, NC).
Calcium imaging. Simultaneous voltage-clamp and intracellular Ca2+ level determinations were made using the perforated patch technique on fura-2-loaded neurons (41). Neurons were loaded with 5 µM fura-2 AM (Molecular Probes) in HBS at room temperature for 1 h. Patch pipettes were loaded with an electrode solution consisting of (in mM) 30 KCl, 65 K2SO4, 10 HEPES, pH 7.4, and 1 EGTA. Amphotericin B (Sigma) at a final concentration of 0.5 µg/ml was used in the internal solution to provide access to the cell. Gigaohm seals were placed on neurons and cells were voltage-clamped at 65 mV. Cell access was monitored by observing the change in cell capacitance and membrane currents during a 15-mV voltage step. The preparation was illuminated with a 75-W xenon light source attached to a beam splitter (Oriel, Stratford, CT) that directed light through a 340- or a 380-nm filter. A dichroic filter (510 nm) directed the illumination to the neurons through a 100x oil-immersion lens (1.3 numerical aperture), and a photomultiplier tube (Oriel) quantified fluorescence emission above 510 nm. Fluorescence emission data from excitation at 340 and 380 nm were collected every 1.55 s.
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RESULTS |
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Temperature responses were elicited by applying heated or chilled HBS as close as possible to the voltage-clamped neuron using a polypropylene Pasteur pipette. A thermocouple with rapid temperature response (Physitemp, Clifton, NJ) placed within 500 µm of the cell was used to estimate the thermal stimulus delivered to the cell. We determined heating and cooling parameters from 30 representative temperature records in which we sought to determine the current response of neurons to a temperature step. Representative temperature traces are presented in Fig. 2, AC, top. The cells were heated from a resting room temperature of 20.4 ± 1.4°C (SD) to a final temperature of 34.5 ± 4.8°C (range 2749°C) at a mean rate of 3.3 ± 1.8°C/s. The duration of the elevated temperature step was variable but always greater than 10 s, during which there was ambient cooling at a rate of 0.17 ± 0.05°C/s. The cells were then cooled with chilled HBS at a rate of 2.0 ± 1.0°C/s back to baseline temperature to show reversal of the response.
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ITs recorded in C. atrox and A. contortix were very similar (Table 1). The magnitude of I
T was 15.6 ± 11.7 pA/°C (SD, n = 13) in TG neurons from C. atrox and 11.2 ± 14.2 pA/°C (n = 36) in TG neurons from A. contortix. The range was 3.9 to 45.4 pA/°C for C. atrox and 2.15 to 63.7 pA/°C for A. contortix. We calculated current density of I
T based on the assumption that TG neurons were spherical, with a surface area determined by the formula 4
r2. The values for surface density are expressed as nA·°C1·cm2 and are presented in Table 1 for the species tested. Current densities showed high variability and were log10 transformed for statistical comparisons (40). There was no statistical difference in I
T current density in TG neurons of C. atrox vs. A. contortix. We pooled data from both snakes to examine the effect of cell size on current density. There was a weak but statistically significant negative correlation (40) between neuron surface area and the log10-transformed current density value (r = 0.49, sr = 0.16, n = 31, t = 3.03, P < 0.05), indicating that smaller cells tend to express more I
T.
We also examined heat-responsive currents in TG neurons from the common garter snake (T. sirtalis; Fig. 2C and Table 1), a snake that does not have specialized heat-sensing organs, but is in the same superfamily (Xenophidia) as the crotaline snakes. TG neurons were isolated and recorded from three different snakes. Temperature-activated currents resembling IT were seen in 3 of 20 TG neurons (15%) from T. sirtalis. It was possible to make a statistical comparison between the proportions of I
T-expressing neurons in the pooled population of C. atrox and A. contortix TG neurons vs. those of T. sirtalis because the sample sizes were within conservative estimates necessary to maintain statistical power for comparisons of unequal samples (40). The crotalines had a significantly greater proportion of I
T-containing neurons in their TG than T. sirtalis (1-tailed test for significant proportions, Z = 6.87, P < 0.01). Additionally, TG neurons from C. atrox or A. contortix had a significantly higher I
T current density than TG neurons from T. sirtalis (see Table 1 for statistical information).
Cooling cells from RT revealed that IT is activated within the range of temperatures that a pit viper would encounter in the wild (38) (Fig. 3A). We examined current-voltage relationships of cells that were first cooled to 1215°C before heating to determine the temperature at which I
T was inactive. Cells cooled to 1215°C showed no further temperature-dependent changes in current, suggesting I
T was inactive in this range (Fig. 3B). The current-temperature relationship of I
T from A. contortix is shown for five cells presented in Fig. 3C. These cells were chosen because they had stable I
T data over a large range of temperatures, and current values were subtracted from the baseline that represented the lowest value over the record. The I
T vs. T relation showed a nonlinear response, with little current increase at lower temperatures (1015°C) transitioning to a more rapid rate of increase at just below 18°C. Regression lines for the two components were predicted by least-squares method minimizing the trend in residuals (40), and the threshold was determined to be 17.8°C for the steeper current gain per unit temperature.
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Previous research established the pharmacological identity of several temperature-sensitive ion channels (7, 20, 23, 25). We exposed IT-expressing TG neurons from A. contortix to 10 µM capsaicin [in 0.001% (vol/vol) ethanol] to determine if a pharmacologically active TRPV1 homologue is present in these cells (7). Cells did not respond to capsaicin nor was the magnitude of response to heat stimulus changed (n = 4). Similarly, amiloride, which blocks temperature-sensitive epithelial Na channels (2), had no effect on the magnitude of I
T in TG neurons (n = 5).
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DISCUSSION |
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Use of in vitro preparations to study thermoreceptors. A generator potential for pit thermoreceptors has been recorded from crotaline snakes including Agkistrodon (34). Terashima and colleagues (34) recorded both voltage spikes and slow potentials using extracellular electrodes inserted just below the pit membrane into the terminal nerve mass. These potentials were proportional to stimulus intensity and duration but drifted in direction due to the instability of the preparation. We chose to use a voltage clamp to obtain more detailed biophysical information on temperature-activated currents, but we were unable to clamp the terminal nerve mass, which probably contains the highest density of cellular structures that cause temperature-dependent currents (i.e., heat-sensitive ion channels). Recordings were taken from cell bodies because of their ease of preparation and because temperature responses of neurons have been shown to be accurately replicated in cultured trigeminal and dorsal root ganglion (DRG) neuron cell bodies (8, 23, 27, 31). This is because heat-sensitive ion channels are expressed and active at the cell body in cultured neurons.
Neurons were subjected to a wide range of temperatures during these recording procedures, and some of these neurons showed no response to temperature change. This is consistent with findings in ganglion cultures from mammalian sensory neurons where there was a clear distinction between temperature-sensitive and -insensitive cells and suggests that there was no artifactual temperature-induced activation of resting leak or voltage-activated currents with thermal stimulus. Cells expressing IT showed characteristic nonlinear relationship between temperature and currents that indicated a threshold just below 18°C. This type of current-temperature relationship has been seen for temperature-activated currents (36) and for isolated temperature-gated ion channels from mammals (7, 25, 39). The transition from slow to rapid current change as a function of temperature is thought to happen at the temperature in which there is a rapid and reversible change in the ion channel, possibly associated with temperature-dependent gating of the channel (36).
We could not record APs in response to heating of TG neurons, although it is clear from studies on intact preparations that heat sensation in the pit organ is transmitted via APs. It is possible that the rate of heat stimulus we presented to these cells was too slow to generate APs. Temperature changes in the low thermal mass pit organ should be nearly instantaneous, even if they are small. With slow changes, however, ion channels responsible for stimulating regenerative changes in membrane potential might be inactivated before the AP could take place or voltage-activated channels responsible for terminating APs might be activated before the IT could significantly change the membrane potential. Two previous studies (3, 9) suggest that pit afferents respond to the rate of temperature change as well as the stimulus intensity. However, changes in AP frequency could be distinguished even with very long temperature rise times (3). It is also possible that the density of I
T in dissociated TG neurons was not sufficient to give rise to AP-producing depolarization of membrane potential or that accessory channels involved in AP production in these cells were not adequately expressed. This could be the result of low or altered expression of putative ion channel(s) responsible for I
T or lowered or altered AP-associated ion channels in dissociated cell culture. Finally, it is possible that the role of I
T is not to produce fast enough or robust enough depolarization for AP activation but instead modulate ongoing changes in membrane potential. This is supported by studies from pit viper trigeminal nerve (3, 9) and TG neurons (33) showing that these elements are spontaneously active and that temperature change is signaled by alterations in ongoing spike activity. We did not see spontaneous APs in our cells, possibly as a result of dissociated culture, but we speculate that one mechanism by which I
T might signal heat is to produce a slow depolarization that increases spike frequency, as has been described for other sensory systems (5, 10).
Neurons expressing a temperature-sensitive current similar to IT were also found in the TG from the common T. sirtalis, a snake that does not have a specialized organ for sensitive heat detection, but at a significantly lower frequency and with lower current density. It is not surprising that we found thermosensitive cells in the TG from this snake, as this ganglion supplies more general temperature sensors to the face (4). It is also possible that some of the temperature-responsive neurons we recorded from the C. atrox and A. contortix TG are actually cutaneous warm receptors that did not send projections to the pit organ but to other parts of the face. If this is the case, then the frequency of cells expressing I
T we observed in the TG is not an accurate estimate of the frequency of warm-receptive neurons sending projections to the pit organ but instead to the entire facial region innervated by the trigeminal nerve. The frequency of TG cells showing various temperature-activated currents that actually send afferents to the pit organ will have to be determined using retrograde labeling from the pit organ in conjunction with physiological recordings in the TG. However, a comparison can be made with the frequency of warm-receptive neurons found in the TG of the T. sirtalis, as both estimates were made using the same sampling procedure. The fact that I
T in T. sirtalis was qualitatively similar to that of pit vipers has interesting implications in the evolution of the pit organ and its thermosensitivity. The pit afferents of crotaline snakes have been shown to have a higher number of warm-sensitive fibers than any known animal (3). Our data on proportion of heat-sensitive neurons in pit vipers vs. T. sirtalis are consistent with these findings. Additionally, our data also showed that the current density of I
T is much higher in pit vipers than in T. sirtalis. It is possible that pit viper thermosensors evolved as specializations of general cutaneous warm receptors like those that would be found in all snakes. The evolution of a specialized pit organ was accompanied by an expansion of numbers of warm-sensitive neurons supplying the area, resulting in very high thermal sensitivity of the pit organ. Additionally, individual neurons may be more sensitive to temperature, and this would result in these neurons having greater electrophysiological responses to temperature change further enhancing sensitivity of an integrated thermoreceptor like the pit.
IT correlates with behavioral data on snake thermosensing.
The concordance in temperature-response characteristics between behavioral or nerve recordings and cultured neuronal preparations has often been used to establish a causal link between temperature sensation and temperature-activated currents (8). Our data from snake TG neurons correlated with features discovered from behavioral, whole nerve, or single-fiber recordings from pit vipers. Most notably, the temperature threshold for I
T in pit viper TG was in good agreement with recordings from the pit afferent nerves that showed loss of spontaneous spiking activity at 1015°C (9). Above this threshold, there was a constitutive inward current, which may be related to "background" activity of nerves seen above 18°C. The temperature-tracking characteristics of I
T may also be related to the nonadapting spike discharge seen in pit afferents when the pit is heated. The detailed investigations of de Cock Bunning et al. (9) demonstrate that rapid adaptation of spiking frequency is a property of rapid cooling in the low thermal mass pit organ and not an adaptation at the nerve itself. Adding water to the pit increased its thermal mass and resulted in a nonadapting discharge of pit afferents. This suggests that the neural response is constant throughout the thermal stimulus and is in agreement with the behavior of I
T. The temperature-tracking characteristics of I
T were also consistent with the slow, continuous temperature-dependent changes seen in the extracellular generator potentials recorded at the pit membrane (34).
Behavioral data also show that pit afferents stop firing above 37°C (9), and this does not correlate well with the behavior of IT, which showed responses even at and above 40°C. This raises the possibility that there may be other temperature-sensitive currents that mediate thermosensitivity at the pit organ. It is possible that there may be a high-threshold temperature-sensitive current that responds at temperatures above 37°C and may be involved with the cessation of the pit response at these temperatures. We already have preliminary data that suggest that the TG may contain neurons that have a transiently activated cooling-sensitive current that may contribute to the sensitivity described in snake thermosensing (30).
Similarities and differences between IT and currents from temperature-sensitive ion channels.
The hypothesis that a heat-sensitive ion conductance, similar to the one found here for the snake, is a transducer of thermal information has also been proposed and investigated for mammalian primary thermosensory neurons in vitro (23, 27, 31). In some neurons, currents evoked at the level of thermal nociception (
42°C) can also be mimicked by capsaicin (7, 23, 27), the noxious ingredient of hot peppers that can elicit a similar thermal sensation and can be partially blocked by antagonists for capsaicin binding (32). This feature has allowed investigators to isolate a temperature-responsive ion channel, TRPV1, by expression cloning (7) and identify a cold-sensitive channel by similar methods (25). Homology screening has revealed a number of temperature-sensitive channels in the transient receptor potential (TRP) family of integral membrane proteins that respond over a broad range of temperatures (7, 13, 25, 39). Although neither the pit organ (26) nor our I
T-expressing TG neurons show capsaicin sensitivity, this does not preclude that I
T could arise from a TRPV1 homolog or ortholog, as shown within chicken (18) and bullfrog (21) DRG neurons, respectively. The similarity of some temperature-response characteristics of I
T to those of members of the TRP family suggests that I
T may arise from a single, temperature-gated ion channel homologous to one of the TRP proteins. I
T shows a similar current-voltage relationship both in terms of its RP and its rectification (7, 13, 39).
In stark contrast to the TRP channels, our data indicated that IT showed no Ca2+ permeability. TRP channels, including the temperature-activated family members, show very high Ca2+ permeability (7, 13, 25, 39). We believe that because a snakes body temperature is often above the threshold of I
T, the current would be active a great deal of time and high Ca2+ permeability could result and Ca2+ induced toxicity like that found for TRPV1 (19). This does not completely rule out a role for a TRP homology in the generation of I
T in snakes, as point mutants of TRPV1 have been shown to have altered Ca2+ permeability (37). It is also possible that the temperature sensitivity of TG neurons is not due to single temperature-sensitive ion channels mediating I
T but that there may be cells that express a population of temperature-dependent ion channels that confer temperature-gated behaviors on these cells, as has been seen with cold-sensitive neurons from the mouse TG (35).
Our investigations suggest that thermosensation at the pit organ may, in part, be mediated by IT, but the behavioral data suggest that the cellular and molecular entities that mediate these processes may be more complex. Thermosensation is probably a complex integrated product of multiple temperature-sensitive currents, possessing unique temperature activation profiles and activation kinetics. The currents we recorded in these TG neurons may represent only part of the complete array of thermosensitive currents in the pit organ. Our continued investigations will focus on elucidating details such as sensitivity and response time of I
T to allow us to further characterize the contribution of I
T to highly sensitive thermodetection in snakes.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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