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Effects of Heating and Cooling on Nerve Terminal Impulses Recorded from Cold-sensitive Receptors in the Guinea-pig Cornea
2 Instituto de Neurociencias, Universidad Miguel Hernandez-Consejo Superior de Investigaciones Cientificas, 03550 San Juan de Alicante, Spain
Address correspondence to James Brock, Prince of Wales Medical Research Institute, Barker St., Randwick, NSW 2035, Australia. Fax: (61) 2 9382 2723; E-mail: j.brock{at}unsw.edu.au
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ABSTRACT |
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Key Words: action potential • thermal transduction • sensory receptor • extracellular recording
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and unmyelinated (C) sensory axons (Hensel et al., 1974
; Hensel, 1981; Heppelmann et al., 2001). Because of the very small diameter of these sensory nerve endings (
1 µm) and their indeterminate location in tissues such as skin, it has not been possible to determine the mechanisms of thermal transduction in cold-sensitive receptors directly. Instead, mechanisms have been inferred from recordings of impulse activity in the centrally projecting axons (Hensel and Zotterman, 1951; Hensel and Iggo, 1971; Kenshalo and Duclaux, 1977; Duclaux et al., 1980; for review see Hensel, 1981) and, more recently, from intracellular calcium measurements and electrophysiological recordings from the soma of cold-sensitive primary sensory neurons in culture (Suto and Gotoh, 1999; Reid and Flonta, 2001a, b; Viana et al., 2002).
These studies showed that, at static temperatures, cutaneous cold–sensitive receptors exhibit an ongoing cyclical pattern of orthodromically propagated action potentials. This activity has been attributed to a rhythmic oscillation in membrane potential at the site of action potential initiation (Hensel, 1981). It was also suggested that thermal transduction results in alterations in the frequency and amplitude of this membrane potential oscillation as well as changes in the overall level of membrane potential (Braun et al., 1980). The net result being that action potential discharge increases with cooling and decreases with heating. Studies of cultured cold–sensitive neurons have shown that these constitute a specific population of primary sensory neurons with distinct electrophysiological characteristics. Specific responses to temperature in cold-sensitive somata have been attributed to the presence of a nonselective cation channel that opens with cold and menthol (Suto and Gotoh, 1999; McKemy et al., 2002; Okazawa et al., 2002; Peier et al., 2002) and to the closure by cold of K+ conductances (Reid and Flonta, 2001a, b; Viana et al., 2002). However, in contrast with the afferent axons of cold-sensitive receptors, spontaneous impulse discharges were seldom observed in cultured cold–sensitive neurons. This difference suggests that the ion channels involved in regulating excitability (including the thermal transduction channels) and impulse generation are heterogeneously distributed in the cell body and the axonal branches of cold-sensitive neurons.
Recently, we described an extracellular recording technique that allows electrical activity to be recorded directly from the terminals of sensory axons in guinea pig cornea (Brock et al., 1998). The cornea is very densely supplied by small-diameter sensory axons that terminate abruptly as they approach the most superficial layer of the corneal epithelium. By applying a suction electrode to the epithelial surface of the cornea, nerve impulses can be recorded and identified as originating in single cold sensory nerve terminals (Brock et al., 1998, 2001), allowing the electrical changes induced by cold stimuli to be studied at the site where the thermal transduction mechanisms are expected to be preferentially located.
The present study investigated whether changes in nerve terminal impulse (NTI)* frequency that occur in cold-sensitive receptors during heating and cooling are associated with concomitant changes in NTI shape that reflect changes in the membrane potential of the nerve terminal. It is demonstrated that both NTI patterning and NTI shape are not simply dependent on the absolute temperature, but vary depending on the rate and direction of temperature change. As the NTIs are recorded directly from the sensory nerve terminals, it is likely that the observed changes in the NTI shape reflect, in part, changes in membrane potential associated with thermal transduction.
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MATERIALS AND METHODS |
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Guinea pigs of both sexes and in the weight range 200–400 g were used. Animals were anesthetized with sodium pentobarbitone (100 mg/kg i.p.) and killed by decapitation. Both eyes were dissected free from their orbits and isolated along with a short length of optic nerve and the associated ciliary nerves. Eyes were mounted in a recording chamber and superfused at 5 ml min-1 with physiological saline of the following composition (mM): Na+ 151; K+ 4.7; Ca2+ 2; Mg2+ 1.2; Cl- 144.5; H2PO3- 1.3; HCO3- 16.3; glucose 7.8. This solution was gassed with 95% O2/5% CO2 to pH 7.4. Under control conditions, the temperature of the bathing solution was maintained at 31–32.5°C. The temperature of the bathing solution was monitored continuously by a thermocouple placed in close apposition to the surface of the cornea. The optic nerve and associated ciliary nerves were drawn into a suction-stimulating electrode. The ciliary nerves were electrically stimulated with a constant voltage stimulator (pulse width 0.1–0.5 ms, 5–30 V).
To record electrical activity from sensory nerve terminals, a glass recording electrode (tip diameter 50 µm) filled with physiological saline was applied to the surface of the corneal epithelium with slight suction (Brock et al., 1998). An Ag/AgCl electrode in the recording chamber served as the indifferent electrode. Electrical activity was recorded through an AC amplifier (Neurolog NL104; Digitimer Ltd; gain x10,000, high pass filter set at 0.1 Hz), digitized at 44 kHz, and stored on magnetic tape using a two channel PCM recorder (A.R. Vetter Co., Inc.). The temperature of the bathing solution was recorded on the second channel. Recordings were only made from sites on the cornea where the NTIs were readily distinguished from the noise (10 µV peak-to-peak when low pass filtered at 3–5 kHz). At many sites on the corneal surface, electrically evoked or spontaneous electrical activity was either absent or too small to be analyzed.
Receptor Identification
The data presented were collected at recording sites where the electrical activity originated from a single nerve terminal. At these sites, electrical stimulation of the ciliary nerves evoked a single all-or-none NTI at the site of recording and the spontaneously occurring orthodromic NTIs could be collided with antidromically propagated, electrically evoked NTIs (see Brock et al., 1998). In the present study, only NTIs that were defined as originating in either polymodal nociceptors or cold-sensitive receptors were analyzed (see Brock et al., 1998). Polymodal nociceptors typically had low levels of ongoing NTI discharge (<1 Hz) and were excited by bath application of a low concentration of capsaicin (0.2–0.5 µM; supplied by Sigma-Aldrich). The cold-sensitive receptors had relatively high levels of ongoing NTI discharge (2–15 Hz) that occurred in a rhythmic manner and this activity was decreased by warming and increased by cooling the solution superfusing the cornea.
Controlling the Temperature of the Bathing Solution
The temperature of the superfusing solution was controlled using a feedback-regulated heater that warmed the solution entering the bath. In most experiments, temperature responses were produced by initially heating the superfusing solution from the normal holding temperature (31–32.5°C) to 37–38°C. This was done by setting the heater target temperature to a temperature 20°C above that required to maintain the normal holding temperature. When the required temperature (38°C) was reached, the heater was switched off and the superfusing solution allowed to cool passively until the temperature reached 28–26°C. The heater was then switched on and the superfusing solution heated back to the normal holding temperature. This was the standard protocol used to identify receptor type and also to investigate the effects of temperature on NTI frequency, NTI shape, and the latency of electrically evoked NTIs. In the experiments comparing the effects of fast and slow heating from the normal holding temperature, fast heating was produced using the standard protocol described above, whereas slow heating was produced by setting the heater target temperature to a temperature 7°C above that required to maintain the normal holding temperature. In the experiments comparing the effects of fast and slow cooling, the bathing solution was first heated from the normal holding temperature to 38°C at a fast rate. Switching the heater off as in the standard protocol produced fast cooling, whereas resetting the solution heater to the temperature required to maintain the normal holding temperature produced slow cooling. We chose to investigate the effects of cooling from an initially raised temperature (38°C) because, without active cooling, this allowed the rate of cooling to be most readily regulated.
Data Analysis
A MacLab data acquisition system (ADInstruments Pty Ltd.) was used to digitize (sampling frequency 20 kHz) the electrophysiological signals previously recorded on tape. Prior to digitizing, the signals were filtered using a low pass filter (cut-off, 3–5 kHz). Subsequent analysis was made with the computer program Igor Pro (Wavemetrics). Measurements of NTI amplitude and shape were taken from averages of 10–40 individual NTIs. Prior to averaging, NTIs were aligned in time at their peak positive amplitude. When NTIs occurred in high frequency, short bursts, their amplitude usually declined during the burst. For this reason only the first NTI in each burst was used to construct the average NTI. The temperature at the time of occurrence of each NTI during temperature responses was measured. The effects of changing temperature on the shape of averaged NTIs were assessed at 1 or 0.5°C intervals above and below the normal holding temperature. To do this analysis, the sets of consecutively occurring NTIs used to construct the averages were selected so that the mean temperature over which they occurred was within ± 0.1°C of these step changes in temperature.
The NTIs are diphasic (positive/negative) with a prominent positive-going component (see Brock et al., 1998). To determine the effects of temperature on the shape of NTIs, the positive amplitude of the NTI and the maximum rate of change of voltage during the initial upstroke and the downstroke of the NTI (maximum dV/dt and minimum dV/dt respectively) were measured. As the amplitude of NTIs changed during the temperature responses, changes in NTI time course were made after the maximum and minimum dV/dt were normalized with respect to NTI amplitude by dividing them by the positive-peak amplitude (for details see Brock et al., 2001).
Statistical comparisons were made with repeated measures ANOVAs or paired t tests as indicated. For the repeated measures ANOVAs, the degrees of freedom were adjusted using Huynh-Feldt adjustment factors. P < 0.05 was considered statistically significant.
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RESULTS |
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).
Fig. 1 shows the effects of the standard heating and cooling protocol (see MATERIALS AND METHODS) on the latency of NTIs evoked at 1 Hz in a single experiment. During heating and cooling, NTI latency decreased and increased respectively (Fig. 1, A and B). Fig. 1 C shows that NTI latency was linearly correlated with temperature and that there was no apparent delay or hysteresis depending on whether the superfusing solution was being heated or cooled. Similar findings were obtained in six other experiments. Since the conduction velocity of mammalian unmyelinated and myelinated axons depends linearly on temperature in the range 26 to 38°C (Paintal, 1965; De Jesus et al., 1973), these findings indicate that the temperature of the eye's surface structures, including the axons forming the ciliary nerves, follow that of the superfusing solution very closely.
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190 s (37°C), NTIs started to occur at relatively fixed intervals. Between 190 and 200 s, single NTIs occurred at intervals of 97 ± 26 ms and, throughout the subsequent period of cooling, the length of the long interval increased (Fig. 2 C). At 210 s, NTIs started to occur in bursts and the number of NTIs in each burst increased as the cooling progressed (Fig. 2 D). These changes in NTI patterning were associated with an increase in the average NTI frequency, above the control value, throughout most of the period of cooling (Fig. 2 B).
At the start of heating back to 31°C, there was an abrupt decrease in the length of the bursts (Fig. 2 D). At 29°C during cooling and heating, the number of NTIs in each cycle was 2.4 ± 1.6 (between 270–280 s) and 1.4 ± 0.7 (between 320 and 330 s), respectively. During heating back to 31°C, there was also a decrease in the long interval. Between 500 and 600 s, when the temperature had returned to 31°C, the average frequency of NTI discharge (7.8 s-1) was slightly higher than that recorded before heating.
Effects of heating and cooling similar to those described above were observed in 83 of 121 cold-sensitive receptors. In the remaining 38 cold-sensitive receptors, the pattern of NTI occurrence during cooling was different to that described above. In these receptors, the bursting pattern of NTI occurrence during cooling changed to a continuous high frequency discharge toward the end of the cooling period (at temperatures <30°C). The bursting pattern of NTI occurrence was restored shortly after the initiation of heating back to the control temperature. Although heating from the control temperature markedly reduced NTI frequency in all receptors, not all receptors were silenced. Furthermore, in those receptors that were silenced, there was considerable variability in the temperature at which NTI activity ceased.
Table I shows various measures of NTI patterning for 20 experiments determined before heating (control temperature), at the control temperature during cooling, and at the end of the temperature response when the temperature was stable. The increase in NTI frequency during cooling was associated with a shortening of the cycle time (i.e., the time between the start of each long interval) and an increase in the number of NTIs in each cycle. The mean interval between NTIs within the bursts (intraburst interval) during cooling did not differ from the control value. At the end of the temperature response, the average NTI frequency and the number of NTIs in each cycle were not significantly different from control values, but the cycle time was shorter and the intraburst interval was longer.
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Effects of Changing the Rate of Heating and Cooling on NTI Frequency and Shape
To investigate whether the response of cold-sensitive receptors to heating and cooling depends on the rate of temperature change, the effects of fast and slow heating and cooling were determined.
Heating
NTI discharge and shape were examined during fast heating (0.104 ± 0.011°C s-1) and slow heating (0.026 ± 0.006°C s-1) from a initial temperature of 32.1 ± 0.1°C (range 31.4–32.6°C). Only receptors that continued to discharge NTIs up to 3°C above the starting temperature, during fast heating, were analyzed (n = 9). Fig. 5, A and B show recordings of temperature and the change in NTI frequency during fast (Fig. 5 A) and slow (Fig. 5 B) heating for a single receptor. Fig. 5 also shows the mean effects of fast and slow heating on NTI frequency (Fig. 5 C), positive NTI amplitude (Fig. 5 D), and the normalized maximum and minimum dV/dt of NTIs (Fig. 5, E and F). Both fast and slow heating produced a temperature-related reduction in NTI frequency and the magnitude of this effect was significantly larger during fast heating (Fig. 5 C). There was also a significant interaction between the rate of heating and temperature on NTI frequency (Fig. 5 C). This interaction indicates that the slope of the relationship between NTI frequency and temperature was different during fast and slow heating. There was also a greater increase in the positive amplitude of NTIs during fast heating compared with slow heating (Fig. 5 D). However, the differential effect of fast and slow heating on the positive amplitude of the NTIs appeared during the initial phase of heating and showed no temperature dependence (Fig. 5 D, see also Fig. 4 D). Both the normalized maximum and minimum dV/dt of the NTIs increased with temperature (Fig. 5, E and F) and there was no significant difference between the effects of slow and fast heating, nor was there a significant interaction between temperature and heating rate for these parameters.
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38°C and then cooled. For each receptor, measurements of NTI frequency and shape during fast cooling (0.080 ± 0.004°C s-1) and slow cooling (0.016 ± 0.001°C s-1) were made at 0.5°C intervals between 4°C and 0.5°C above the starting temperature (32.1 ± 0.1°C, range 31.6–32.5°C). Fig. 6, A and B, show recordings of temperature and the change in NTI frequency during fast (Fig. 6 A) and slow (Fig. 6 B) cooling for a single receptor. Fig. 6 also shows the mean effects of fast and slow cooling on NTI frequency (Fig. 6 C), positive NTI amplitude (Fig. 6 D), and the normalized maximum and minimum dV/dt of NTIs (Fig. 6, E and F). Fast cooling produced a greater increase in NTI frequency than slow cooling. However, during cooling NTI frequency did not significantly change over the range of temperatures tested. There was a significant interaction between the effects of cooling rate and temperature on NTI frequency (Fig. 6 C), indicating that the relationship between NTI frequency and temperature differed for fast and slow cooling. Cooling was also associated with a temperature-related decrease in the positive amplitude and the normalized maximum and minimum dV/dt of the NTIs (Fig. 6, D–F). In addition, rapid cooling produced a greater decrease in all three of these parameters than did slow cooling (Fig. 6, D–F). The interaction of the effects of cooling rate and temperature on the positive amplitude and the normalized maximum and minimum dV/dt of the NTIs was not significant.
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), the observed changes in NTI shape may be produced by changes in the level of ongoing activity. To investigate the possibility that changes in NTI frequency may alter NTI shape, ciliary nerve stimulation was used to evoke antidromic NTIs at different frequencies. The effects of stimulation frequency on NTI shape were investigated at 29, 31.5, and 35°C. Fig. 7 A shows the sequential average of 10 NTIs evoked during trains of 50 stimuli at 10, 20, and 40 Hz in a single receptor at 31.5°C. During the trains of stimuli there was a stimulation-dependent increase in the latency and positive amplitude of the electrically evoked NTIs that increased with the frequency of stimulation. For nine cold-sensitive receptors, Fig. 7 B shows the mean effect of stimulation frequency on the positive amplitude of NTIs at 31.5°C. At all frequencies of stimulation tested, the amplitude of NTIs increased during the trains of stimuli (repeated measures ANOVA, P < 0.01 at all frequencies). Similar effects of stimulation on the positive amplitude of NTIs were also observed at 29 and 35°C during trains of stimuli at 10, 20, and 40 Hz (repeated measures ANOVA, P < 0.01 for all frequencies at 29 and 35°C).
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DISCUSSION |
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; Heinz et al., 1990). These previous studies investigated impulse activity in the centrally projecting axons of cutaneous cold–sensitive receptors both at static temperatures and after rapid step changes in temperature. Cutaneous cold–sensitive receptors have a cyclical pattern of NTI discharge, with single or short bursts of impulses occurring at regular intervals. At static temperatures, the length of the cycle period and the number of impulses per cycle are inversely related to temperature. A step decrease in temperature evokes a high frequency discharge of impulses, whereas a step increase in temperature produces a marked reduction, or cessation, of impulse activity. In both cases, after the change in temperature, the receptors adapt to a new static level of activity within 1–2 min. In most studies, it has been reported that the static discharge rate at a given temperature, after adaptation, is the same if that temperature is reached by cooling or heating (see Heinz et al., 1990), i.e., the static discharge rate signals the absolute temperature. However, in rat and monkey facial cold–sensitive receptors, adaptation at a particular temperature results in a higher level of static activity after a step decrease in temperature compared with a step increase in temperature (Dubner et al., 1975; Davies et al., 1985; c.f. Heinz et al., 1990).
In the present study, where the cornea was bathed with physiological solution and the electrode was applied close to the site of thermal transduction, it was not possible to produce step changes in temperature in the receptive field similar to those used in previous studies on cutaneous cold–sensitive receptors. Instead, the effects of more gradual changes in temperature affecting both the nerve terminal and preterminal axon were investigated. During these temperature responses, the NTI frequency measured at the same absolute temperature was greater during cooling than during heating. In addition, the magnitude of the effect of heating and cooling on NTI frequency increased with the rate of change of temperature. This effect of altering the rate of temperature change may be explained by the previously reported adaptation of the dynamic response to heating and cooling observed in cutaneous cold–sensitive receptors. While not shown here, when, after rapid heating, the temperature is held at 37–38°C for 1 min, the heating-induced decrease in NTI activity in corneal cold–sensitive receptors usually displays some adaptation during the period when temperature is no longer changing (unpublished data).
At 31–32°C, NTIs recorded from cold-sensitive receptors are produced by electrotonic invasion of the nerve terminal from a point more proximal in the axon where action potentials fail or are initiated (Brock et al., 2001). The extracellularly recorded NTIs are proportional to net membrane current, which is determined primarily by the capacitive current produced when the impulse invades the nerve terminal (Smith, 1988). Temperature is known to affect the rate of change of membrane potential during an action potential by altering the kinetics of voltage-activated ion channels (Russ and Siemen, 1996); the time course speeding with heating and slowing with cooling (Hodgkin and Katz, 1949; Li et al., 2002). Thus, it would be expected that NTIs would increase in amplitude and speed in time course when the temperature is raised. However, this effect of temperature on action potential time course cannot explain the differential effects of heating and cooling on the shape of NTIs recorded at the same absolute temperature.
The differential effects of heating and cooling on NTI shape were associated with changes in NTI frequency and might therefore reflect activity-dependent changes in membrane excitability. This possibility was investigated by determining the effects of stimulation frequency on the shape of electrically evoked NTIs. In corneal cold–sensitive receptors, the mean NTI frequency at 31–32°C during cooling was 13 Hz. Trains of electrical stimuli at frequencies between 10 and 40 Hz produced an activity-dependent increase in NTI amplitude. In addition, during the trains of stimuli, the latency of the electrically evoked NTI increased and, after trains at 20–40 Hz, there was a cessation of ongoing NTI activity that lasted for several seconds (unpublished data). It has been suggested that the slowing of conduction velocity produced by repetitive stimulation can be explained by a hyperpolarization of the axon produced by an increase in Na+/K+ ATPase activity (Rang and Ritchie, 1968; Morita et al., 1993; Serra et al., 1999). Activity-dependent hyperpolarization is also consistent with the cessation of spontaneous activity observed in cold-sensitive receptors after repetitive stimulation. Furthermore, as axonal hyperpolarization would be expected to increase the amplitude of the invading action potential (Morita et al., 1993), this can explain the activity-dependent increase in NTI amplitude. However, since the increase in NTI frequency produced by cooling was associated with a decrease in NTI amplitude, activity-dependent changes in membrane excitability, similar to those observed during repetitive electrical stimulation, cannot explain the differential effects of heating and cooling on NTI shape.
The effects of heating and cooling on NTI frequency and shape might be explained if heating hyperpolarizes the nerve terminal and cooling depolarizes the nerve terminal. The cyclical pattern of NTI activity in cold-sensitive receptors suggests that at some point within the nerve terminal arbor there is a pacemaker. The electronic spread of potential from the nerve terminals may regulate the frequency of NTI activity by interacting with this pacemaker. As voltage-dependent conductances are likely to underlie the pacemaker, the "receptor" potential may regulate the pacemaker currents as well as the likelihood of the membrane potential reaching threshold for action potential initiation. As the conductances involved in pacing would themselves be modulated by temperature, it seems probable that their temperature dependence would also contribute to the behavior of the cold-sensitive receptors. At present, the pacemaking conductances in cold receptors are unknown and, therefore, it is not possible to predict the effects of changing membrane potential or temperature on NTI patterning. However, it is likely that hyperpolarization of the nerve terminal would decrease NTI activity whereas depolarization of the nerve terminal would increase NTI activity.
Hyperpolarization and depolarization of the nerve terminal may also explain the observed effects of heating and cooling on NTI shape. At 31–32°C, hyperpolarization of the nerve terminal with focally applied extracellular current produces a marked increase in the amplitude of the negative-going component of cold-sensitive receptor NTIs (Carr et al., 2002). This change is caused by an increase in inward current and is abolished when the recording electrode is perfused with the local anesthetic, lidocaine. Under control conditions, locally applied lidocaine has only a small effect on the time course of cold-sensitive receptor NTIs (Brock et al., 2001; Carr et al., 2002). The simplest explanation is that under control conditions the nerve terminal is depolarized relative to the more proximal axon and, as a result, most of the Na+ channels present are inactivated. Hyperpolarization with extracellular current relieves Na+ channel inactivation and thereby allows active invasion of the nerve terminal. If the nerve terminals are normally depolarized relative to the more proximal axon, the spread of potential from the nerve ending would be expected to produce a progressive decrease in the number of inactivated Na+ channels proximal to the nerve terminal. If this is the case, heating-induced hyperpolarization would be expected to relieve Na+ channel inactivation proximal to the terminal and, as a result, the point at which the action potential fails to propagate actively would move toward the site of recording. Conversely, cooling-induced depolarization would be expected to increase the proportion of inactivated Na+ channels proximal to the nerve terminal and, as a result, the point at which the action potential fails to propagate actively would move away from the site of recording.
The effects of changing membrane potential at the nerve terminal on NTI shape were investigated numerically using a compartmental model for a length of axon with Hodgkin-Huxley action potential kinetics (Hodgkin and Huxley, 1952). NTIs were represented as net membrane current across the terminal compartment. Membrane potential changes that are expected to inactivate Na+ channels were mimicked by reducing the maximum Na+ conductance as a sigmoidal function of length in the terminal compartments. Fig. 9 shows the effect on simulated NTIs of shifting the sigmoid-scaled maximum Na+ conductance toward (mimicking depolarization) and away (mimicking hyperpolarization) from the nerve terminal. Mimicking the effects of changing the membrane potential on the Na+ conductance of the nerve terminal in this way produced an increase in amplitude and a speeding of time course of the NTI with hyperpolarization and a decrease in amplitude and slowing in time course of the NTI with depolarization. These changes in the amplitude and time course of the simulated NTIs are similar to those observed for cold-receptor NTIs at any particular ambient temperature during heating and cooling respectively (see Fig. 3 C). It is difficult to model the changes in NTI shape more precisely, since the type and density of ionic conductances in the nerve terminals are not known. However, the similarity of the changes in NTI shape produced by heating and cooling with those simulated by changing the spatial distribution of Na+ channels provides qualitative support for the idea that the nerve terminal depolarizes during cooling and hyperpolarizes during heating. This effect of changing temperature on membrane potential appears to be related to the process of thermal transduction in cold receptors, since for polymodal receptors, no differential effects of heating and cooling on NTI shape were observed (Fig. 8 D).
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; Heinz et al., 1990). In this respect it is important to note that the cold and menthol-induced increase in inward current in cultured cold–sensitive dorsal root ganglion neurons shows adaptation (Reid et al., 2002). Adaptation of a cold-induced current in the nerve terminals could presumably contribute to the differential effects of fast and slow cooling on frequency and NTI shape for cold receptors. Indeed, there are several candidate mechanisms that may individually or in concert contribute to the differential changes in NTI activity and shape during heating and cooling. We assume that it is the adaptation of the thermal transduction mechanism(s) in cold-sensitive receptors that is responsible for the differential effects on NTI frequency and shape when the receptor is warmed versus when it is cooled over the same absolute temperature range. In conclusion, the findings of the present study suggest that the differential effects of heating and cooling on the configuration of cold-sensitive receptor NTIs reflect membrane potential changes associated with thermal transduction. The findings indicate that the nerve terminal membrane potential depolarizes with cooling and hyperpolarizes with heating. Consequently, to understand the mechanism underlying thermal transduction in cold-sensitive receptors, it is necessary to explain the effects of both heating and cooling.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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Lawrence G. Palmer served as editor.
Submitted: 6 February 2003
Revised: 24 March 2003
Accepted: 24 March 2003
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) and cooling (
). (D) The normalized maximum and minimum dV/dt of averages of successive sets of 20 NTIs. NTIs were larger in amplitude and faster in time course during heating compared with those recorded at the same temperature during cooling.
, *), the direction of temperature change (
), and their interaction (
) were compared. The error bars are SEMs. ns = not significant. *, 
cm. An axon diameter of 1 µm and compartment lengths of 50 µm were used. The action potentials were modeled at 6.3°C. The forward Euler method of integration was used with time steps of 0.5 µs.