Barium ions inhibit the dynamic response of guinea-pig corneal cold receptors to heating but not to cooling

Abstract

An in vitro preparation of the guinea-pig cornea was used to study the effects of the K⁺ channel blockers 4-aminopyridine (4-AP), tetraethylammonium (TEA) and Ba²⁺ on nerve terminal impulses (NTIs) recorded extracellularly from cold sensory receptors. These receptors have an ongoing discharge of NTIs that is increased by cooling and decreased by heating. The K⁺ channel blocker 4-AP reduced the negative amplitude of the diphasic (positive–negative) NTIs, whereas TEA and Ba²⁺ prolonged the duration of the negative component. As the shape of the NTI is determined by the first derivative (dV/dt) of the membrane voltage change, these changes in the negative component are consistent with the blockade of K⁺ channels that contribute to action potential repolarization. Only TEA changed the basal activity of the receptors, increasing the likelihood of burst discharges. Ba²⁺ selectively reduced the response of the receptors to heating, whereas neither 4-AP nor TEA modified the response to heating or to cooling. The findings indicate that K⁺ channels blocked by 4-AP, TEA and Ba²⁺ contribute to action potential repolarization in corneal cold receptors, and that ionic mechanisms that underlie the reduction in NTI frequency in response to heating differ from those that increase activity in response to cooling.

In mammals, cold thermal receptors on the body surface fire spontaneously at neutral temperatures and increase their discharge frequency with small temperature decreases (Hensel, 1981). In humans, these receptors are believed to be responsible for eliciting the sensory experiences of cold. While these receptors are cold responsive, the largest changes in activity are produced by dynamic changes in temperature; peak increases or decreases in firing frequency occurring during cooling and heating, respectively (e.g. see Dykes, 1975; Carr et al. 2003). Therefore these receptors might more appropriately be considered thermal receptors, as they can potentially signal both cooling and heating.

At static temperatures between 25 and 40°C, action potentials occur in a periodic manner, with single or short bursts of impulses occurring at regular intervals. Over this range of temperatures, the frequency and number of impulses per burst are inversely related to temperature, whereas the cycle frequency increases with temperature. This pattern of activity is suggested to originate from a cyclical oscillation in membrane potential at the site of impulse initiation (Braun et al. 1980).

The mechanisms that underlie the temperature-induced changes in impulse frequency remain poorly understood but it is likely that cooling and heating, respectively, depolarize and hyperpolarize the nerve terminal (Carr et al. 2003). Studies in cultured sensory neurones have indicated that temperature-induced changes in membrane potential are produced by gating of the cooling-activated transient receptor potential channel TRPM8 (McKemy et al. 2002; Peier et al. 2002; de la Pena et al. 2005), as well as thermal modulation of a background K⁺ conductance (Reid & Flonta, 2001; Viana et al. 2002). It has also been suggested that thermal modulation of the membrane Na⁺,K⁺-ATPase contributes to thermal transduction in cold receptors (Pierau et al. 1975; Schafer & Braun, 1990). As the mechanisms that generate the ongoing activity of cold receptors are themselves temperature sensitive (Braun et al. 1980), it is very likely that they contribute to the process of thermal transduction, although this possibility has not received much attention (Schafer et al. 1991). While the underlying mechanisms that generate the rhythmic discharge of action potentials are unknown, it is likely that K⁺ channels play a role in regulating this activity.

We have used a technique that allows electrical activity to be recorded directly from single nerve terminals in guinea pig cornea (Brock et al. 1998) to investigate the effects of broad-spectrum K⁺ channel blockers (4-aminopyridine, 4-AP; tetraethylammonium, TEA; and Ba²⁺) on the configuration of nerve terminal impulses (NTIs) in cold receptors, and their ongoing activity during heating and cooling. At the concentrations used, the K⁺ channels blocked by these agents include those producing both transient and slowly inactivating K⁺ currents (I_K(A) and I_K(D): 4-AP), delayed rectifier currents (TEA and Ba²⁺), Ca²⁺-activated K⁺ currents (TEA and Ba²⁺) and inward rectifier currents (Ba²⁺). All these agents changed the configuration of NTIs in a manner consistent with the blockade of K⁺ channels, but only TEA changed the basal activity of the receptors, while Ba²⁺ selectively interfered with their response to heating. The findings suggest that separate ionic mechanisms underlie the response of corneal cold receptors to cooling and to heating.

Methods

All experimental procedures conformed to the National Health and Medical Research Council of Australia guidelines and were approved by the University of New South Wales Animal Care and Ethics Committee.

Guinea pigs of both sexes in the weight range 250–450 g were anaesthetized with sodium pentobarbitone (100 mg kg⁻¹i.p.) and killed by exsanguination. Both eyes were isolated along with a short length of optic nerve and the associated ciliary nerves. Eyes were mounted either in a single or two-compartment recording chamber. In the two-compartment recording chamber, the eye was mounted in a hole in the dividing wall so that the cornea and conjunctiva were separated from the rest of the eye, and was held in place by pinning the conjunctiva to the silicone resin-coated surface of the dividing wall. The chambers were perfused at 3–5 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 basal conditions, the temperature of the bathing solution was maintained at 31.5–32.5°C. The temperature of the solution superfusing the cornea was monitored continuously by a thermocouple placed in close apposition to its surface. The optic nerve and associated ciliary nerves were drawn into a suction-stimulating electrode. The ciliary nerves were stimulated electrically with a constant voltage stimulator (pulse width 0.1–0.5 ms, 5–30 V).

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 amplified (×1000; Geneclamp 500; Axon Instruments), filtered (high pass 1 Hz, low pass 5 kHz; 432 Wavetek) and digitized (sampling frequency 20 kHz; PowerLab data acquisition system, ADInstruments Pty Ltd, Castle Hill, NSW, Australia). Recordings were only made from sites on the cornea where the NTIs were readily distinguished from the noise (∼10 μV peak-to-peak).

To deliver agents locally at the site of recording, a polyethylene tube was positioned inside the recording electrode, close to the tip (see Brock & Cunnane, 1995). The electrode was continually perfused at ∼50 μl min⁻¹ with Hepes-buffered saline of the following composition (mm): Hepes 10; NaCl 151; KCl 4.7; CaCl₂ 2; MgCl₂ 1.2; glucose 7.8. The pH of this solution was adjusted to 7.4 using NaOH. Agents were added to the Hepes-buffered saline at the required concentration. Perfusion of the recording electrode with Hepes-buffered saline alone has previously been shown to be without effect on cold receptor NTIs (see Brock et al. 2001). TEA and 4-AP were supplied by Sigma. All salts used were of analytical grade.

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 and the spontaneously occurring orthodromic NTIs collided with antidromically propagated, electrically evoked NTIs (see Brock et al. 1998). Only NTIs that were defined as originating in cold receptors were analysed (see Brock et al. 1998). The cold receptors had relatively high levels of ongoing NTI discharge (6–15 Hz) which occurred rhythmically and was decreased by warming and increased by cooling the solution superfusing the cornea.

Data analysis

Data were analysed using the computer program Igor Pro (Wavemetrics, Lake Oswego, OR, USA). Measurements of NTI amplitude and shape were taken from averages of 50–100 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 declined during the burst. For this reason only the first NTI in each burst was used to construct the average NTI.

The NTIs were diphasic (positive–negative) with a prominent positive component (see Fig. 2). The positive and negative amplitude of the NTI, the maximum rate of change of voltage during the initial upstroke and the downstroke of the NTI (maximum and minimum dV/dt) and the half-widths of the positive and negative components were measured. Prior to assessing the effects produced by each K⁺ channel blocker using Student's paired t tests, the measured changes were normalized to the values measured just prior to their addition. Comparisons between the effects of the K⁺ channel blockers were made with one-way ANOVA followed by Tukey HSD post hoc tests.

Figure 2. 4-Aminopyridine, TEA and Ba²⁺ changed the configuration of NTIs.

A–D, averaged NTIs recorded before (upper) and during (middle) application of 4-aminopyridine (4-AP; 1 mm; A), TEA (20 mm; B), Ba²⁺ (5 mm; C) and 4-AP plus TEA (1 mm+ 20 mm; D). The lower panel in A–D shows these NTIs overlaid (bold line is drug treated).

Figure 1 shows the typical pattern of nerve activity in corneal cold receptors at the basal temperature. Single or short bursts of impulses occur at regular intervals and the distribution of intervals between NTIs is bimodal (Fig. 1B) with long and short intervals, the latter occurring during bursts of NTIs. To assess the effects of the K⁺ channel blockers on the patterning of NTI activity the mean frequency of NTI discharge, the long interval (Fig. 1A and B), the cycle time (i.e the time between ends of the long intervals; Fig. 1A), the number of NTIs per cycle (Fig. 1C), the number of NTIs per burst and the relative frequency of bursts (i.e. number of bursts/number of cycles) were compared before and 10–15 min following their addition using Student's paired t tests.

Figure 1. Patterning of nerve terminal impulses (NTIs) in a corneal cold receptor at the basal temperature (∼32°C).

A, ongoing NTI discharge. The lines above the trace indicate the cycles (upper) and the long intervals (lower). The asterisks indicate the bursts. B and C, frequency distribution of intervals between NTIs (B) and the number of NTIs per cycle (C) during a 40 s recording period. Single NTIs or bursts of two NTIs occurred at regular intervals.

The dynamic changes in NTI frequency in response to heating and to cooling were assessed before and ∼15 min following the addition of the K⁺ channel blockers. Prior to statistical comparison, the changes in NTI frequency during the temperature responses were normalized to values measured just prior to initiating heating. The effects of each K⁺ channel blocker on the responses to heating and to cooling were compared separately using multivariate repeated measures ANOVA. For these comparisons, P values were adjusted using Greenhouse-Geisser's correction.

Unless otherwise indicated, data are presented as means ±s.e.m. SPSS version 11 for Macintosh (SPSS, Inc., Chicago, IL, USA) was used for all statistical comparisons and P values < 0.05 were considered significant.

Results

Effects of K⁺ channel blockers on NTI shape

Figure 2 shows effects of locally applied 4-AP (1 mm), TEA (20 mm) and Ba²⁺ (5 mm) on NTI shape and Table 1 shows effects of these agents on the measured NTI parameters. Each of these agents had a characteristic effect on NTI shape. Locally applied 4-AP reduced the negative amplitude of the NTI but was without effect on the positive amplitude of the NTI or any of the measures of NTI time course (Fig. 2A, Table 1). TEA produced a small decrease in the positive amplitude of the NTI and increased the half-width of the negative component of the NTI, but was without effect on negative amplitude of the NTI or on any other measures of NTI time course (Fig. 2B, Table 1). Ba²⁺ also reduced the positive amplitude and increased the half-width of the negative component of the NTI but, in addition, increased the half-width of the positive component and reduced the maximum dV/dt of the NTI (Fig. 2C, Table 1). Despite these apparent differences between the effects of TEA and Ba²⁺, there were no differences when the changes produced by these agents in the two sets of nerve terminals were compared. The effect of TEA and Ba²⁺ on the half-width of the negative component was primarily due to slowing of its decay (Fig. 2B and C).

Table 1.

Normalized changes in the measured NTI parameters produced by 4-aminopyridine (4-AP; 1 mm), TEA (20 mm), Ba²⁺ (5 mm), Cs⁺ (5 mm) and raised K⁺ concentration (15 mm)

	+Amplitude	−Amplitude	Maximum dV/dt	Minimum dV/dt	+Half-width	−Half-width	n
4-AP	1.00 ± 0.03	0.64 ± 0.07†a	0.99 ± 0.03	1.03 ± 0.03	0.99 ± 0.03	0.89 ± 0.15c, d	11
TEA	0.91 ± 0.02†	1.00 ± 0.10a,b	1.00 ± 0.03	1.00 ± 0.04	1.03 ± 0.03	1.59 ± 0.21*, c	10
Ba2+	0.87 ± 0.03†	1.07 ± 0.06	0.92 ± 0.02†	0.94 ± 0.03	1.06 ± 0.02	*1.51 ± 0.14†	9
4-AP + TEA	0.93 ± 0.03	0.60 ± 0.07‡b	0.98 ± 0.02	0.97 ± 0.07	1.02 ± 0.04	2.12 ± 0.23†d	9
Cs+	0.87 ± 0.02‡	0.66 ± 0.06†	1.03 ± 0.04	0.84 ± 0.04	1.05 ± 0.03	1.81 ± 0.17†	7
Raised K+	0.93 ± 0.05	0.87 ± 0.06	0.87 ± 0.02†	0.87 ± 0.03*	1.15 ± 0.04*	1.07 ± 0.05	8

Normalized data are presented as means ±s.e.m. Significant differences for the paired comparisons with NTIs recorded just prior to drug application:

^*P < 0.05

^†P < 0.01

^‡P < 0.001). Significant differences for pairwise comparisons between 4-AP, TEA and 4-AP plus TEA data:

^cP < 0.05

^a,bP < 0.01

^dP < 0.001). For these multiple comparisons, the data were first compared by one-way ANOVA and then by Tukey HSD post hoc tests.

The effects of 4-AP and TEA differed significantly from each other and appeared to sum when they were applied together (Fig. 2D, Table 1).

Effects of K⁺ blockers on NTI occurrence

The NTI shape changes produced by 4-AP, TEA and Ba²⁺ indicate that the K⁺ channels blocked by these agents are likely to be present in the cold-sensitive nerve endings (see Discussion). To determine their effects on the ongoing NTI activity and on responses to thermal stimulation, the K⁺ channel blockers were applied to the whole cornea by their addition to the solution perfusing the front chamber of the divided recording chamber. When applied in this manner, 4-AP (0.5 mm, n = 8), TEA (20 mm, n = 8) and Ba²⁺ (3 mm, n = 7) produced similar changes in the shape of NTIs to those produced by their local application (results not shown).

Under basal conditions (31.5–32.5°C), prior to applying the K⁺ channel blockers, NTIs occurred rhythmically, with single or short bursts of usually two but up to five NTIs occurring at regular intervals (see Fig. 1). Table 2 shows the effects of 4-AP, TEA and Ba²⁺ on the frequency and patterning of NTI occurrence. Ba²⁺ and 4-AP had no detectable effect on the ongoing activity of the nerve terminals (Table 2). In contrast, TEA increased the frequency of NTI occurrence, and this change was associated with an increase in both the number of impulses per burst and the frequency of burst occurrence (Table 2).

Table 2.

Changes in the frequency and patterning of NTI occurrence produced by 4-AP (0.5 mm), TEA (20 mm) and Ba²⁺ (3 mm)

	Frequency (impulses s−1)	Long interval (ms)	Impulses per cycle	Impulses per burst	Relative frequency of cycles with bursts	Cycle time (ms)	n
Control	7.1 ± 1.4	0.22 ± 0.05	1.19 ± 0.10	2.19 ± 0.07	0.20 ± 0.10	0.22 ± 0.05	8
4-AP	6.5 ± 1.2	0.24 ± 0.07	1.13 ± 0.12	2.20 ± 0.13	0.24 ± 0.14	0.25 ± 0.07	8
Control	7.6 ± 0.45	0.17 ± 0.02	1.28 ± 0.11	2.02 ± 0.01	0.29 ± 0.11	0.17 ± 0.02	8
TEA	10.2 ± 1.40*	0.15 ± 0.01	1.61 ± 0.24	2.40 ± 0.20*	0.41 ± 0.12*	0.16 ± 0.01	8
Control	6.8 ± 0.5	0.18 ± 0.02	1.25 ± 0.21	2.16 ± 0.11	0.15 ± 0.11	0.18 ± 0.02	7
Ba2+	7.2 ± 0.6	0.17 ± 0.02	1.26 ± 0.19	2.32 ± 0.15	0.17 ± 0.10	0.18 ± 0.03	7

Data presented as means ±s.e.m.

^*P < 0.05, significant difference for the paired comparison between data recorded before and during drug application.

Effects of K⁺ channel blockers on responses to thermal stimulation

The solution superfusing the cornea was first heated from the basal temperature (31–32.5°C) to 36–38°C (maximum rate of heating 0.18 ± 0.07°C s⁻¹, mean ±s.d.), then cooled to 29–28°C (maximum rate of cooling −0.16 ± 0.06°C s⁻¹) and then heated back to the basal temperature (see Fig. 3A). Figure 3A–D shows a typical response of a corneal cold receptor to this thermal stimulation protocol and Fig. 4 shows the changes in NTI frequency during heating and cooling for the 23 cold receptors investigated (measured just prior to adding the K⁺ channel blockers). The frequency of NTI discharge is not simply related to the ambient temperature but is also dependent on the rate of change of temperature (see Carr et al. 2003). As a result, the peak increase in NTI frequency occurred at temperatures close to the basal temperature during cooling and the largest difference between the effects of heating and cooling was observed at temperatures above the basal temperature (Fig. 4). Subsequently, the effects of the K⁺ blockers were compared during heating to 4°C above the basal temperature and cooling back from this temperature to the basal temperature.

Figure 3. Typical thermal responses of a corneal cold receptor before and during application of Ba²⁺.

Responses to heating and cooling before (A–D) and during (E–H) application of Ba²⁺ (3 mm). A and E, temperature of the bathing solution recorded close to the corneal surface. B and F, effect of changing temperature on the frequency of NTIs. F, the arrow indicates the sharp reduction in NTI frequency at the start of heating. C and G, intervals between successive NTIs. D and H, number of NTIs that occurred during each cycle (i.e. between successive long intervals). In the absence of Ba²⁺, heating silenced the receptor, and cooling increased NTI frequency; the latter change was associated with a shortening of the long interval and an increase in the number of impulses per cycle. In the presence of Ba²⁺ (3 mm), the receptor did not silence during heating but the changes in NTI activity produced by cooling were similar to those in the absence of this agent.

Figure 4. Changes in NTI frequency during the thermal responses.

Effects of heating and cooling on NTI frequency in the cold receptors (n = 23) used to determine the effects of the K⁺ channel blockers. The change in NTI frequency and the change in temperature are normalized to values measured just prior to initiating heating. The filled square indicates the starting point and the arrows indicate the direction of the change in temperature. The largest differences between the effects of heating and cooling on NTI frequency were at temperatures above the basal temperature.

Figure 5 shows the normalized change in NTI frequency during heating and cooling in the absence and in the presence of the K⁺ channel blockers. In the presence of 4-AP (n = 8, Fig. 5A) and TEA (n = 8, Fig. 5B), the effects of heating and cooling on NTI frequency were not significantly changed. Furthermore, the changes in the patterning of NTI occurrence produced by thermal stimulation were similar in the absence and in the presence of 4-AP and TEA (results not shown).

Figure 5. Ba²⁺ selectively changes responses to heating.

Changes in the response of cold receptors to heating and cooling produced by 4-AP (0.5 mm; A), TEA (20 mm; B) and Ba²⁺ (3 mm; C). The change in NTI frequency and the change in temperature are normalized to values measured just prior to initiating heating. Temperature responses were elicited before (warming ○, cooling □) and during (warming •, cooling ▪) application of the K⁺ channel blocker. Prior to applying the K⁺ channel blocker, heating decreased NTI activity and cooling increased NTI activity. Both 4-AP and TEA had no significant effect on the responses to heating or cooling (repeated measures ANOVA). Ba²⁺ selectively reduced the response to heating, producing a significant difference between the grouped NTI frequency data recorded before and during its application (vertical double arrowhead). In addition, there was significant interaction between the effects of Ba²⁺ and temperature on NTI frequency (horizontal double arrowhead). *P < 0.05, **P < 0.01.

By contrast, while Ba²⁺ (n = 7, Fig. 5C) did not change the response to cooling this agent did change the response to heating. In all receptors treated with Ba²⁺ (3 mm), the changes in NTI occurrence during heating and cooling were similar and Fig. 3 shows a representative example. In the absence of Ba²⁺, heating produced a marked decrease in NTI frequency and cooling increased NTI activity (Fig. 3A and B). While bursts of two NTIs occurred infrequently at the basal temperature, their frequency increased during cooling, contributing to the overall increase in NTI activity (Fig. 3C and D). In the presence of Ba²⁺, the resting activity was not greatly different from that in the absence of this agent and at the start of heating there was a sharp reduction in NTI frequency (indicated by the arrow in Fig. 3F). However, shortly after the initiation of heating, the receptor started to discharge bursts of up to six NTIs (Fig. 3G and H). These bursts initially occurred at intervals of about 3 s but their frequency increased with the increase in temperature (Fig. 3G), leading to an overall maintenance of NTI frequency. Almost immediately upon cooling, the pattern of NTI discharge in the presence of Ba²⁺ returned to that observed in the absence of this agent (Fig. 3F–H).

Effects of K⁺-induced depolarization and Cs⁺

Ba²⁺ interferes with a variety of ion channels and often produces depolarization through blockade of K⁺ channels that are open at the resting membrane potential. To determine whether the changed response to heating in the presence of Ba²⁺ was due to depolarization, we investigated the effects of depolarizing the nerve terminals by raising the K⁺ concentration of the solution superfusing the cornea. In addition, to investigate whether blockade of inwardly rectifying K⁺ channels by Ba²⁺ contributes to the changed response to heating, the effects of this ion were compared with those of Cs⁺, which also blocks these channels. In these experiments a slower rate of heating was used (maximum rate 0.11 ± 0.04°C s⁻¹, mean ±s.d.). Using this heating protocol, the reduction in NTI frequency was smaller than that produced by the faster heating rate (cf. Figs 5 and 6) and in corneas treated with 3 mm Ba²⁺ (n = 7), no change in the NTI frequency was observed during heating to 2°C above the basal temperature (Fig. 6A). In these experiments, 1 mm Ba²⁺ did not significantly change the response to heating (Fig. 6A), demonstrating that the effects of this ion were concentration dependent.

Figure 6. Neither raised K⁺ concentration nor Cs⁺ changed responses to heating.

Changes in the response of cold receptors to heating produced by Ba²⁺ (1 and 3 mm; A), raised K⁺ concentration (15 mm; B), and Cs⁺ (5 mm; C). The change in NTI frequency and the change in temperature are normalized to values measured just prior to initiating heating. In both B and C, the insets show averaged NTIs recorded before and during (bold line) application of a raised K⁺ concentration (B) and Cs⁺ (C). At 3 mm, Ba²⁺ abolished the reduction in NTI frequency with heating, and produced a significant difference between the grouped NTI frequency data recorded before and during its addition (vertical double arrowhead). At 1 mm, Ba²⁺ did not significantly change the response to heating (P = 0.06). Neither raising the K⁺ concentration nor Cs⁺ changed the response to heating, but both these agents changed the configuration of NTIs (see also Table 1). **P < 0.01.

Raising the K⁺ concentration from 4.7 to 15 mm (n = 8) did not change NTI frequency (control, 9.2 ± 1.2 Hz; 15 mm K⁺, 8.8 ± 1.6 Hz, P = 0.37) but it did slow the maximum and minimum dV/dt of the NTI and increase the half-width of its positive component (Fig. 6B, Table 1). In the presence of 15 mm K⁺ there was no change in the inhibition of NTI activity observed during heating (Fig. 6B). Raising the concentration of K⁺ to 30 mm (n = 8) inhibited NTI activity (control, 8.4 ± 1.1 Hz; 30 mm K⁺, 3.4 ± 1.3 Hz; P < 0.05), producing a more irregular pattern of firing. In the presence of 30 mm K⁺, heating still inhibited NTI activity (results not shown).

Cs⁺ (5 mm, n = 7) did not significantly change NTI frequency (control, 11.1 ± 1.0 Hz; 5 mm Cs⁺, 9.2 ± 0.7 Hz; P = 0.18) but it did reduce the positive and negative amplitude of the NTI and the minimum dV/dt and increase the half-width of the negative component of the NTI (Fig. 6C, Table 1). In the presence of Cs⁺, there was no change in the inhibition of NTI activity during heating (Fig. 6C).

Discussion

The NTIs recorded from the nerve terminals of cold receptors are diphasic (positive–negative) with the prominent positive component. These extracellularly measured changes in potential are proportional to the net membrane current, with positive and negative deflections from baseline being produced by net outward and inward current, respectively (Smith, 1988; Brock et al. 2001). Our previous studies indicated that, at the basal temperature (31–32°C), the nerve terminals of cold receptors are passively invaded by nerve impulses initiated at a site proximal to the nerve terminal (Brock et al. 2001; Carr et al. 2002). If it is assumed that there is no active ionic current, the membrane current will be composed of both a capacitive current and a resistive ionic current and the configuration of the NTI will be the first derivative of the membrane voltage change. In this case, the NTI will be positive during depolarization and negative during repolarization (see Fig. 7). While a regenerative Na⁺ current does not to appear to be triggered in the nerve terminal, voltage-activated K⁺ channels may be present and their opening may contribute to speeding repolarization of the nerve ending.

Figure 7. Modelled changes in the first derivative of the action potential produced by prolonging repolarization.

A, action potentials were calculated using a compartmental model for a length of axon incorporating the Hodgkin-Huxley equations. To simulate the effects of blocking K⁺ channels, the maximum K⁺ conductance in the terminal portion of the axon was reduced. B, prolonging action potential repolarization reduced the amplitude and slowed the time course of the negative component of the first derivative (dV/dt) of the action potential.

Figure 7 shows the effects of slowing action potential repolarization on the time course of the capacitive current (i.e. the first derivative of the action potential). The effects observed for these modelled NTIs are similar to those produced by the combined application of 4-AP and TEA (Fig. 2D), with both a reduction in the amplitude and a slowing in the time course of the negative component. However, when applied on its own, 4-AP reduced the negative amplitude of the NTI but did not detectably change the overall time course of this component. This effect would be produced by blocking K⁺ channels that selectively speed the initial phase of repolarization, resulting in a reduction in the peak amplitude of the inward capacitive current. This effect is most likely due to blockade of fast, transient voltage-activated K⁺ channels (I_K(A) channels). In contrast, TEA or Ba²⁺ when applied alone did not reduce the negative amplitude of the NTI but these agents did prolong the duration of the negative component of the NTI. Blockade of K⁺ channels (delayed rectifier and Ca²⁺-activated K⁺ channels) that speed a later stage of repolarization would prolong the inward capacitive current. Therefore the effects of all three K⁺ channel blockers on the negative component of the NTI can be explained by blockade of K⁺ channels that contribute to repolarization.

In addition to changing the negative component of the NTI, both TEA and Ba²⁺ reduced the positive amplitude of the NTI and, in addition, Ba²⁺ slowed the upstroke of the NTI (i.e. the maximum dV/dt) and increased the half-width of the positive component of the NTI. A depolarization of the nerve terminal axon may decrease the voltage change produced by the invading nerve impulse and this change may explain the decrease in positive amplitude of the NTI. For Ba²⁺, the effects on the time course can probably be explained by blockade of background K⁺ channels (Sperelakis et al. 1967; Cohen et al. 1983) and the resultant increase in membrane resistance. Such a change would increase the membrane time constant and thereby slow the upstroke of the NTI, a component of the NTI that is determined primarily by passive membrane properties (see Brock et al. 2001; Carr et al. 2002).

Despite the finding that both 4-AP and Ba²⁺ changed the configuration of NTIs, neither of these agents changed NTI frequency or the patterning of NTI discharge at the basal temperature. TEA increased the NTI frequency by about 25% and this change was due to an increase in both the number of impulses per burst and the frequency of burst occurrence. This finding suggests that activation of TEA-sensitive K⁺ channels limits the ability of the cold neurones to discharge in bursts (Utzschneider et al. 1993). As none of the K⁺ channel blockers significantly changed the cycle time (Table 2), K⁺ channels blocked by these agents do not appear to play a major role in the oscillation of membrane potential that produces the ongoing rhythmic generation of action potentials in cold receptors (Braun et al. 1980).

Neither 4-AP nor TEA changed the response of the cold receptors to heating or cooling. In contrast, Ba²⁺ selectively interfered with the response of cold receptors to heating. Under normal circumstances, heating from the basal temperature (∼32°C) almost immediately reduces NTI discharges, silencing them almost completely with temperature increases of 3–4°C (see Figs 3 and 4). In the presence of Ba²⁺, when the receptors were heated at the faster rate (see Figs 3 and 5), there was still a reduction in NTI activity at the onset of the heating but, as temperature continued to rise, NTI activity increased. With the slower rate of heating, the decrease in NTI activity at the start of heating was not observed in the presence of Ba²⁺ (see Fig. 6). Normally the magnitude of the decrease in NTI activity depends on the rate of heating (cf. Figs 5 and 6; Carr et al. 2003), with slower rates of heating producing smaller reductions in NTI activity. This effect of altering the rate of heating can be explained by the previously reported adaptation of the dynamic responses of cold receptors (see Carr et al. 2003). Therefore the difference in the effects of Ba²⁺ with slower and faster heating rates can probably be accounted for by the speed with which these receptors adapt during heating. In contrast to the effects of heating, the increase in NTI activity and the change in patterning of NTI occurrence during cooling were similar in the presence and the absence of Ba²⁺.

The mechanisms that underlie the temperature-induced changes in NTI activity in cold receptors remain to be elucidated, but it is likely that cooling and heating, respectively, depolarize and hyperpolarize the nerve terminal (Carr et al. 2003). The cooling-activated transient receptor potential channel TRPM8 is also activated by menthol, and this agent increases the NTI frequency in all corneal cold receptors that have been tested (10–100 μm, n = 16, authors' unpublished observations), indicating that they are likely to possess these channels. As Ba²⁺ did not change the response to cooling, it seems unlikely that the effects of this ion can be attributed to blockade of TRPM8 channels. However, it is possible that Ba²⁺ changes the gating of these channels.

We can find no reports that Ba²⁺ directly changes the activity of the Na⁺,K⁺-ATPase, but it is known that this ion reduces the background K⁺ conductance in many cell types (e.g. Sperelakis et al. 1967; Cohen et al. 1983). As indicated above, the effects of Ba²⁺ on the positive component of the NTI can be attributed to a decrease in background K⁺ conductance, but this agent did not change NTI activity at the basal temperature. Similarly, raising the K⁺ concentration of the bathing solution from 5 to 15 mm did not change NTI activity, indicating that a small depolarization does not stimulate corneal cold receptors. Moreover, raising the K⁺ concentration did not change the reduction in NTI activity produced by heating, indicating that the changed response to heating in the presence of Ba²⁺ cannot simply be attributed to a depolarization of the sensory nerve terminal. The effect of Ba²⁺ on the thermal response of cold receptors is also unlikely to be due to blockade of inward rectifier K⁺ channels, as Cs⁺, which blocks these channels, did not change the response to heating.

Recently two-pore domain K⁺ channels (TREK-1, TREK-2 and TRAAK) have been described that are strongly activated by heating (Maingret et al. 2000; Kang et al. 2005). In expression systems, TREK-1 and TREK-2 are activated at temperatures above ∼25°C, whereas TRAAK is activated at temperatures above ∼31°C (Kang et al. 2005). Channels with properties similar to TREK-1, TREK-2 and TRAAK are present in some rat cultured DRG sensory neurones where they are also activated by heating (Kang et al. 2005). As millimolar concentrations of Ba²⁺ would be expected to reduce currents generated by TREK-1, TREK-2 and TRAAK channels (Fink et al. 1996, 1998; Bang et al. 2000), blockade of these thermally sensitive two-pore domain K⁺ channels could explain how, in corneal cold receptors, this ion produces a marked reduction in the inhibition of sensory discharges produced by heating.

In conclusion, TEA, 4-AP and Ba²⁺ all changed the shape of NTIs in a manner consistent with blockade of K⁺ channels that contribute to shaping action potentials in the terminals of cold receptors. However, only Ba²⁺ changed the thermal response, selectively inhibiting the firing rate reduction exhibited by cold receptors in response to heating. This finding suggests that different ionic mechanisms underlie the dynamic response of corneal cold receptors to heating and to cooling.