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

J Physiol 575.2 (2006) pp 573–581 573

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

James Brock1, M. Carmen Acosta2, Amr Al Abed1, Svetlana Pianova1 and Carlos Belmonte2

1Prince of Wales Medical Research Institute, University of New South Wales, Randwick, NSW 2031, Australia2Instituto de Neurociencias de Alicante, Universidad Miguel Hernandez-CSIC, Sant Joan d’Alacant, Spain

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.

(Received 21 March 2006; accepted after revision 19 June 2006; first published online 22 June 2006)

Corresponding author J. Brock: Prince of Wales Medical Research Institute, Barker St, Randwick, NSW 2031, Australia.

Email: [email protected]

In mammals, cold thermal receptors on the bodysurface fire spontaneously at neutral temperatures andincrease their discharge frequency with small temperaturedecreases (Hensel, 1981). In humans, these receptorsare believed to be responsible for eliciting the sensoryexperiences of cold. While these receptors are coldresponsive, the largest changes in activity are producedby dynamic changes in temperature; peak increases ordecreases in firing frequency occurring during cooling andheating, respectively (e.g. see Dykes, 1975; Carr et al. 2003).Therefore these receptors might more appropriately beconsidered thermal receptors, as they can potentially signalboth cooling and heating.

At static temperatures between 25 and 40◦C, actionpotentials occur in a periodic manner, with single or shortbursts of impulses occurring at regular intervals. Overthis range of temperatures, the frequency and number ofimpulses per burst are inversely related to temperature,whereas the cycle frequency increases with temperature.This pattern of activity is suggested to originate from acyclical oscillation in membrane potential at the site ofimpulse initiation (Braun et al. 1980).

The mechanisms that underlie the temperature-inducedchanges in impulse frequency remain poorly understoodbut it is likely that cooling and heating, respectively,depolarize and hyperpolarize the nerve terminal (Carret al. 2003). Studies in cultured sensory neurones haveindicated that temperature-induced changes in membranepotential are produced by gating of the cooling-activatedtransient receptor potential channel TRPM8 (McKemyet al. 2002; Peier et al. 2002; de la Pena et al. 2005), as wellas thermal modulation of a background K+ conductance(Reid & Flonta, 2001; Viana et al. 2002). It has alsobeen suggested that thermal modulation of the membraneNa+,K+-ATPase contributes to thermal transduction incold receptors (Pierau et al. 1975; Schafer & Braun, 1990).As the mechanisms that generate the ongoing activity ofcold receptors are themselves temperature sensitive (Braunet al. 1980), it is very likely that they contribute to theprocess of thermal transduction, although this possibilityhas not received much attention (Schafer et al. 1991). Whilethe underlying mechanisms that generate the rhythmicdischarge of action potentials are unknown, it is likely thatK+ channels play a role in regulating this activity.

C© 2006 The Authors. Journal compilation C© 2006 The Physiological Society DOI: 10.1113/jphysiol.2006.110130

574 J. Brock and others J Physiol 575.2

We have used a technique that allows electrical activity tobe recorded directly from single nerve terminals in guineapig cornea (Brock et al. 1998) to investigate the effects ofbroad-spectrum K+ channel blockers (4-aminopyridine,4-AP; tetraethylammonium, TEA; and Ba2+) on theconfiguration of nerve terminal impulses (NTIs) in coldreceptors, and their ongoing activity during heating andcooling. At the concentrations used, the K+ channelsblocked by these agents include those producing bothtransient and slowly inactivating K+ currents (IK(A) andIK(D): 4-AP), delayed rectifier currents (TEA and Ba2+),Ca2+-activated K+ currents (TEA and Ba2+) and inwardrectifier currents (Ba2+). All these agents changed theconfiguration of NTIs in a manner consistent withthe blockade of K+ channels, but only TEA changedthe basal activity of the receptors, while Ba2+ selectivelyinterfered with their response to heating. The findingssuggest that separate ionic mechanisms underlie theresponse of corneal cold receptors to cooling and toheating.

Methods

All experimental procedures conformed to the NationalHealth and Medical Research Council of Australiaguidelines and were approved by the University of NewSouth Wales Animal Care and Ethics Committee.

Guinea pigs of both sexes in the weight range250–450 g were anaesthetized with sodium pento-barbitone (100 mg kg−1

i.p.) and killed by exsanguination.Both eyes were isolated along with a short length of opticnerve and the associated ciliary nerves. Eyes were mountedeither in a single or two-compartment recording chamber.In the two-compartment recording chamber, the eye wasmounted in a hole in the dividing wall so that the corneaand conjunctiva were separated from the rest of the eye, andwas held in place by pinning the conjunctiva to the siliconeresin-coated surface of the dividing wall. The chamberswere perfused at 3–5 ml min−1 with physiological saline ofthe 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 bathingsolution was maintained at 31.5–32.5◦C. The temperatureof the solution superfusing the cornea was monitoredcontinuously by a thermocouple placed in close appositionto its surface. The optic nerve and associated ciliary nerveswere drawn into a suction-stimulating electrode. Theciliary nerves were stimulated electrically with a constantvoltage 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 surfaceof the corneal epithelium with slight suction (Brock et al.1998). An Ag–AgCl electrode in the recording chamberserved 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 dataacquisition system, ADInstruments Pty Ltd, Castle Hill,NSW, Australia). Recordings were only made from siteson the cornea where the NTIs were readily distinguishedfrom the noise (∼10 μV peak-to-peak).

To deliver agents locally at the site of recording, apolyethylene tube was positioned inside the recordingelectrode, close to the tip (see Brock & Cunnane, 1995). Theelectrode was continually perfused at ∼50 μl min−1 withHepes-buffered saline of the following composition (mm):Hepes 10; NaCl 151; KCl 4.7; CaCl2 2; MgCl2 1.2; glucose7.8. The pH of this solution was adjusted to 7.4 usingNaOH. Agents were added to the Hepes-buffered salineat the required concentration. Perfusion of the recordingelectrode with Hepes-buffered saline alone has previouslybeen shown to be without effect on cold receptor NTIs (seeBrock 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 siteswhere the electrical activity originated from a singlenerve terminal. At these sites, electrical stimulation ofthe ciliary nerves evoked a single all-or-none NTI andthe spontaneously occurring orthodromic NTIs collidedwith antidromically propagated, electrically evoked NTIs(see Brock et al. 1998). Only NTIs that were defined asoriginating in cold receptors were analysed (see Brocket al. 1998). The cold receptors had relatively high levelsof ongoing NTI discharge (6–15 Hz) which occurredrhythmically and was decreased by warming and increasedby cooling the solution superfusing the cornea.

Data analysis

Data were analysed using the computer program Igor Pro(Wavemetrics, Lake Oswego, OR, USA). Measurementsof NTI amplitude and shape were taken from averagesof 50–100 individual NTIs. Prior to averaging, NTIswere aligned in time at their peak positive amplitude.When NTIs occurred in high frequency, short bursts, theiramplitude declined during the burst. For this reason onlythe first NTI in each burst was used to construct the averageNTI.

The NTIs were diphasic (positive–negative) with aprominent positive component (see Fig. 2). The positiveand negative amplitude of the NTI, the maximum rate ofchange of voltage during the initial upstroke and the down-stroke of the NTI (maximum and minimum dV /dt) andthe half-widths of the positive and negative componentswere measured. Prior to assessing the effects produced by

C© 2006 The Authors. Journal compilation C© 2006 The Physiological Society

J Physiol 575.2 Effects of K+ channel blockers on cold receptors 575

each K+ channel blocker using Student’s paired t tests, themeasured changes were normalized to the values measuredjust prior to their addition. Comparisons between theeffects of the K+ channel blockers were made with one-wayANOVA followed by Tukey HSD post hoc tests.

Figure 1 shows the typical pattern of nerve activity incorneal cold receptors at the basal temperature. Single orshort bursts of impulses occur at regular intervals and thedistribution of intervals between NTIs is bimodal (Fig. 1B)with long and short intervals, the latter occurring duringbursts of NTIs. To assess the effects of the K+ channelblockers on the patterning of NTI activity the meanfrequency of NTI discharge, the long interval (Fig. 1A andB), the cycle time (i.e the time between ends of the longintervals; Fig. 1A), the number of NTIs per cycle (Fig. 1C),the number of NTIs per burst and the relative frequencyof bursts (i.e. number of bursts/number of cycles) werecompared before and 10–15 min following their additionusing Student’s paired t tests.

The dynamic changes in NTI frequency in responseto heating and to cooling were assessed before and∼15 min following the addition of the K+ channelblockers. Prior to statistical comparison, the changes inNTI frequency during the temperature responses werenormalized to values measured just prior to initiatingheating. The effects of each K+ channel blocker onthe responses to heating and to cooling were comparedseparately using multivariate repeated measures ANOVA.For these comparisons, P values were adjusted usingGreenhouse-Geisser’s correction.

Unless otherwise indicated, data are presented asmeans ± s.e.m. SPSS version 11 for Macintosh (SPSS, Inc.,Chicago, IL, USA) was used for all statistical comparisonsand 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 andTable 1 shows effects of these agents on the measuredNTI parameters. Each of these agents had a characteristiceffect on NTI shape. Locally applied 4-AP reduced thenegative amplitude of the NTI but was without effect onthe positive amplitude of the NTI or any of the measuresof NTI time course (Fig. 2A, Table 1). TEA produced asmall decrease in the positive amplitude of the NTI andincreased the half-width of the negative component ofthe NTI, but was without effect on negative amplitudeof the NTI or on any other measures of NTI time course(Fig. 2B, Table 1). Ba2+ also reduced the positive amplitudeand increased the half-width of the negative componentof the NTI but, in addition, increased the half-widthof the positive component and reduced the maximum

dV /dt of the NTI (Fig. 2C, Table 1). Despite these apparentdifferences between the effects of TEA and Ba2+, therewere no differences when the changes produced by theseagents in the two sets of nerve terminals were compared.The effect of TEA and Ba2+ on the half-width of thenegative component was primarily due to slowing of itsdecay (Fig. 2B and C).

The effects of 4-AP and TEA differed significantly fromeach other and appeared to sum when they were appliedtogether (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 arelikely to be present in the cold-sensitive nerve endings (seeDiscussion). To determine their effects on the ongoingNTI activity and on responses to thermal stimulation, theK+ channel blockers were applied to the whole cornea bytheir addition to the solution perfusing the front chamberof the divided recording chamber. When applied in thismanner, 4-AP (0.5 mm, n = 8), TEA (20 mm, n = 8) andBa2+ (3 mm, n = 7) produced similar changes in the shapeof NTIs to those produced by their local application (resultsnot shown).

Under basal conditions (31.5–32.5◦C), prior to applyingthe K+ channel blockers, NTIs occurred rhythmically,

Figure 1. Patterning of nerve terminal impulses (NTIs) in acorneal 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 thenumber of NTIs per cycle (C) during a 40 s recording period. SingleNTIs or bursts of two NTIs occurred at regular intervals.



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 11TEA 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 10Ba2+ 0.87 ± 0.03† 1.07 ± 0.06 0.92 ± 0.02† 0.94 ± 0.03 1.06 ± 0.02 ∗1.51 ± 0.14† 94-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 9Cs+ 0.87 ± 0.02‡ 0.66 ± 0.06† 1.03 ± 0.04 0.84 ± 0.04 1.05 ± 0.03 1.81 ± 0.17† 7Raised 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 todrug application: ∗P < 0.05, †P < 0.01, ‡P < 0.001). Significant differences for pairwise comparisons between 4-AP, TEA and 4-AP plusTEA data: cP < 0.05, a,bP < 0.01, dP < 0.001). For these multiple comparisons, the data were first compared by one-way ANOVA andthen by Tukey HSD post hoc tests.

with single or short bursts of usually two but up tofive NTIs occurring at regular intervals (see Fig. 1).Table 2 shows the effects of 4-AP, TEA and Ba2+ on thefrequency and patterning of NTI occurrence. Ba2+ and4-AP had no detectable effect on the ongoing activity ofthe nerve terminals (Table 2). In contrast, TEA increasedthe frequency of NTI occurrence, and this change wasassociated with an increase in both the number of impulsesper burst and the frequency of burst occurrence (Table 2).

Figure 2. 4-Aminopyridine, TEA and Ba2+ changed the configuration of NTIsA–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 NTIsoverlaid (bold line is drug treated).

Effects of K+ channel blockers on responsesto thermal stimulation

The solution superfusing the cornea was first heated fromthe basal temperature (31–32.5◦C) to 36–38◦C (maximumrate 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 basaltemperature (see Fig. 3A). Figure 3A–D shows a typical



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)

RelativeLong Impulses Impulses frequency of Cycle

Frequency interval per per cycles with time(impulses s−1) (ms) cycle burst bursts (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 84-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 8TEA 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 7Ba2+ 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 beforeand during drug application.

Figure 3. Typical thermal responses of a corneal cold receptorbefore 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 bathingsolution recorded close to the corneal surface. B and F, effect ofchanging temperature on the frequency of NTIs. F, the arrow indicatesthe sharp reduction in NTI frequency at the start of heating. C and G,intervals between successive NTIs. D and H, number of NTIs thatoccurred during each cycle (i.e. between successive long intervals). Inthe absence of Ba2+, heating silenced the receptor, and coolingincreased NTI frequency; the latter change was associated with ashortening of the long interval and an increase in the number ofimpulses per cycle. In the presence of Ba2+ (3 mM), the receptor didnot silence during heating but the changes in NTI activity produced bycooling were similar to those in the absence of this agent.

response of a corneal cold receptor to this thermalstimulation protocol and Fig. 4 shows the changes in NTIfrequency during heating and cooling for the 23 coldreceptors investigated (measured just prior to adding theK+ channel blockers). The frequency of NTI discharge isnot simply related to the ambient temperature but is alsodependent on the rate of change of temperature (see Carret al. 2003). As a result, the peak increase in NTI frequencyoccurred at temperatures close to the basal temperatureduring cooling and the largest difference between theeffects of heating and cooling was observed at temperaturesabove the basal temperature (Fig. 4). Subsequently, theeffects of the K+ blockers were compared during heatingto 4◦C above the basal temperature and cooling back fromthis temperature to the basal temperature.

Figure 5 shows the normalized change in NTI frequencyduring heating and cooling in the absence and in the

Figure 4. Changes in NTI frequency during the thermalresponsesEffects of heating and cooling on NTI frequency in the cold receptors(n = 23) used to determine the effects of the K+ channel blockers. Thechange in NTI frequency and the change in temperature arenormalized to values measured just prior to initiating heating. Thefilled square indicates the starting point and the arrows indicate thedirection of the change in temperature. The largest differencesbetween the effects of heating and cooling on NTI frequency were attemperatures above the basal temperature.



presence of the K+ channel blockers. In the presence of4-AP (n = 8, Fig. 5A) and TEA (n = 8, Fig. 5B), the effectsof heating and cooling on NTI frequency were notsignificantly changed. Furthermore, the changes in thepatterning of NTI occurrence produced by thermal

Figure 5. Ba2+ selectively changes responses to heatingChanges in the response of cold receptors to heating and coolingproduced 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 arenormalized to values measured just prior to initiating heating.Temperature responses were elicited before (warming �, cooling �)and during (warming •, cooling �) application of the K+ channelblocker. Prior to applying the K+ channel blocker, heating decreasedNTI activity and cooling increased NTI activity. Both 4-AP and TEA hadno significant effect on the responses to heating or cooling (repeatedmeasures ANOVA). Ba2+ selectively reduced the response to heating,producing a significant difference between the grouped NTI frequencydata recorded before and during its application (vertical doublearrowhead). In addition, there was significant interaction between theeffects of Ba2+ and temperature on NTI frequency (horizontal doublearrowhead). ∗P < 0.05, ∗∗P < 0.01.

stimulation were similar in the absence and in the presenceof 4-AP and TEA (results not shown).

By contrast, while Ba2+ (n = 7, Fig. 5C) did not changethe response to cooling this agent did change the responseto heating. In all receptors treated with Ba2+ (3 mm), thechanges in NTI occurrence during heating and coolingwere similar and Fig. 3 shows a representative example. Inthe absence of Ba2+, heating produced a marked decrease inNTI frequency and cooling increased NTI activity (Fig. 3Aand B). While bursts of two NTIs occurred infrequentlyat the basal temperature, their frequency increased duringcooling, contributing to the overall increase in NTI activity(Fig. 3C and D). In the presence of Ba2+, the restingactivity was not greatly different from that in the absenceof this agent and at the start of heating there was a sharpreduction in NTI frequency (indicated by the arrow inFig. 3F). However, shortly after the initiation of heating,the receptor started to discharge bursts of up to sixNTIs (Fig. 3G and H). These bursts initially occurred atintervals of about 3 s but their frequency increased withthe increase in temperature (Fig. 3G), leading to an overallmaintenance of NTI frequency. Almost immediately uponcooling, the pattern of NTI discharge in the presence ofBa2+ 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 oftenproduces depolarization through blockade of K+ channelsthat are open at the resting membrane potential. Todetermine whether the changed response to heating inthe presence of Ba2+ was due to depolarization, weinvestigated the effects of depolarizing the nerve terminalsby raising the K+ concentration of the solution super-fusing the cornea. In addition, to investigate whetherblockade of inwardly rectifying K+ channels by Ba2+

contributes to the changed response to heating, the effectsof this ion were compared with those of Cs+, which alsoblocks these channels. In these experiments a slower rateof heating was used (maximum rate 0.11 ± 0.04◦C s−1,mean ± s.d.). Using this heating protocol, the reductionin NTI frequency was smaller than that produced bythe faster heating rate (cf. Figs 5 and 6) and in corneastreated with 3 mm Ba2+ (n = 7), no change in the NTIfrequency was observed during heating to 2◦C above thebasal 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 wereconcentration dependent.

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 themaximum and minimum dV /dt of the NTI and increasethe half-width of its positive component (Fig. 6B, Table 1).



In the presence of 15 mm K+ there was no change inthe 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 moreirregular 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 changeNTI frequency (control, 11.1 ± 1.0 Hz; 5 mm Cs+,9.2 ± 0.7 Hz; P = 0.18) but it did reduce the positive andnegative amplitude of the NTI and the minimum dV /dtand increase the half-width of the negative component ofthe NTI (Fig. 6C, Table 1). In the presence of Cs+, therewas no change in the inhibition of NTI activity duringheating (Fig. 6C).

Discussion

The NTIs recorded from the nerve terminals of coldreceptors are diphasic (positive–negative) with theprominent positive component. These extracellularlymeasured changes in potential are proportional to the netmembrane current, with positive and negative deflectionsfrom baseline being produced by net outward and inwardcurrent, respectively (Smith, 1988; Brock et al. 2001). Ourprevious studies indicated that, at the basal temperature(31–32◦C), the nerve terminals of cold receptors arepassively invaded by nerve impulses initiated at a siteproximal to the nerve terminal (Brock et al. 2001; Carret al. 2002). If it is assumed that there is no active ioniccurrent, the membrane current will be composed of botha capacitive current and a resistive ionic current and theconfiguration of the NTI will be the first derivative ofthe membrane voltage change. In this case, the NTI willbe positive during depolarization and negative duringrepolarization (see Fig. 7). While a regenerative Na+

current does not to appear to be triggered in the nerveterminal, voltage-activated K+ channels may be presentand their opening may contribute to speedingrepolarization of the nerve ending.

Figure 7 shows the effects of slowing action potentialrepolarization on the time course of the capacitive current(i.e. the first derivative of the action potential). The effectsobserved for these modelled NTIs are similar to thoseproduced by the combined application of 4-AP and TEA(Fig. 2D), with both a reduction in the amplitude and aslowing in the time course of the negative component.However, when applied on its own, 4-AP reduced thenegative amplitude of the NTI but did not detectablychange the overall time course of this component. Thiseffect would be produced by blocking K+ channelsthat selectively speed the initial phase of repolarization,resulting in a reduction in the peak amplitude of the inwardcapacitive current. This effect is most likely due to blockadeof fast, transient voltage-activated K+ channels (IK(A)

channels). In contrast, TEA or Ba2+ when applied alone

did not reduce the negative amplitude of the NTI but theseagents did prolong the duration of the negative componentof the NTI. Blockade of K+ channels (delayed rectifierand Ca2+-activated K+ channels) that speed a later stageof repolarization would prolong the inward capacitivecurrent. Therefore the effects of all three K+ channelblockers on the negative component of the NTI can beexplained by blockade of K+ channels that contribute torepolarization.

Figure 6. Neither raised K+ concentration nor Cs+ changedresponses to heatingChanges in the response of cold receptors to heating produced byBa2+ (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 temperatureare normalized to values measured just prior to initiating heating. Inboth B and C, the insets show averaged NTIs recorded before andduring (bold line) application of a raised K+ concentration (B) and Cs+(C). At 3 mM, Ba2+ abolished the reduction in NTI frequency withheating, and produced a significant difference between the groupedNTI frequency data recorded before and during its addition (verticaldouble arrowhead). At 1 mM, Ba2+ did not significantly change theresponse to heating (P = 0.06). Neither raising the K+ concentrationnor Cs+ changed the response to heating, but both these agentschanged the configuration of NTIs (see also Table 1). ∗∗P < 0.01.



In addition to changing the negative component of theNTI, both TEA and Ba2+ reduced the positive amplitudeof the NTI and, in addition, Ba2+ slowed the upstrokeof the NTI (i.e. the maximum dV /dt) and increasedthe half-width of the positive component of the NTI. Adepolarization of the nerve terminal axon may decrease thevoltage change produced by the invading nerve impulseand this change may explain the decrease in positiveamplitude of the NTI. For Ba2+, the effects on the timecourse can probably be explained by blockade of back-ground 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 constantand thereby slow the upstroke of the NTI, a component ofthe NTI that is determined primarily by passive membraneproperties (see Brock et al. 2001; Carr et al. 2002).

Despite the finding that both 4-AP and Ba2+ changedthe configuration of NTIs, neither of these agents changedNTI frequency or the patterning of NTI discharge at thebasal temperature. TEA increased the NTI frequency byabout 25% and this change was due to an increase inboth the number of impulses per burst and the frequencyof burst occurrence. This finding suggests that activationof TEA-sensitive K+ channels limits the ability of thecold neurones to discharge in bursts (Utzschneider et al.1993). As none of the K+ channel blockers significantlychanged the cycle time (Table 2), K+ channels blockedby these agents do not appear to play a major role inthe oscillation of membrane potential that produces theongoing rhythmic generation of action potentials in coldreceptors (Braun et al. 1980).

Neither 4-AP nor TEA changed the response of thecold receptors to heating or cooling. In contrast, Ba2+

selectively interfered with the response of cold receptorsto heating. Under normal circumstances, heating from thebasal temperature (∼32◦C) almost immediately reducesNTI discharges, silencing them almost completely withtemperature increases of 3–4◦C (see Figs 3 and 4). In the

Figure 7. Modelled changes in the first derivative of the actionpotential produced by prolonging repolarizationA, action potentials were calculated using a compartmental model fora length of axon incorporating the Hodgkin-Huxley equations. Tosimulate 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 amplitudeand slowed the time course of the negative component of the firstderivative (dV /dt) of the action potential.

presence of Ba2+, when the receptors were heated at thefaster rate (see Figs 3 and 5), there was still a reduction inNTI activity at the onset of the heating but, as temperaturecontinued to rise, NTI activity increased. With the slowerrate of heating, the decrease in NTI activity at the startof heating was not observed in the presence of Ba2+ (seeFig. 6). Normally the magnitude of the decrease in NTIactivity depends on the rate of heating (cf. Figs 5 and 6; Carret al. 2003), with slower rates of heating producing smallerreductions in NTI activity. This effect of altering the rateof heating can be explained by the previously reportedadaptation of the dynamic responses of cold receptors (seeCarr et al. 2003). Therefore the difference in the effectsof Ba2+ with slower and faster heating rates can probablybe accounted for by the speed with which these receptorsadapt during heating. In contrast to the effects of heating,the increase in NTI activity and the change in patterningof NTI occurrence during cooling were similar in thepresence and the absence of Ba2+.

The mechanisms that underlie the temperature-inducedchanges in NTI activity in cold receptors remain tobe elucidated, but it is likely that cooling and heating,respectively, depolarize and hyperpolarize the nerveterminal (Carr et al. 2003). The cooling-activated transientreceptor potential channel TRPM8 is also activated bymenthol, and this agent increases the NTI frequency in allcorneal cold receptors that have been tested (10–100 μm,n = 16, authors’ unpublished observations), indicatingthat they are likely to possess these channels. As Ba2+ didnot change the response to cooling, it seems unlikely thatthe effects of this ion can be attributed to blockade ofTRPM8 channels. However, it is possible that Ba2+ changesthe gating of these channels.

We can find no reports that Ba2+ directly changes theactivity of the Na+,K+-ATPase, but it is known that thision reduces the background K+ conductance in manycell types (e.g. Sperelakis et al. 1967; Cohen et al. 1983).As indicated above, the effects of Ba2+ on the positivecomponent of the NTI can be attributed to a decrease inbackground K+ conductance, but this agent did not changeNTI activity at the basal temperature. Similarly, raisingthe K+ concentration of the bathing solution from 5 to15 mm did not change NTI activity, indicating that a smalldepolarization does not stimulate corneal cold receptors.Moreover, raising the K+ concentration did not change thereduction in NTI activity produced by heating, indicatingthat the changed response to heating in the presence ofBa2+ cannot simply be attributed to a depolarization ofthe sensory nerve terminal. The effect of Ba2+ on thethermal response of cold receptors is also unlikely to bedue to blockade of inward rectifier K+ channels, as Cs+,which blocks these channels, did not change the responseto 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; Kanget al. 2005). In expression systems, TREK-1 and TREK-2are activated at temperatures above ∼25◦C, whereasTRAAK is activated at temperatures above ∼31◦C (Kanget al. 2005). Channels with properties similar to TREK-1,TREK-2 and TRAAK are present in some rat culturedDRG sensory neurones where they are also activated byheating (Kang et al. 2005). As millimolar concentrationsof Ba2+ would be expected to reduce currents generatedby TREK-1, TREK-2 and TRAAK channels (Fink et al.1996, 1998; Bang et al. 2000), blockade of these thermallysensitive two-pore domain K+ channels could explain how,in corneal cold receptors, this ion produces a markedreduction in the inhibition of sensory discharges producedby heating.

In conclusion, TEA, 4-AP and Ba2+ all changed theshape of NTIs in a manner consistent with blockade of K+

channels that contribute to shaping action potentials in theterminals of cold receptors. However, only Ba2+ changedthe thermal response, selectively inhibiting the firingrate reduction exhibited by cold receptors in responseto heating. This finding suggests that different ionicmechanisms underlie the dynamic response of corneal coldreceptors to heating and to cooling.

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Acknowledgements

This work was supported by National Health and Medical

Research Council of Australia Project Grant 113920. J.A.B. is a

National Health and Medical Research Council Senior Research

Fellow. We thank Elspeth McLachlan and Richard Carr for their

comments on the manuscript.


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