Acute temperature sensitivity in optic nerve axons ...

Acute temperature sensitivity in optic nerve axons explained by an electrogenic membrane potential Tom A. Coates, Oscar Woolnough, Joseph M. Masters, Gulsum Asadova, Charmilie Chandrakumar & Mark D. Baker Pflügers Archiv - European Journal of Physiology European Journal of Physiology ISSN 0031-6768 Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-015-1696-2

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Author's personal copy Pflugers Arch - Eur J Physiol DOI 10.1007/s00424-015-1696-2

NEUROSCIENCE

Acute temperature sensitivity in optic nerve axons explained by an electrogenic membrane potential Tom A. Coates & Oscar Woolnough & Joseph M. Masters & Gulsum Asadova & Charmilie Chandrakumar & Mark D. Baker

Received: 2 September 2014 / Revised: 17 February 2015 / Accepted: 17 February 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract Classical work in squid axon reports resting membrane potential is independent of temperature, but our findings suggest that this is not the case for axons in mammalian optic nerve. Refractory period duration changes over 10 times between 37 °C and room temperature, and afterpotential polarity is also acutely temperature sensitive, inconsistent with changes in temperature impacting nerve function only through altered rates of ion channel gating kinetics. Our evidence suggests that the membrane potential is enhanced by warming, an effect reduced by exposure to ouabain. The temperature dependence can be explained if axonal Na+/K+ ATPase continuously expels Na + ions that enter axons largely electroneutrally, thereby adding a substantial electrogenic component to the membrane potential. Block of the Na+ transporter NKCC1 with bumetanide increases refractoriness, like depolarization, indicating that this is a probable route by which Na+ enters, raising the expectation that the rate of electroneutral Na+ influx increases with temperature and suggesting a temperature-dependent transmembrane Na+ cycle that contributes to membrane potential. Keywords Optic nerve . Membrane potential . Recovery cycle . Na+/K+-ATPase . NKCC1 . Ouabain . Bumetanide Abbreviations QTRAC A computerized threshold-tracking programme available from Digitimer Ltd ATX-II 47 Amino acid peptidyl toxin from sea anemone, Anemonia sulcata T. A. Coates : O. Woolnough : J. M. Masters : G. Asadova : C. Chandrakumar : M. D. Baker (*) Neuroscience and Trauma Centre, Blizard Institute, Barts and the London School of Medicine, Queen Mary University of London, 4 Newark Street, London E12AT, UK e-mail: [email protected]

HEPES NKCC1 NHE, NHE1 DAP

2-[4-(2-Hydroxyethyl)piperazin-1yl]ethanesulfonic acid Bumetanide-sensitive Na-K-2Cl co-transporter Na-H ion exchanger Depolarizing afterpotential

Introduction The mechanisms responsible for setting the normal resting membrane potential of central white matter axons have not been fully elucidated although they must impact axonal excitability and play a key role in nervous system signalling. Experiments on squid axon [21] indicated that heating the nerve from near 0 °C has very little effect on membrane potential, although over the warmest temperatures investigated (>30 °C), membrane potential was progressively and slightly reduced, consistent with an increased Na+ leakage. The dependence of ionic reversal potentials on the absolute temperature as formulated in the Nernst equation is predicted to have only tiny effects on membrane potential over temperature ranges even as wide as 10 or 20 °C, and in the light of these considerations, changes in the rates of channel gating [21] are usually assumed to account for the effects of the small alterations of temperature on membrane excitability. In contrast, work in Aplysia neurons [10] has revealed a temperaturedependent resting potential, attributed to a Na+-pump-mediated electrogenic component, where warming can increase the resting potential by as much as 50 %. In this preparation, the membrane potential ionic dependence behaves similarly to the Goldman, Hodgkin, and Katz (GHK) equation [23] only when the Na+-pump is inhibited. Subsequent work by Hakozaki and

Author's personal copy Pflugers Arch - Eur J Physiol

colleagues [17] on these same cells has confirmed an oubainsensitive component of the resting membrane potential, but also revealed that the temperature-dependent effects are unlikely to be mediated by changes in pump activity alone. They report that a K+ channel activation involving temperaturesensitive G-protein regulation can also produce an ouabaininsensitive hyperpolarization in these cells. These considerations reveal two possible means of conferring a warmingdependent increase in membrane potential, namely a temperature-sensitive electrogenic component and warmingdependent increase in K+ conductance. Given the very limited information available regarding the response of resting properties of mammalian nerve to changing temperature, and their potential importance in brain function, we have investigated the properties of axons in mammalian optic nerve ex vivo over a range of temperatures between room and close to physiological. Changing the temperature modifies ion transport rates including the rate of electrogenic Na+ pumping [14, 29, 39], and the Na+/K+-ATPase (Na+-pump) is present in the optic nerve [16, 48]. Impulse-dependent increased pump activity has been described for peripheral nerve, with mammalian unmyelinated afferents in vagus undergoing transient pump-mediated hyperpolarizations of up to 30 mV at room temperature [37]. One unknown is the degree to which on-going electrogenic pump activity normally contributes to the resting potential of central axons. It would be reasonable to expect that the electrogenic component of resting potential would be larger than that in peripheral myelinated axons, because of the larger surface area to volume ratio of the smaller central fibres and the likelihood of proportionally greater Na+ influx per unit time through both impulse activity and resting leakage, perhaps by a variety of mechanisms. Evidence consistent with the Na+-pump contributing substantially to axonal membrane potential, in vivo, over many minutes, comes from studies of the post-ischaemic state in the periphery, where axons are hyperpolarized relative to rest after Na+ loading and subexcitable due to pump activity [8]. Because of the large activity-dependent effects, it is commonly assumed that the predominant route of Na+ influx is always via ion channels. One important factor concerning a role for the Na+-pump in setting the axonal resting membrane potential is the normal mechanism by which Na+ ions leak into the axons. This is because if the Na+ entry was predominantly via open Na+ channels (as expected during activity), then the stoichiometry of the pump would rule it out as a significant contributor to resting potential, because 3 units of charge would be transferred across the membrane from outside to inside through the channels in order to allow the pump to pass 1 unit of charge outward. The pump would be unable to keep up with the inwardly directed current, and the membrane potential would have to be maintained by the resting K+ conductance with the normally outwardly directed movement of K+ ions. However,

just as the resting membrane potential of central axons is unknown, there is rather limited data on the contribution of ion channels to resting Na+ leakage. On exposure to high concentrations of oubain, optic nerve axons depolarize and an early component of that depolarization cannot be blocked by tetrodotoxin [35]. This implies that the membrane potential has a resting electrogenic component and that some Na+ entry may not involve Na+ channels. The electroneutral anion-cation cotransporter, NKCC1, is known to be functionally expressed in central axons [28], reviewed in [4], and may therefore be involved in the resting Na+ influx, without any obligatory transmembrane charge movement. The transporter rate would be expected to increase on warming, resulting in Na+ influx increasing with increasing temperature where extracellular Cl− and K+ ion concentrations are not rate limiting. (Maximal transport rates for ion antiporters running off the Na+ gradient in central axons, including those where ion exchange is electroneutral, would also be expected to increase). This line of argument would suggest that if NKCC1 is involved in temperature-dependent Na+ influx, it is unlikely to be the only transport system involved. The functional role of NKCC1 may be investigated using a selective blocker, the loop diuretic bumetanide (e.g., reviewed by Löscher et al. [31]). If this transporter provided a significant fraction of the resting Na+ influx, then membrane potential would be predicted to acquire temperature sensitivity and bumetanide to depolarize the axons.

Methods Isolating optic nerve Wistar rats (male, 320–420 g) and C57bl/6 mice (either sex, >10 weeks) were used in this study. Animals were killed by exposure to a rising concentration of CO2 followed by cervical dislocation, in accordance with UK Home Office guidelines. Following decapitation, the skull was exposed and the upper surface carefully removed using scissors. The olfactory bulbs were identified and separated from the brain using a scalpel blade, allowing the brain to be lifted out of the skull and everted, revealing the pair of optic nerves that were immediately severed from the brain at the chiasm. The eyeballs were gently and gradually pulled from their orbits, without crushing the nerves, releasing the optic nerves through the eye sockets. The optic nerves and eyeballs were placed in a 35-mm Petri dish containing oxygenated quasi-physiological buffer solution, cleaned of adherent muscle and connective tissue, and then kept on ice for around 15 min until use. Two nerve baths were developed that allowed recording from the differently sized rodent optic nerves (Fig. 1a). The rat optic nerve bath utilized a hollow barrier that the nerve was gently pulled across, between two chambers filled with buffer


solution. Once the nerve was in position, the barrier was filled with petroleum jelly or a cocktail of petroleum jelly and paraffin. The mouse nerve bath consisted of a single chamber filled with buffer solution, bounded on one side by a narrow plastic barrier through which the nerve was threaded, leaving the eyeball only in an adjacent small grease-filled chamber. Stable recordings could be achieved from these baths for hours. Example action potentials recorded from mouse nerve are shown in Fig. 1c.

The quasi-physiological solution used for recording contained (in mM) NaCl 140, hemi Na HEPES 10, CaCl2 2.1, MgCl2

2.12, KCl 2.5, and glucose 10, adjusted to 7.2–3 using HCl. Where a raised K + solution was required, NaCl was equimolarly replaced with KCl. The applied solutions were oxygenated both before and after passing through a heating jacket (HPT-2, ALA Scientific) and into the bath. The temperature in the bath, close to the optic nerve, was kept at a selected value using a heater control (TC10, npi) employing negative feedback from a local thermistor and continuously recorded. Flow rate was close to 2 ml min −1 . When recording afterpotentials and extracellular membrane potential (demarcation potential), the solution in the left-hand chamber of the rat nerve bath was replaced with 60 mM K+ buffer solution, with equimolar replacement of Na+. For some demarcation potential recordings, the left-hand chamber also contained

Fig. 1 Stimulation and recording arrangements. a Rat optic nerve mounted across a paraffin barrier between two chambers filled with recording buffer. Nerve penetrates barrier surface on either side, and the barrier was subsequently filled with petroleum jelly. Non-polarizable electrodes in the left (x) and right hand chamber (y) form the differential recording pair. b Mouse optic nerve, differential recording made between a platinum wire electrode touching the eyeball (x) in a grease-filled chamber, and a non-polarizable electrode in aqueous solution in the main bath (y). Optic nerve enters the right hand chamber through a small hole in a plastic barrier. In both a and b, solution enters the recording buffer chamber through a jacketed cannula and is removed on

the other side of the bath by suction applied through a broken glass pipette, maintaining constant volume. Recording buffer oxygenated in the bath. c Examples of supramaximal compound action potential and a near 50 % maximal, tracked response (upper and lower panel, respectively) evoked by 200-μs duration current pulses in mouse optic nerve. Supramaximal action potential composed of an early (FM, i.e., fast, medium) response comprising upswing and peak, and an S, i.e., slow, response peak, indicative of involvement of different axonal subtypes within the nerve. The tracked (i.e., 50 % maximal response) waveform was without exception dominated by the FM component, as the S component required a greater stimulus current for recruitment

Solutions and drugs


50 μM ouabain. In this state, the difference in potential across the dividing barrier represents a fraction of the membrane potential of the healthy nerve. Ouabain and drugs were obtained from Tocris (R&D systems, Bristol, UK) and salts for recording solutions from Sigma-Aldrich (Dorset UK).

Data analysis Statistical comparisons were made in SPSS version 18 and in Microsoft Excel. The probability values returned for comparisons are stated in the text or appropriate figure legend. Wherever possible, data are plotted as means ± s.e.m.

Electrophysiological stimulating and recording We have applied computerized threshold-tracking to both adult rat and mouse isolated nerve, in response to 200-μs current pulses. The software employed was QTRAC-S (Digitimer; Hugh Bostock, Institute of Neurology, UCL), controlling a constant current stimulator (DS4, Digitimer, Welyn Garden City, UK) and maintaining a response near 50 % of maximal (Fig. 1c). Tracking was always done in the positive direction only, as this is the action current signal brought about when the impulses enter the barrier. Also, we have recorded the characteristics of action potentials and afterpotentials, extracellularly, after depolarizing the distal end of the nerve with raised K+, over a range of temperatures between room (22– 24 °C) and 37 °C. Finally, we have recorded the extracellular membrane potential changes (demarcation potential) that accompany changes in temperature (up to 42 °C) and the effects of bath-applied ouabain. The action potential was recorded using a differential amplifier (DP-311, Warner Instruments) and band-pass filter (10 Hz–3 KHz). When recording afterpotentials and demarcation potential with the same amplifier, no high-pass filtering was used. When generating a recovery cycle, a control and a conditioned stimulus protocol were alternated. In the conditioned case, a conditioning stimulus that remained 1.5 or 2 times that of the most recent estimate of current required to produce an adequate test response was applied at a variable latency before the test stimulus and the value of current appropriate to produce the test response taken as a record of the postconditioning changes in threshold. The advance of the variable latency was made conditional on the fulfillment of an adequate test response, allowing a reliable estimate of threshold to be derived for a range of inter-stimulus intervals (ISIs) and the derivation of a recovery cycle (Fig. 2). When recovery cycle data were analysed, the test threshold was expressed as a fractional increase or decrease from the control threshold recorded at the same time, so that any slow changes in threshold, unrelated to the applied protocol, were accounted for. Channel advance time in rat nerve was 1 s, but in mouse, this was increased to 1.6 s, in order to allow for the extraordinarily long cool refractory period. The axons are said to be refractory during the period in which threshold is elevated relative to control, and the duration of the relative refractory period during the recovery cycle is here defined as the earliest measured time that the mean change in excitability is within 1×s.e.m. of zero or when the mean change in excitability becomes negative with respect to control, indicating superexcitability.

Results Temperature-sensitive recovery cycles We used an ex vivo isolated optic nerve preparation and extracellular stimulation and recording to continuously track the threshold of the most excitable axons. Application of a conditioning and test stimuli with varying inter-stimulus intervals allowed the measurement of recovery cycles, which at warmer temperatures exhibited both refractoriness and superexcitability (e.g., [47]). No changes in evoked action potential frequency occurred during periods of warming and cooling, ruling out the effects of impulse activity-dependent increases in pump current. In order to generate recovery cycles, the nerves were held at a uniform temperature for 10 s of minutes, and this was assumed to represent a steady state. Action potentials recorded from mouse nerve were usually of a lower amplitude than the responses recorded from rat, and we attribute much of the variation in response amplitude in nerves of both species to the degree of success in making a high-resistance recording seal around the nerve. So the absolute amplitudes of the responses reveal little about the state of the optic nerve axons (and were only used to scale changes in resting membrane potential). At room temperature, we found there was less superexcitability (p=0.016954, n=6 optic nerves from 6 rats, paired t test), and the refractory period was far longer than at warmer temperatures, lasting around 10 times the duration found at 30 °C (Fig. 3a, b), much longer than refractory periods reported in the literature for peripheral myelinated axons. This finding was surprising, because in this case there was less than a 10 °C change in temperature involved and the compound action potential duration changed by a factor of less than 2 (Fig. 3c). The refractory period in mouse axons at room temperature was even longer than that found in rat (Fig. 3b), and we therefore sought an explanation for this phenomenon that must severely curtail high-frequency impulse transmission in cool nerve. NaV1.6 is known to be the major Na+ channel in myelinated optic nerve axons ([5, 6, 9, 27]; reviewed in [30]). The duration of the refractory period at room temperature can be explained by the time taken for Na+ channels (NaV1.6) to recover from inactivation (also called ‘repriming’, Fig. 3d) where the resting potential is assumed to be near −60 mV [20]. The voltage dependence of NaV1.6 repriming can also explain the dramatic difference of the refractory period at warmer temperatures, but only if the


Fig. 2 Recovery cycle data obtained from mouse nerve. a Examples of control and conditioned recordings in QTRAC. In the conditioned case, a conditioning stimulus was applied before the test stimulus and the interstimulus-interval (ISI) were made to increment while the thresholdtracking software maintained a constant test response amplitude. b Raw recovery cycle data for the same nerve as in a. Upper panel, the conditioned response stimulus current approaches and matches the control towards the end of a cycle as the ISI increases (bottom panel). The long duration refractory period is typical at room temperature. Response amplitudes (middle panel) show how the tracking software takes time to match the conditioned response amplitude with the target,

evident at the start of the recording cycle (indicated as ‘tracking’ open bars), a necessary requirement before the ISI is incremented. (These periods of tracking are also evident in the middle panels of figure c and d). c, d Raw recovery cycle data for another mouse nerve at 35 °C throughout. c With normal extracellular K + , the nerve shows superexcitability (represented by an asterisk), d with 11.25 mM K+ superexcitability is lost, consistent with axonal depolarization. Inset in c the start of the ISI ramp in more detail, showing the ISI increments only after the conditioned response is at the set amplitude, matching the control response

membrane responds to warming by hyperpolarizing. Briefly, the refractory period near room temperature is at least an order of magnitude longer than the duration of a recorded biphasic action potential, whereas in near-physiological temperature, the refractory period is comparable with the duration of an action potential. Both rodent nerves behaved in a very similar way to altered bath temperature, although it is apparent that subtle differences could exist between species (possibly related to idiosyncratic ion channel properties or to different physical properties, for example, the rat nerve is larger likely making O2 diffusion less adequate at higher temperatures). We

then ascertained that depolarizing the mouse optic nerve axons with high external K+ at a continuous temperature of 35 °C caused the loss of superexcitability and shifted the recovery cycle rightwards, consistent with cooling depolarizing the axons (Fig. 4a). We were able to rule out the possibility that the long duration refractory period at room temperature was caused by the activation of slow K+ channels because exposure to millimolar concentrations of tetraethylammonium (TEA) ions did not reduce the duration of refractoriness (Fig. 4b). In fact, exposing the optic nerve to TEA ions appeared to increase the refractory period slightly, consistent


Fig. 3 Temperature dependence of recovery cycles. Recovery cycles recorded between 37 °C and room temperature in rat a and mouse b, exhibiting large changes in refractory period (up to 3 orders of magnitude) and the loss of superexcitability with cooling. The values for duration of the relative refractory period in rat nerve were 7, 9, and 90 ms for 37, 30, and 22–24 °C, respectively. For mouse nerve, these values were 3, 6, 18, and >1000 ms for 37, 35, 30, and 22–24 °C, respectively. Long duration of refractory period when cool may be accounted for by a depolarization-dependent slow escape from Na+ channel inactivation (repriming). Changes in action potential kinetics (c) are far smaller (upward deflection duration 4.8 ms at 23 °C and 2.9 ms at 30 °C), similar to expected Q10 for Na+ channel gating. Warming is associated with little change (in this example a slight fall)

in maximal action potential amplitude. d Long refractory period duration at room temperature may be explained by the time taken for Na+ channels to recover from inactivation (n=8, means±s.e.m.), where the maximum change in threshold is taken as 90 % of the control value and the components of the recovery from fast (τ=15 ms) and slow inactivation (τ=1000 ms) are taken as 86.7 and 13.3 % of the total, respectively (smooth curve). The far shorter refractory period at 37 °C may be explained by the rapid Na+ channel repriming at a more negative potential. Smooth curve drawn with a time constant of 0.825 ms (derived from the published value for −100 mV at 21 °C, i.e., 2.5 ms), with a Q10 of 2 and maximum threshold change taken as 100 % of the control value. Time constants derived from voltage-clamp data [18]

with a depolarization resulting from channel block and supporting slow Na+ channel repriming (Fig. 3d) as the probable mechanism for prolonged refractoriness. While these findings on refractory period alone do not prove the possibility that central axons have membrane potentials that are temperature dependent, the change in refractory period we report is very substantial and difficult, maybe impossible, to explain in another way. In addition, other lines of evidence suggest the membrane potential must be changing.

ouabain and hence with a much reduced resting membrane potential; see BMethods^) and measured the height of the afterpotential at the latency at which it was maximally negative (i.e., hyperpolarizing) at room temperature (Fig. 5a, b). We found that the depolarizing afterpotential (DAP) recorded when the axons were warm was reversibly replaced by a hyperpolarizing afterpotential when the axons were cool. This finding also suggests a temperature-sensitive membrane potential [1] and a high impedance internode when the axons were warm [2]. The temperature dependence of the afterpotential polarity appears to mimic the effects of intraaxonal direct polarization in rat spinal root axons [1]. As it is impossible directly to record membrane potential in optic nerve axons, we chose to record demarcation potential in rat nerve (i.e., the potential difference measured between the

Membrane potential recording We made monophasic recordings of compound action potentials and afterpotentials in rat nerves (i.e., with the nerve on the left-hand chamber depolarized with high K+ exposed to


functional nerve in the right-hand chamber and the depolarized nerve in the left-hand chamber), estimating the change in membrane potential assuming the maximal compound action potential represented an intracellular change of 100 mV. Our recordings were consistent with warming hyperpolarizing the axons and cooling depolarizing the axons (Fig. 6a–c), and on average, our estimates of the effect of warming rat optic nerve from room temperature over a period of 2 min added 40 mV to the resting potential, although in some nerves the temperature-dependent component seemed considerably larger. As the nerve bath incorporated a closely positioned reference (earth) and active electrode in the warmed bath chamber, it is unlikely that the demarcation potentials recorded were artefactually generated by temperaturedependent changes in electrode junction potential. Finally, we were able partially to suppress the warming-associated hyperpolarization with high concentrations of ouabain (≥50 μM), consistent with a low-ouabain affinity Na+-pump α1-isozyme (e.g.,[19]) known to be present in optic nerve [16] being involved in the phenomenon (Fig. 6d, e). Resolving the effect of warming on the maximal compound action potential

Fig. 4 Depolarized and cool mouse axons have similar long refractory periods. a Effects of high external K+ on recovery cycles. Depolarizing mouse optic nerve axons with 11.25 mM K+ partially replicates the effect of cooling (n=4), comparing the lowest threshold recorded, p=0.01355, paired t test. Examples of raw data sets used to generate the plot are shown in Fig. 2. b Cool axons have long refractory periods, independent of the activation of kinetically slow K+ channels, as 5 mM TEA ions appear unable to shorten refractoriness (mean values plotted, n=2) Fig. 5 Monophasic recordings of action potentials in rat optic nerve. Recording action potentials after exposing the nerve in the left-hand chamber to high K+ and ouabain shows a reversible change in afterpotential polarity (p=0.000736, n=6, paired t test), consistent with a more negative membrane potential and higher axonal input resistance at the warmer temperatures

One finding initially did not appear to support the proposed temperature-dependent membrane potential, because on warming, the compound action potential maximal height always either did not change or was reduced slightly (cf Fig. 3c). If resting membrane potential became more negative with warming, and no other temperature-dependent factor influenced action potential height, then action potential amplitude would be predicted to increase. This discrepancy was resolved by applying 50 nM ATX-II to rat optic nerve, a site 3 Na+ channel neurotoxin that slows the rate of Na+ channel fast inactivation (reviewed in [11]; [43]) . In the presence of the

Author's personal copy Pflugers Arch - Eur J Physiol Fig. 6 Effect of changing temperature on rat optic nerve extracellular membrane potential. a The temperature record and, above, corresponding extracellular membrane potential plus unavoidable drift in raw record, the latter approximated by a straight line. Correcting for the sloping baseline, average membrane potential changes for the two periods of warming are shown in b, assuming the peak maximal compound action potential (recorded at room temperature, immediately before the first period of warming) corresponds with an intracellular change of 100 mV. Average change in membrane potential recorded over the first minutes of warming (c) (p=0.018 at 2.1 min after starting warming, n=5 nerves from n=5 rats, one-sample t test). d, e Temperaturedependent changes in membrane potential with repeated warming in the same nerve are partly blocked by high concentrations of ouabain (50–300 μM, p=0.033, n=5 nerves, one-sample t test, making a comparison with zero change)

toxin, action potentials became prolonged (Fig. 7a), and the maximal amplitude was smaller when the nerve was close to 30 °C than when at 37 °C, by 14.7 % (p=0.043) (Fig. 7a, b). This result implies that fast Na+ channel inactivation is normally preferentially speeded by warming (confirming a larger

temperature coefficient for Na+ channel inactivation than for activation [3, 13], reviewed in [42]), curtailing the action currents, and acting to reduce any effect of increasing resting membrane potential on the action potential amplitude. Hodgkin and Katz [22] also saw a greater temperature

Fig. 7 Partial block of Na+ channel fast inactivation uncovers a fall in action potential amplitude on cooling. a ATX-II (50 nM) prolongs maximal compound action potentials (biphasic recordings) and increases amplitude at a constant temperature (before and after exposure to ATX-II, fine and heavy lines, respectively); components of the upswing

(F, M) generated a stepped increase during the periods of their recruitment (differences in amplitude plotted as lower trace and indicated ΔV in (a) the left panel). b Cooling from near 37 to near 30 °C increases action potential latency and the width, but reduces the amplitude by 14.7±5.8 % (p=0.043, n=3 nerves from 3 rats, one-sample t test)


coefficient for squid axon action potential repolarization than for upswing, although repolarization depends upon both Na+ channel inactivation and K+ channel activation occurring together. The larger action potential at the warmer temperature strongly supports a temperature-dependent resting potential, and the difference in amplitude is compatible with our other estimates of the slope of the relation. Bumetanide acts on optic nerve axon recovery cycles Our data suggest that optic nerve axons have temperaturesensitive membrane potentials, and the sensitivity is probably caused by changes in the resting activity of the Na+-pump, as we expected NKCC1 to be expressed in central nerve fibres. We tested whether bumetanide might be able to depolarize axonal membrane potential through the depletion of intraaxonal Na+ and thus indirectly reduce the resting activity of the pump at a constant temperature. Recovery cycles were altered by bumetanide in a manner similar to that expected for depolarization, with increased refractoriness and decreased superexcitability (Fig. 8 a–d), although the effects of bumetanide could only be reversed partially by washing. While this may be a reflection of the drug’s high lipophilicity and membrane partitioning, it may also be because transporter block can act only indirectly on membrane potential and we had no way of knowing that intra-axonal Na+ had returned to preexposure levels. For this reason, although we may have correctly implicated the NKCC1 transporter, further study of the relationship between the transporter and the temperature dependence of the optic nerve axons will be required to be able to form a more definitive view of its importance. However, in order to provide an insight into whether another alternative route for Na+ entry may have contributed in our preparation, we added amiloride to the bumetanide already in the mouse nerve bath (Fig. 8c, d), where amiloride is a blocker of Na+-H+ ion exchange (NHE1) in white matter (e.g., [25, 32, 34, 38]). The influx of Na+ by this mechanism is predicted to be temperature dependent [12]. While our findings suggest that HNE may contribute to temperature-dependent Na+ fluxes in axons, the increased refractoriness in the presence of both drugs was not significantly different from the result in bumetanide alone. Although no further significant change in the recovery cycle was seen, on average, the refractoriness increased and the presence of both antagonists produced a recovery cycle similar to that previously found for mouse nerve in high K+ (Fig. 8c).

Discussion We have provided evidence suggesting that the membrane potential of optic nerve axons is temperature sensitive, and this may be explained if transmembrane Na+ movements at

rest are mediated by the Na+-pump and electroneutral transport (Fig. 8e). For example, optic nerve axons could have membrane potentials of around −60 mV at room temperature (assuming Na+ channel repriming determines the rate of recovery from refractoriness (Fig. 3)) and a temperaturedependent hyperpolarization as the nerve warms to 37 °C, so that the normal resting potential at body temperature may be −100 mVor perhaps even more negative. There is no necessity to propose that temperature sensitivity at temperatures below room will mean that membrane potential collapses entirely with further cooling, because the dependence of pump rate on temperature is known to be highly non-linear [14], and with a quiescent pump the membrane potential will be maintained by the membrane K+ permeability. We suggest the reason that the optic nerve compound action potential does not consistently increase in amplitude with warming is because Na+ channel inactivation has a higher temperature coefficient than activation, and the action currents are progressively truncated with increasing temperature. A Na+-pump-mediated membrane potential implies the requirement of a direct and continuous energy expenditure to maintain axonal membrane potential and also implicates white matter functional properties in the effects of cooling on the brain. The functional effects of a resting potential that can change by around 4 mV/°C, near-physiological temperature, may help explain Uhthoff’s observations on changes in body temperature impacting symptom severity in multiple sclerosis [41] because in multiple sclerosis (MS) the safety factor for conduction in affected fibres may be diminished and modest cooling would be predicted to depolarize axons, increasing excitability and aiding impulse transmission. Recently, cooling has been used to recruit damaged and possibly demyelinated axons following spinal cord injury that are unable to function at normal physiological temperatures [24] and here too a temperature-dependent membrane potential may contribute to the finding. Previously, temperature-dependent effects on the security of conduction have been ascribed to changes in Na+ channel gating kinetics only ([22]; reviewed in [42]). Greater cooling (to ~30 °C) will also dramatically change axonal refractory period, eliminating high-frequency burst impulse transmission and helping to explain the loss of consciousness associated with low core body temperatures (30–32 °C) [46]. By the same token, axonal pump inhibition through reduced levels of ATP in anoxia and ischaemia would affect refractoriness in a similar way, albeit without slowed channel gating kinetics, and might impact consciousness through an inability of white matter to carry high-frequency trains. Conversely, warming will hyperpolarize axons and make them less excitable by increasing transmembrane Na+ cycling, potentially contributing to impulse conduction failure, especially at sites of high capacitative load, such as regions of demyelination. Certainly, loss of consciousness on brain cooling is likely to involve processes other than


maintenance of axonal membrane potential, including reduction of pre-synaptic Ca2+ currents, impacting the probability of transmitter release (e.g., Borst and Sakmann [7]). Although the recordings of Borst and Sakmann may in principle have

revealed a temperature-dependent resting potential in the presynapse, they used voltage-clamped calyx of Held pre-synapses, employing a two-electrode voltage-clamp technique and imposing an action potential voltage profile. So if the resting

Fig. 8 Bumetanide acts on superexcitability and refractoriness in a manner similar to cooling. Exposure to 5–50 μM bumetanide reduces superexcitability and increases refractoriness (a) (p= 0.01586, n= 4 nerves from 4 rats, paired t test), consistent with a depolarized axonal membrane potential. Data collected using 5 and 50 μM bumetanide could not be distinguished and are pooled. The horizontal bar indicates that raw data at two latencies in the recovery cycles were averaged for each nerve, and then subsequently, the means of these averaged values were compared. The effects of bumetanide were difficult to fully reverse even with prolonged washing (10 s of minutes, b). c, d Exposure of mouse optic nerve to bumetanide and a combination of 5 μM bumetanide and 10 μM amiloride reduced superexcitability and increased refractoriness. Continuous line in c indicates the effect of raising extracellular K+ to 11.25 mM (average data from Fig. 4). Data at 9 ms ISI in c plotted in d, plus the effect of bumetanide alone. Addition

of antagonists significantly increased refractoriness (p