Gordon Reid

Action potentials and membrane currents in the human node of Ranvier

Action potentials and membrane currents were recorded in single human myelinated nerve fibres under current- and voltage-clamp conditions at room temperature. Nerve material was obtained from patients undergoing nerve graft operations. Successful recordings were made in 11 nerve fibres. In Ringers solution, large transient Na currents were recorded, which could be blocked completely with tetrodotoxin. Partial block of these currents with 3 nM tetrodotoxin was used to reduce the voltage-clamp error due to series resistance. Outward K currents were very small in intact nerve fibres, but had a large amplitude in fibres showing signs of paranodal demyelination. In isotonic KCl, the K current could be separated into three components: two fast components (Kf1 and Kf2) and one slow component (Ks). Time constants and steady-state activation and inactivation of Na permeability and of fast and slow K conductance were measured within the potential range of −145 mV to +115 mV. From these parameters, the corresponding rate constants were calculated and a mathematical model based on the Frankenhaeuser-Huxley equations was derived. Calculated action potentials closely matched those recorded. Single calculated action potentials were little affected by removing the fast or slow K conductance, but the slow K conductance was required to limit the repetitive response of the model to prolonged stimulating currents.

A cold- and menthol-activated current in rat dorsal root ganglion neurones: properties and role in cold transduction

Skin temperature is sensed by peripheral thermoreceptors. Using the neuronal soma in primary culture as a model of the receptor terminal, we have investigated the mechanisms of cold transduction in thermoreceptive neurones from rat dorsal root ganglia. Cold‐sensitive neurones were pre‐selected by screening for an increase in [Ca2+]i on cooling; 49 % of them were also excited by 0.5 μm capsaicin. Action potentials and voltage‐gated currents of cold‐sensitive neurones were clearly distinct from those of cold‐insensitive neurones. All cold‐sensitive neurones expressed an inward current activated by cold and sensitised by (‐)‐menthol, which was absent from cold‐insensitive neurones. This current was carried mainly by Na+ ions and caused a depolarisation on cooling accompanied by action potentials, inducing voltage‐gated Ca2+ entry; a minor fraction of Ca2+ entry was voltage‐independent. Application of (‐)‐menthol shifted the threshold temperatures of the cold‐induced depolarisation and the inward current to the same extent, indicating that the cold‐ and menthol‐activated current normally sets the threshold temperature for depolarisation during cooling. The action of menthol was stereospecific, with the (+)‐isomer being a less effective agonist than the (‐)‐isomer. Extracellular Ca2+ modulated the cold‐ and menthol‐activated current in a similar way to its action on intact cold receptors: lowered [Ca2+]o sensitised the current, while raised [Ca2+]o antagonised the menthol‐induced sensitisation. During long cooling pulses the current showed adaptation, which depended on extracellular Ca2+ and was mediated by a rise in [Ca2+]i. This adaptation consisted of a shift in the temperature sensitivity of the channel. In capsaicin‐sensitive neurones, capsaicin application caused a profound depression of the cold‐activated current. Inclusion of nerve growth factor in the culture medium shifted the threshold of the cold‐activated current towards warmer temperatures. The current was blocked by 50 μm capsazepine and 100 μm SKF 96365. We conclude that the cold‐ and menthol‐activated current is the major mechanism responsible for cold‐induced depolarisation in DRG neurones, and largely accounts for the known transduction properties of intact cold receptors.

ThermoTRP channels and cold sensing : what are they really up to?

Cooling is sensed by peripheral thermoreceptors, the main transduction mechanism of which is probably a cold- and menthol-activated ion channel, transient receptor potential (melastatin)-8 (TRPM8). Stronger cooling also activates another TRP channel, TRP (ankyrin-like)-1, (TRPA1), which has been suggested to underlie cold nociception. This review examines the roles of these two channels and other mechanisms in thermal transduction. TRPM8 is activated directly by gentle cooling and depolarises sensory neurones; its threshold temperature (normally ~26–31°C in native neurones) is very flexible and it can adapt to long-term variations in baseline temperature to sensitively detect small temperature changes. This modulation is enabled by TRPM8’s low intrinsic thermal sensitivity: it is sensitised to varying degrees by its cellular context. TRPM8 is not the only thermosensitive element in cold receptors and interacts with other ionic currents to shape cold receptor activity. Cold can also cause pain: the transduction mechanism is uncertain, possibly involving TRPM8 in some neurones, but another candidate is TRPA1 which is activated in expression systems by strong cooling. However, native neurones that appear to express TRPA1 respond very slowly to cold, and TRPA1 alone cannot account readily for cold nociceptor activity or cold pain in humans. Other, as yet unknown, mechanisms of cold nociception are likely.

Physiology: Cold current in thermoreceptive neurons

We sense the temperature of our skin and surroundings using specific thermoreceptors, which are sensitive to cold and warmth, but little is known about how these receptors transduce temperature into electrical activity. We have discovered an inward ionic current that is activated by moderate cooling in a small number of rat sensory neurons. This current has features that are found in intact cold receptors, including sensitization by menthol, adaptation upon sustained cooling, and modulation by calcium, and is likely to be important in cold sensing.

Two populations of cold-sensitive neurons in rat dorsal root ganglia and their modulation by nerve growth factor

Cold sensing in mammals is not completely understood, although significant progress has been made recently with the cloning of two cold‐activated ion channels, TRPM8 and TRPA1. We have used rat DRG neurons in primary culture and calcium fluorimetry to identify distinct populations of cold‐sensitive neurons, which may underlie different functions. Menthol sensitivity clearly separated two classes of cold‐responding neurons. One group was menthol‐sensitive (MS), was activated at warmer temperatures and responded faster and with a larger increase in intracellular calcium concentration during cooling; the fraction of MS neurons in culture and their cold sensitivity were both increased in the presence of nerve growth factor. Neurons in the menthol‐insensitive (MI) group required stronger cooling for activation than MS cells and neither their proportion nor their cold sensitivity were significantly altered by nerve growth factor. The two groups of cold‐sensitive neurons also had different pharmacology. A larger fraction of MS cells were capsaicin‐sensitive and coexpression of menthol and capsaicin sensitivity was observed in the absence of NGF. MI neurons were not stimulated by the super‐cooling agent icilin or by the irritant mustard oil. Taken together these findings support a picture in which TRPM8 is the major player in detecting gentle cooling, while TRPA1 does not seem to be involved in cold sensing by MI neurons, at least in the temperature range between 32 and 12 °C.

Cold transduction by inhibition of a background potassium conductance in rat primary sensory neurones.

Transduction in cutaneous cold receptors is poorly understood at present. We have studied this question using dorsal root ganglion (DRG) neurones in primary culture as a model of the otherwise inaccessible receptor terminal. Whole-cell recordings during cooling from 32 to 20 degrees C revealed a large depolarization (>8mV) in 22 of 88 DRG neurones (25%), sometimes accompanied by action potentials. In cold-sensitive neurones cooling inhibited a time-independent background K+ current (Icold) which was resistant to tetraethylammonium and 4-aminopyridine. Ouabain elicited a substantially smaller depolarization than cooling, and no action potentials. We conclude that excitation by cooling in this model is primarily due to inhibition of Icold and that the previously suggested role of the Na+/K+ adenosine triphosphatase is secondary. We suggest that Icold may underlie cold transduction in cutaneous thermoreceptors.

Changes in excitability and accommodation of human motor axons following brief periods of ischaemia.

1. The mechanism of post‐ischaemic ectopic impulse generation in nerve is not known, and previous measurements of excitability changes in human motor axons have appeared to conflict. We have used automatic threshold tracking and different stimulus‐response combinations to follow the effects on excitability of brief (5‐10 min) periods of ischaemia, too short to induce motor fasciculations. Excitability changes have been compared at different sites in axons innervating hand, arm and foot muscles. 2. Threshold was determined as the percutaneous stimulus current required to excite a single motor unit, or to evoke a constant multiunit response, after rectifying and integrating the electromyogram (EMG). Three different waveforms of stimulus current were compared: short (less than or equal to 2 ms) pulses, long (100‐200 ms) pulses to measure rheobase, and 100 ms current ramps. We also measured accommodation by recording the effects of subthreshold depolarizing currents on excitability. 3. Ischaemic and post‐ischaemic excitability changes were greatest in the proximal parts of the longest motor axons, and greater if the sphygmomanometer cuff was inflated over, rather than proximal to, the stimulating site. 4. Using integrated EMG responses from abductor digiti minimi, the ulnar nerve stimulated above the elbow became rapidly much less excitable after ischaemia when tested with short pulses, but more excitable when tested with current ramps. The rheobase rose briefly, but then fell, often below resting level, always staying below the pulse and ramp thresholds. 5. The latency of the response to a rheobasic stimulus altered in parallel with the threshold to short current pulses, and increased dramatically after ischaemia. This latency increase was associated with a prolonged phase of ‘negative accommodation’, i.e. the continued increase in excitability to a maintained subthreshold depolarizing current. 6. Changes in excitability and accommodation similar to those occurring after ischaemia were recorded following high frequency trains of stimuli. They were attributed primarily to hyperpolarization by the electrogenic sodium pump, since comparable changes could be induced by passing a steady hyperpolarizing current through the stimulating electrode. 7. Threshold and latency recordings from single motor units during and after ischaemia resembled in most respects the multiunit responses, but single unit rheobase did not show a post‐ischaemic fall below the resting level. Repetitive firing contributed to the low multiunit thresholds recorded with long current pulses during the post‐ischaemic period. 8. We conclude that human motor nerves become simultaneously both more and less excitable than normal after 10 min of ischaemia, depending on the choice of stimulus and response.(ABSTRACT TRUNCATED AT 400 WORDS)

Quantitative measures of sympathetic skin response in diabetes: relation to sudomotor and neurological function.

The sympathetic skin response (SSR) at the foot to a deep inspiration was measured in 68 randomly selected diabetic patients and 46 age matched normal subjects and compared with other quantitative measures of neurological and sudomotor function. SSR was obtained in all but three diabetic patients. The upper limit of normal for the onset latency was 2202 ms and the lower limit for the amplitude of the first wave 92 microV. Ten diabetic patients had measurable but prolonged latencies, and 11 had measurable but low amplitudes. There were no significant associations between latency, height, and age, but in insulin dependent patients there was a significant diminution of response amplitude with increasing duration of diabetes. Latency was weakly associated with Marstock thermal thresholds, respiratory RR variation, and common peroneal nerve conduction velocity. SSR amplitude was associated with the density of pilocarpine activatable sweatspots in the same region of the foot. Patients with abnormal latencies were significantly older and had reduced thermal sensation than those with normal latencies. Median coefficients of variation for repeat testing in diabetic patients were 9% for latency and 13% for amplitude. The test is objective and reproducible, but latency measurements reflect conduction in a long multineuronal pathway and are not purely a measure of peripheral C fibre function; amplitude measurements reflect the density of spontaneously activable sweat glands and are therefore a valid measure of peripheral sympathetic activity, though they depend more on temperature than do latencies (mean change over the range 32-34 degrees C; 8.5% degrees C for amplitude, -2.5%/degrees C for latency).

Human axons contain at least five types of voltage-dependent potassium channel

1 We investigated voltage‐gated potassium channels in human peripheral myelinated axons; apart from the I, S and F channels already described in amphibian and rat axons, we identified at least two other channel types. 2 The I channel activated between ‐70 and ‐40 mV, and inactivated very slowly (time constant 13.1 s at ‐40 mV). It had two gating modes: the dominant (‘noisy’) mode had a conductance of 30 pS (inward current, symmetrical 155 mM K+) and a deactivation time constant (τ) of 25 ms (‐80 mV); it accounted for most (≈50‐75 %) of the macroscopic K+ current in large patches. The secondary (‘flickery’) gating mode had a conductance of 22 pS, and showed bi‐exponential deactivation (τ= 16 and 102 ms; ‐80 mV); it contributed part of the slow macroscopic K+ current. 3 The I channel current was blocked by 1 μM α‐dendrotoxin (DTX); we also observed two other DTX‐sensitive K+ channel types (40 pS and 25 pS). The S and F channels were not blocked by 1 μM DTX. 4 The conductance of the S channel was 7‐10 pS, and it activated at slightly more negative potentials than the I channel; its deactivation was slow (τ= 41.7 ms at ‐100 mV). It contributed a second component of the slow macroscopic K+ current. 5 The F channel had a conductance of 50 pS; it activated at potentials between ‐40 and +40 mV, deactivated very rapidly (τ= 1.4 ms at ‐100 mV), and inactivated rapidly (τ= 62 ms at +80 mV). It accounted for the fast‐deactivating macroscopic K+ current and partly for fast K+ current inactivation. 6 We conclude that human and rat axonal K+ channels are closely similar, but that the correspondence between K+ channel types and the macroscopic currents usually attributed to them is only partial. At least five channel types exist, and their characteristics overlap to a considerable extent.

A comparison of two methods for measuring thermal thresholds in diabetic neuropathy.

Thermal thresholds can be measured psychophysically using either the method of limits or a forced-choice method. We have compared the two methods in 367 diabetic patients, 128 with symptomatic neuropathy. The Sensortek method was chosen for the forced-choice device, the Somedic modification of the Marstock method for a method of limits. Cooling and heat pain thresholds were also measured using the Marstock method. Somedic thermal thresholds increase with age in normal subjects, but not to a clinically significant degree. In diabetics Marstock warm threshold increased by 0.8 degrees C/decade, Sensortek by 0.1 degrees C/decade. Both methods had a high coefficient of variation in normal subjects (Sensortek 29%, Marstock warm 14%, cool 42%). The prevalence of abnormal thresholds was similar for both methods (28-32%), though Marstock heat pain thresholds were less frequently abnormal (18%). Only 15-18% of patients had abnormal results in both tests. Sensortek thresholds were significantly lower on repeat testing, and all thresholds were higher in symptomatic patients. Both methods are suitable for clinical thermal testing, though the method of limits is quicker. In screening studies the choice of a suitable apparatus need not be determined by the psychophysical basis of the test.