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 Ringer's 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.

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.

SUMMARY1. We have investigated the origin of post-ischaemic ectopic discharges in human nerve by recording changes in electrical excitability following periods of ischaemia (15-20 min) sufficient to induce spontaneous motor fasciculations. The ulnar nerve was stimulated beneath a pressure cuff on the upper arm, and compound motor action potentials recorded from abductor digiti minimi.2. On releasing the cuff after 15 min of ischaemia, thresholds to short current pulses increased in two distinct phases: a slow phase followed by a rapid rise to a peak threshold. The rapid rise was too fast to track (i.e. 100 % threshold increase in less than 4 s), and was sometimes followed after 30-40 s by an equally rapid fall. Small polarizing currents affected the timing of the rapid threshold increase, as if it was occurring at a particular membrane potential.3. By recording complete stimulus-response curves every few seconds, we found that the rapid threshold changes were associated with a bimodal distribution of thresholds. Most fibres were found in either a high-threshold or low-threshold state, and these two states converged over a period of about 10 min.4. Spontaneous motor fasciculations were only recorded after the rapid rise in threshold and when the fibres existed in two threshold states. The spontaneous activity was not responsible for inducing the two states, since they could also be recorded in its absence.5. A computer model of a human motor axon node and internode was constructed, incorporating channel types demonstrated in other axons, and channel densities adjusted to match the responses of human axons to depolarizing and hyperpolarizing current pulses. An increase in extracellular potassium concentration produced a region of negative slope conductance in the current-voltage relationship of the model, and the appearance of two stable states with enhanced activity of the electrogenic sodium pump.6. Transitions between the two stable states of the model could account qualitatively for the rapid threshold changes recorded from post-ischaemic axons. In the model, spontaneous action potentials occurred following some transitions from the high potential state to the low potential state. We suggest that postischaemic motor fasciculations in man also involve transitions between two MS 9120 H. BOSTOCK AND OTHERS equilibrium states, occurring in axons with high extracellular potassium and high electrogenic pump activity.

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 degrees C.

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.