Many damage-sensing neurons express tetrodotoxin (TTX)-resistant voltage-gated sodium channels. Here we examined the role of the sensory-neuron-specific (SNS) TTX-resistant sodium channel alpha subunit in nociception and pain by constructing sns-null mutant mice. These mice expressed only TTX-sensitive sodium currents on step depolarizations from normal resting potentials, showing that all slow TTX-resistant currents are encoded by the sns gene. Null mutants were viable, fertile and apparently normal, although lowered thresholds of electrical activation of C-fibers and increased current densities of TTX-sensitive channels demonstrated compensatory upregulation of TTX-sensitive currents in sensory neurons. Behavioral studies demonstrated a pronounced analgesia to noxious mechanical stimuli, small deficits in noxious thermoreception and delayed development of inflammatory hyperalgesia. These data show that SNS is involved in pain pathways and suggest that blockade of SNS expression or function may produce analgesia without side effects.

ABSTRACT-A variety of different isoforms of voltage-sensitive Na+ channels have now been identified. The recent three-dimensional analysis of Na + channels has unveiled a unique and unexpected structure of the Na + channel protein. Na + channels can be classified into two categories on the basis of their amino acid sequence, NaV1 isoforms currently comprising nine highly homologous clones and NaX that possesses structure diverging from Na V 1, especially in several critical functional motifs. Although the functional role of Na V 1 isoforms is primarily to form an action potential upstroke in excitable cells, recent biophysical studies indicate that some of the Na V 1 isoforms can also influence subthreshold electrical activity through persistent or resurgent Na + currents. Na V 1.8 and Na V 1.9 contain an amino acid sequence common to tetrodotoxin resistant Na + channels and are localized in peripheral nociceptors. Recent patch-clamp experiments on dorsal root ganglion neurons from Na V 1.8-knock-out mice unveiled an additional tetrodotoxin-resistant Na + current. The demonstration of its dependence on Na V 1.9 provides evidence for a specialized role of Na V 1.9, together with NaV1.8, in pain sensation. Although NaX has not been successfully expressed in an exogenous system, recent investigations using relevant native tissues combined with gene-targeting have disclosed their unique "concentration"-sensitive but not voltage-sensitive roles. In this context, these emerging views of novel functions mediated by different types of Na + channels are reviewed, to give a perspective for future research on the expanding family of Na + channel clones.

Small dorsal root ganglion neurons express preferentially the Na+ channel isoform Na(v)1.9 that mediates a tetrodotoxin-resistant (TTX-R) Na+ current. We investigated properties of the Na+ current mediated by Na(v)1.9 (I(NaN)) using the whole-cell, patch-clamp recording technique. To isolate I(NaN) from heterogeneous TTX-R Na+ currents that also contain another type of TTX-R Na+ current mediated by Na(v)1.8, we used Na(v)1.8-null mutant mice. When F- was used as an internal anion in the patch pipette solution, both the activation and inactivation kinetics for I(NaN) shifted in the hyperpolarizing direction with time. Such a time-dependent shift of the kinetics was not observed when Cl- was used as an internal anion. Functional expression of I(NaN) declined with time after cell dissociation and recovered during culture, implying that Na(v)1.9 may be regulated dynamically by trophic factors or depend on subtle environmental factors for its survival. During whole-cell recordings, the peak amplitude of I(NaN) increased dramatically after a variable delay, as if inactive or silent channels had been "kindled". Such an unusual increase of the amplitude could be prevented by adding ATP to the pipette solution or by recording with the nystatin-perforated patch-clamp technique, suggesting that the rupture of patch membrane affected the behaviour of Na(v)1.9. These peculiar properties of I(NaN) may provide an insight into the plasticity of Na+ channels that are related to pathological functions of Na+ channels accompanying abnormal pain states.

1The ionic mechanism underlying the effect of (-)-baclofen in the hippocampus was investigated using guinea-pig brain slices.2 (-)-Baclofen either perfused or applied directly by microiontophoresis hyperpolarized the membrane and decreased the membrane input resistance of pyramidal cells in a dose-dependent manner.3 The value of the reversal potential for the baclofen-induced hyperpolarization, as estimated from the current-voltage relationships, was about -95 mV. 4 The reversal potential of the baclofen-induced hyperpolarization measured directly coincided with that for the post-burst hyperpolarization which is known to result from an activation of Ca2+-activated K+ conductance. 5The amplitude of the baclofen-induced hyperpolarization was increased in low K+ (1.24mM) medium whereas the hyperpolarization was decreased or abolished in high K+ (12.4 and 25 mM). Low Cl-(10.2 mM) medium had no noticeable effect on the baclofen-induced hyperpolarization. 6 The effect of baclofen was antagonized by a low dose of 4-aminopyridine (5 x 10-6 M) whereas it was unaffected by picrotoxin (2 x 10-5M).7 These results strongly suggest that the effect of baclofen is mediated by an increase in K+ conductance.

1 Effects of imipramine and haloperidol on voltage-gated sodium channels were investigated in guinea-pig isolated ventricular myocytes by the whole-cell patch clamp technique. Some additional experiments were also performed with chlorpromazine for the purpose of comparison. 2 All test drugs in micromolar concentrations suppressed the amplitude of peak sodium current associated with step depolarization from a holding potential of -140 mV in a reversible manner. The order of potency was chlorpromazine > imipramine > haloperidol. 3 Dose-response curves obtained with a holding potential of -140 mV were best fitted by 2:1 stoichiometry in all three drugs and were shifted in the direction of lower concentrations when a holding potential of -90 mV was used. 4 The drug-induced block was not associated with any change in the time courses of sodium current activation and inactivation. 5 Steady-state sodium channel inactivation curve was shifted in the direction of more negative potentials by the drugs. 6 All three drugs also produced marked use-dependent block as demonstrated by a cumulative increase in the block during a train of depolarizing pulses. 7 The use dependence was due to a higher affinity of the drugs for the inactivated state of sodium channels than the resting state and to a very slow repriming of the drug-bound sodium channels from inactivation. 8 The steady-state and use-dependent block of voltage-gated sodium channels by psychotropic drugs may contribute to their cardiotoxic and perhaps antiarrhythmic effect.

SUMMARY1. Brain slices of the guinea-pig hypothalamus were used to determine the effects of vasopressin on intracellular potentials in neurones of the supraoptic nucleus.2. Vasopressin (0-05-1 i.u./ml.) depolarized the membrane without apparent change in the input resistance and decreased the spontaneous firing rate. This action of vasopressin was retained in the medium containing 0 mM-Ca2+, 12 mMMg2+ and 0-3 mM-EGTA.3. Amplitude of the vasopressin-induced depolarization was voltage-independent. 4. The vasopressin-induced depolarization was blocked at a temperature of 15 0C and by ouabain in a dose of 10-4 M.5. Dibutyryl cyclic AMP (2 mM) produced electrophysiological effects similar to those seen with vasopressin, and actions of both agents were potentiated by either papaverine (10-4 M) or theophylline (10-2 M).6. Contents of cyclic AMP in tissues incubated with vasopressin were significantly higher than in cases of incubation with normal Krebs solution.7. We conclude that vasopressin directly modulates the activity of supraoptic neurones, possibly through activation of adenylate cyclase.

Small (<25 microm in diameter) neurons of the dorsal root ganglion (DRG) express multiple voltage-gated Na(+) channel subtypes, two of which being resistant to tetrodotoxin (TTX). Each subtype mediates Na(+) current with distinct kinetic property. However, it is not known how each type of Na(+) channel contributes to the generation of action potentials in small DRG neurons. Therefore, we investigated the correlation between Na(+) currents in voltage-clamp recordings and corresponding action potentials in current-clamp recordings, using wild-type (WT) and Na(V)1.8 knock-out (KO) mice, to clarify the action potential electrogenesis in small DRG neurons. We classified Na(+) currents in small DRG neurons into three categories on the basis of TTX sensitivity and kinetic properties, i.e., TTX-sensitive (TTX-S)/fast Na(+) current, TTX-resistant (TTX-R)/slow Na(+) current, and TTX-R/persistent Na(+) current. Our concurrent voltage- and current-clamp recordings from the same neuron revealed that the action potentials in WT small DRG neurons were mainly dependent on TTX-R/slow Na(+) current mediated by Na(V)1.8. It was surprising that a large portion of TTX-S/fast Na(+) current was switched off in WT small DRG neurons due to a hyperpolarizing shift of the steady-state inactivation (h (infinity)), whereas in KO small DRG neurons which are devoid of TTX-R/slow Na(+) current, the action potentials were generated by TTX-S/fast Na(+) current possibly through a compensatory shift of h (infinity) in the positive direction. We also confirmed that TTX-R/persistent Na(+) current mediated by Na(V)1.9 actually regulates subthreshold excitability in small DRG neurons. In addition, we demon strated that TTX-R/persistent Na(+) current can carry an action potential when the amplitude of this current was abnormally increased. Thus, our results indicate that the action potentials in small DRG neurons are generated and regulated with a combination of multiple mechanisms that may give rise to unique functional properties of small DRG neurons.

The effects of (—)‐baclofen on evoked potentials in the hippocampus were examined through intracellular recordings from guinea‐pig brain slices. The evoked responses were recorded in two fibre connections within the hippocampus: the Schaffer collateral/commissural‐CA1 pyramidal cell, and the mossy fibre—CA3 pyramidal cell. The Schaffer collateral/commissural‐CA1 response was suppressed by (—)‐baclofen in concentrations over 2 × 10−5m, whereas (+)‐baclofen, an inactive isomer, in a concentration of 10−4m had no effect on the response. A compound action potential of Schaffer collateral/commissural axons was unaffected by (—)‐baclofen even at 10−4m, a concentration that almost completely depressed the evoked response in the CA1 pyramidal cell. The mossy fibre—CA3 response was not inhibited by (—)‐baclofen (10−4m). The depressant action of (—)‐baclofen on the Schaffer collateral/commissural—CA1 response was unaffected by bicuculline (10−4m), whereas the direct membrane effects of (—)‐baclofen were antagonized by bicuculline (10−5m). It is suggested that (—)‐baclofen may modulate neuronal transmission through presynaptic recognition sites possibly by decreasing transmitter release from nerve terminals and also may directly regulate the endogenous neuronal excitability through an activation of the postsynaptic recognition sites.