The biophysical and pharmacological properties of presumptive serotonergic neurones recorded intracellularly from the dorsal raphe nucleus in the in vitro slice preparation

Presumptive serotonergic neurones recorded intracellularly from the dorsal raphe nucleus in the in vitro slice preparation had a high input resistance Rm (203 ± 7MO), a linear current-voltage relationship in the hyperpolarising direction and a long membrane time constant r (29 ± 1.4ms). Spontaneous rhythmically firing action potentials (0.5-2Hz) were seen at the resting membrane potential (-59 ± 0.8mV). The rhythmicity resulted from a four stage pacemaker potential cycle; 1) a slow depolarising prepotential leading to 2) a broad (2ms) overshooting action potential followed by 3) a rapid afterhyperpolarisation (AHP, 10-20mV) and 4) a gradual (=200ms) return to the resting membrane potential. Several time and voltage dependent ionic conductances were found to underlie the pacemaker potentials. A Co^-+ sensitive low threshold (-60 to -55mV) depolarising prepotential was seen to be mediated by a transient inactivating inward calcium current. The subsequent action potential consists of a) a TTX sensitive, fast sodium spike and b) a TTX insensitive, high threshold (-40mV) component. The latter potential was blocked by Co^+ as was the following AHP. The AHP was found to reverse direction between -80 to -90mV consistent with a potassium mediated event. In voltage clamp depolarisation evoked a transient inward current followed by a TEA-sensitive outward current. The AHP was therefore assumed to be mediated via a calcium dependent potassium conductance, Ik(Ca)• Apamin, a known inhibitor of Ik(Ca),blocked the AHP irreversibly. 5-HT appeared to have no direct effect on AHP size or duration. A second more transient outward current was observed that required hyperpolarisation from the resting membrane potential to remove channel inactivation. This outward current was insensitive to TEA but 4-aminopyridine (4-AP) caused a 60% reduction in amplitude. The inactivation and activation properties of the current (Vhalf -74 and -49mV respectively) were characteristic of 1^. 5-HT had no apparent effect on 1^. A third non-inactivating outward current was observed with depolarising voltage commands to -40mV, or more positive. Transient hyperpolarising voltage step commands produced an inward current relaxation and on termination of the pulse an outward relaxation was observed. This current, reminiscent of %, could act as a rectifier and oppose prolonged membrane depolarisations. 5-HT (50-100/xM) caused a membrane hyperpolarisation (4-18mV) and a concomitant decrease in Rm (15-20%) and r (24-32%). The reversal potential for the 5HT effect was -91mV. This was close to the K+ equilibrium potential. Chloride loaded cells showed a depolarising response to lOmM GABA application whereas 5HT under the same experimental conditions still evoked a hyperpolarisation. Baclofen, believed to activate a G protein mediated potassium conductance also caused a longlasting hyperpolarisation together with a decrease in Rm and rm which reversed at -90mV. The selective 5-HT^ agonists 8hydroxy-2-(di-n-propylamino)teralin (8-OHDPAT), dipropylcarboxamidotryptamine (DP-5-CT) and the novel anxiolytic buspirone caused a marked longlasting hyperpolarisation with an associated decrease in Rm and r at concentrations lower than required for an equivalent response to 5-HT. All three compounds had reversal levels similar to that of 5-HT. Methysergide (IOO/2M) and S2 (lOOnM5/iM) , a purported 5HT]^ antagonist, were ineffective in antagonising the 5HT induced response. Spiperone (30/^M) caused a 60% reduction in the 5HT induced response. Furthermore spiperone caused a gradual increase in Rm and r possibly by blocking a local tonic release of 5HT or dopamine. In conclusion this study has demonstrated that DR neurones posses the necessary voltage gated conductances needed to maintain an intrinsic pacemaker activity. Exogenous application of 5HT does not affect any presently characterised conductance but appears to produce inhibition via an increase in potassium conductance. It is postulated that this response is mediated primarily by the activation of a 5HT^ receptor-G protein complex. Hence the anxiolytic action of buspirone may be attributable at least in part to the activation of this same receptor G protein complex.