Pflfigers Arch (1990) 416 : 473 -- 475 Short communication Joumal of Physiology 9 Springer-Verlag 1990 Tolbutamide reverses membrane hyperpolarisation induced by activation of D2 receptors and GABAB receptors in isolated substantia nigra neurones J. Roeper*, A. H. Hainsworth, and F. M. Ashcroft UniversityLaboratory of Physiology, Parks Road, Oxford, OX1 3PT, UK Received March 19/Receivedafter revisionApril 17/AcceptedApril 24, 1990 ABSTRACT A high density of binding sites for sulphonylureas is found in the substantia nigra. These binding sites may be linked to ATP-regulated K-channels (K-ATP channels) as sulphonylureas selectively inhibit these channels in pancreatic g-cells. We have studied the effect of the sulphonylurea tolbutamide on the electrical properties of freshly isolated neurones from guinea-pig substantia nigra, using the perforated patch technique. In spontaneously firing cells, both the D2 agonist quinpirole and the GABAB agonist baclofen abolished firing, hyperpolarised the cell and increased the whole-cell conductance. Tolbutamide reversed all three effects, but was without effect in the absence of quinpirole or baclofen. We suggest that D2 and GABAB receptors activate a K-channel that is blocked by tolbutamide. Keywords: substantia nigra, tolbutamide, dopamine, GABA INTRODUCTION In heart and pancreatic B-cell membranes, sulphonylureas act as specific inhibitors of ATP- regulated K-channels (K-ATP channels; Ashcroft & Ashcroft 1990). High affinity binding sites for these drugs have recently been described in the brain, the highest concentration being located within the substantia nigra (Mourre et al 1989). The physiological function of sulphonylurea-binding sites in the substantia nigra is unknown. In the glucose-sensing neurones of the ventromedial hypothalamus, however, sulphonylureas produce membrane depolarization and initiate electrical activity by blocking K-ATP channels (Ashford et al 1990). Intracellular recordings from pars compacta neurones of the substantia nigra have established that activation of either D2 receptors or GABAB receptors * Present address: Physiologisches Institut, Universit/its-Kranken- haus Eppendorf, UniversitfitHamburg, D-2000 Hamburg, Federal Republic of Germany Offprint requests to." F. M. Ashcroft produces membrane hyperpolarization and cessation of firing by activating an outward K-current (Lacey et al 1987; 1988). There is good evidence that both these receptors may be linked to a common K-channel whose activation involves a GTP-binding protein (Lacey et al 1988). The precise identity of this K-channel is not known. We report here that the sulphonylurea tolbutamide is able to reverse the membrane hyperpolarization and the increase in outward current induced by D2 or GABAB receptor agonists in dissociated neurones from guinea-pig substantia nigra. METHODS Coronal slices (400 gm) of adult male guinea-pig mid-brain were prepared using standard methods (Lacey et al 1987). The substantia nigra (SN) was excised and incubated in external solution containing 12 U/ml papain at 30 ~ for 10-15 mins. Subsequently, these pieces of SN were maintained in papain-free solution at 30 ~ for up to 6 hr and neurones were dissociated by trituration as required. The external (bath) solution contained (in mM): 135 NaC1, 5 KC1, 2 MgSO 4, 2 CaC12, 5 HEPES (pH 7.4 with NaOH), 10 glucose. Whole-cell currents and membrane potentials were recorded using the perforated patch configuration of the patch clamp method (Horn & Marty 1988) with an EPC-7 patch clamp amplifier and stored on video-tape. The pipette solution contained (in mM): 55 KC1, 70 K2SO 4, 7 MgCI2, 2 glutathione, 10 HEPES (pH 7.4 with NaOH), osmolarity 280 mosm. The tip of the pipette was first filled with this solution by capillary action and the pipette then backfilled with the same solution supplemented with 100 gg/ml nystatin (in 0.2% DMSO). Perforation was monitored by the increase in our ability to compensate for the cell capacitance and by the decrease in the series resistance (Gs). Perforation was usually adequate (Gs<50Ms within 5 mins of seal formation. The bath was continuously perfused and exchange of the bath solution was complete within about 2 min. Tolbutamide was dissolved in DMSO (final concentration 0.1%); DMSO had no effect alone. All experiments were carried out at 30-32 ~