Strychnine-Induced Potassium Current in Isolated Dorsal Root Ganglion Cells of the Rat K

Home , Strychnine

Br. J. Pharmacol. (1991), 102, 492-496 C) Macmillan Press Ltd, 1991 Strychnine-induced potassium current in isolated dorsal root ganglion cells of the rat K. Aibara, M. Oonuma & 1N. Akaike Department of Neurophysiology, Tohoku University School of Medicine, Sendai 980, Japan

1 Effects of strychnine (Str) on the dissociated dorsal root ganglion (DRG) cells of the rat have been investigated in whole-cells configuration by a conventional patch-clamp technique. 2 y-Aminobutyric acid (GABA)-induced Cl- current (Icl) increased sigmoidally with increasing concentration. The half-maximal response (K.) was 3 x 10- M and the Hill coefficient was 1.5. Both Str and bicuculline inhibited the GABA-induced Ic, in a concentration-dependent manner. 3 Str itself could elicit the current at concentrations over 1O-IM, at which concentrations the GABA response was completely suppressed. The concentration-response curve for the Str-induced current was bell-shaped, and a nearly maximum response occurred at 3 x 10-4M. A transient 'hump' current appeared immediately after the wash-out of external solution containing high concentrations of Str over 3 x 10-4M. 4 The Str-induced outward current and a transient 'hump' current were augmented by the removal of extracellular K+ and were suppressed by the substitution of intracellular K+ for Cs . But the current was not sensitive to extracellular Na+, Ca2 + and CI-. 5 The reversal potential of Str-induced current (Estr) was -75mV, which was close to the K+ equilibrium potential (EK =-76.3 mV). The change of Esr for a ten fold change in extracellular K' concentration was 58 mV, indicating that the membrane behaves like a K+ electrode in the presence of Str. The reversal potential of the 'hump' current was also close to EK. 6 The Str-induced outward current was antagonized by K+ channel blockers such as Ba2+, tetraethylammonium (TEA)-chloride, and 4-aminopyridine (4-AP) in a concentration-dependent manner. 7 The Str-induced K+ current was not affected by internal perfusion of bis(y-aminophenoxy)ethane-N, N,N',N-tetraacetic acid (BAPTA), indicating that the Str response does not result in the activation of K+ conductance by the intracellular Ca2+.

Introduction tion with 0.33 mgml-' DISPASE (Godo Shusei) and 4mg ml 1 collagenase (Sigma) at 310C for 40 min. In a culture Glycine is an important inhibitory neurotransmitter in mam- dish (35mm Falcon #3801) filled with a standard external malian CNS neurones, especially at the motoneurones and solution (see below), the DRG neurones were dissociated interneurones in the spinal cord, and strychnine (Str) acts as a mechanically by gentle pipetting. The dissociated DRG cells selective and competitive antagonist of the glycine receptor. adhered to the bottom of the dish within 30min and were The convulsant action of Str is due to interference with the then prepared for the experiments at room temperature. postsynaptic inhibition mediated by glycine. Hence, Str has been used as a valuable pharmacological tool for studies of Solutions glycinergic inhibition (Curtis, 1969; Kuno & Weakly, 1972; Johnston, 1978; Zarbin et al., 1981). However, in dissociated The solutions used in the present experiments were composed frog dorsal root ganglion neurones (Yakushiji et al., 1987) and as follows. Incubation solution (mM): NaCl 124, KC1 5, CaCl2 dissociated rat Purkinje cells (Kaneda et al., 1989), Str was 2.4, KH2PO4 1.2, MgSO4 1.3, NaHCO3 26 and glucose 10. able to inhibit not only the glycine-induced Cl- current Standard external solution: NaCl 150, KCI 5, CaCl2 2, MgCl2 ('cl) 1, glucose 10, and HEPES 10. Na'-free external solution: but also the GABA-induced Ic, in a competitive manner at an almost equal concentration-range of the drug. NaCl in external solution was replaced with equimolar exter- Recently, while we investigated the effect of Str on y- choline chloride. K+-free external solution: KCI in the aminobutyric acid (GABA)-induced in dissociated rat nal solution was replaced with CsCl. Ca2+-free external solu- Ic, was dorsal root ganglion (DRG) cells, we observed that Str itself tion: CaCI2 in the external solution replaced with CoCl2. Low in the external could induce a response at concentrations over 10- sM. In the Cl- external solution: 80mm NaCI solu- Na-isethionate. The of present study, therefore, we examined the electrical and phar- tion was replaced with equimolar pH macological properties of the Str-induced current in disso- all external solutions was adjusted to 7.4 with Tris-base. Inter- ciated DRG cells of the rat under voltage-clamp conditions. nal solution (mM): KCI 50, K-gluconate 70, NaCl 30, CaCl2 0.25, MgCl2 1, Mg-ATP 2, HEPES 10, and EGTA 5. K+-free internal solution: KCI and K-gluconate in the internal solution were replaced with CsCl. The pH of both internal solu- Methods tions was adjusted to 7.2 with Tris-base. Preparation Electrical measurements Dorsal root ganglia (DRG) were dissected from 2 weeks-old Ionic current was measured in whole-cell voltage-clamp mode Wistar rats under ether anaesthesia. The isolated ganglia were by the use of conventional patch-clamp technique (Hamill et kept in an incubation solution (see below) bubbled with 95% al., 1981). The resistance between the glass pipette filled with 02 and 5% CO2 at 31°C for 40-60min and then the ganglia internal solution and the reference electrode in bathing were successively treated enzymatically in the incubation solu- medium was 4 to 8 MCI. Both electrodes were led to a patch- clamp amplifier (List Medical, EPC-7), and the junction Author for correspondence. potential was compensated with an offset circuit. The current STRYCHNINE-INDUCED POTASSIUM CURRENT 493 and voltage were monitored on a storage oscilloscope GABA Str (Toshiba, 10M63) after being low-pass filtered at 1 kHz, and 1.0r 3 x 10'5 M recorded simultaneously on a pen-recorder (Sanei, RECTI- / 3 x 10-6 M HORIZ-8K) and a video cassette recorder (Mitsubishi, 1 nA / -.-z U 20 s HV-F32) in a digital mode by a digital audio processor (Sony, a) PCM-501ES). 0.5 (U) 3x 10-5 Drugs cc3 Drugs used in the present experiments were y-aminobutyric acid (GABA), 4-aminopyridine (4-AP) and tetra- 0[ 11 I11 1 1 ethylammonium (TEA)-chloride [Tokyo Kasei], strychnine 10-6 10-5 1O-4 hydrochloride (Str), bicuculline methioide (Bic) and bis(y- Concentration (M) aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, Sigma). All drugs were dissolved in the external or internal Figure 2 Inhibition of y-aminobutyric acid (GABA)-induced Ic, by use. test solution was exchanged strychnine (Str; 0) and bicuculline (A). VH was - 50mV. The prep- solution just before External aration was pretreated with antagonists for 30 s. All responses induced by a rapid application technique termed 'Y-tube' method, by a mixture of 3 x 10- M GABA plus one of the antagonists at which allows the exchange of solutions surrounding an iso- various concentrations were normalized to the peak current induced lated DRG cell within 10 to 20ms (Akaike et al., 1989; by 3 x 10- M GABA alone. Abscissa scale shows antagonist concen- Murase et al., 1989). tration. Each point shows the average of 4-6 experiments. The inset shows the inhibition of GABA-induced la by Str of various concentrations. All recordings shown in the inset were obtained from the Results same cell. Note that Str itself induces the outward current (inset). GABA-induced response increased in a sigmoidal fashion with increasing GABA con- The isolated DRG cells were perfused with standard external centration. In the GABA concentration-response curve, a half- and internal solutions containing 161 and 82.5 mm Cl-, maximal response (K.) was 3 x 10 sM and the Hill coefficient respectively. The holding potential (VH) was -50 mV. GABA was 1.5. was applied every 2 min as desensitization did not occur with this time interval. ofconvulsants on GABA-induced The values of a transient peak and a successive steady-state Effects Ic, current components of the GABA-induced Cl- current (Ic) Figure 2 shows the concentration-dependency of inhibitory are plotted as a function of GABA concentration in Figure 1. actions of strychnine (Str) and bicuculline (Bic) on the peak In the figure all responses were normalized to the peak I current component elicited by 3 x 10-5M GABA. In this response induced by 10- M GABA. The threshold concentra- experiment, the dissociated DRG cells were pretreated with tion of GABA was 3 x 10-6M and the GABA-induced Ic, Str or Bic for 30s, and successive applications of 3 x 10- M GABA plus one of the antagonists at various concentrations 7 were made. All responses were normalized to the peak current induced by 3 x 10- M GABA alone. The concentrations for 6 half-inhibition (IC50) were 4.0 x 106M for Str and 1.8 x 10-6M for Bic. Unexpectedly, as shown in Figure 2 5 (inset), Str itself induced outward currents at concentrations higher than 10- M. On the other hand, Bic itself did not elicit a)4 any current even at the extremely high concentration of 6 10-4M. Z 3- cr - r- Strychnine-induced current (Is,,) 2 I/ Experiments were carried out at a VH of -50 mV. Str was 20 s applied every 5min, at which interval a constant response to Str could be repeatedly evoked for 1 h or more. The threshold 0 concentration for the Str-induced current (ISr) was around I III I11 I11 10 -M. The Is,, increased with increasing drug concentration 10-6 10-5 1o-4 10-3 in the range 10-' to 3 x 10-4M. Further increases in the Str GABA concentration (M) concentration over 1O- ' M decreased the peak current, and the peak current rapidly subsided to a steady plateau level during Figure 1 y-Aminobutyric acid (GABA)-induced Cl- current (Qcj). Concentration-response relationship for GABA, in which all GABA continuous application of the drug. Moreover, the 'wash-out' responses at various concentrations were normalized to the peak of external solution containing Str of concentrations higher response induced by 10- M GABA (symbol marked with asterisk). than 3 x 1O-4M always induced a transient outward 'hump' Holding potential (VH) was -5OmV; [Cl-]; = 82.5mM; [CI-]O = current (Figure 3a). However, this 'hump' current was never 161 mM. Each point shows the average of 4-6 experiments, and bars induced by continuous applications for more than 1 min of Str indicate + I s.e. when larger than symbol. A continuous theoretical at concentrations below 3 x 10-4M. Figure 3b shows the Str- curve was drawn according to the following equation with n = 1.5, induced peak currents plotted as a function of Str concentra- Ka= 3 x 10-5, and I... = 6.5: tion. A maximal response during a continuous application of Cn the drug was regarded as the peak current. When all Str I = Imax Cn responses were normalized in relation to the peak current induced by 1O-4M Str, the concentration-response curve was where I is the observed GABA-activated current, Img, is the maximum 10-8- current, C is the GABA concentration, K. is the GABA concentration bell-shaped. Glycine over the concentration range that evoked the half-maximal response, and n is the Hill coefficient. 10-M did not induce any responses; glycine had no effect on The inset shows the GABA-induced Ic s. Currents were obtained istr. from the same cell. The cell was exposed to various concentrations of The inset of Figure 4 shows the currents induced by 10-M GABA for periods indicated by a horizontal bar above each response. Str at various VH levels. In the current-voltage (I-V) relation- 494 K. AIBARA et al.

a Or _ 11L_0 t I \ 400 pA _ _- _ _ 20s -20-- 3 X 10i M 10-4 3 X 104 10-3 3 X 10-3 Str b 2 -40 _. 5 E 'EK

1/) L- -60--

. 1* a) -80 L

0 -100 L I I11111111 1 -11 fill I I I l 11 1o-5 10-4 10-3 1o-2 1 3 10 30 100 Str concentration (M) lK+lo (mM) Figure 3 (a) Strychnine (Str)-induced outward currents in a disso- Figure 5 Effect of extracellular K+ concentration ([K+]d) on the ciated DRG cell. VH was -50 mV. The cell was exposed to various reversal potential of strychnine (Str)-induced current (Esgr). Disso- concentrations of Str for period indicated by a horizontal bar below ciated DRG cells were perfused with external solutions containing K+ each response. (b) Concentration-peak response curve for Str. The x of various concentrations. The intracellular K+ concentration ([K+]i) axis denotes the Str concentration, and the y axis represents the rela- was 120 mm throughout this experiment. Each point shows the tive peak Str response (Isj,). A maximal response during a continuous average of 4-5 experiments. A straight line shows EK. application of the drug was regarded as a peak response. Each point shows the average of 4-6 experiments. All Str responses were normalized to the peak current induced by 10-4M Str (see symbol marked with asterisk). The continuous curve was drawn by eye. of Str also reversed at almost EK (not shown). The results indicate that the Istr and the 'hump' current are carried by passing through K+ channels. ship of 10-4M Str-induced response, the responses at various When the dissociated DRG cells were perfused with an VH were normalized to the peak response induced by Str at a internal solution containing 120 mm K+ and the external solu- VH of -50mV. The reversal potential of Istr (Estr) estimated tions containing various K+ concentrations (i.e. [K+]. = 2.5, from an intersect on the voltage axis in the I-V curve was 5, 10, 30, and 60mM), the measured Estr values were -75.0 + 2.0 mV (mean + s.e.mean, n = 5). The experimental -87.3 + 2.1 mV (n = 5), -75.0 + 2.0 mV (n = 5), Estr value was quite close to the K+ equilibrium potential -54.3 + 0.9 mV (n = 4), -29.0 + 1.5 mV (n = 4), and (EK = -76.3 mV) calculated from the Nernst equation -12.0 + 1.5mV (n = 4) for 2.5, 5, 10, 30, and 60mM [K+]0, knowing the extra- and intracellular K+ concentrations. respectively (Figure 5). In the Est, and [K+] relationship, the Moreover, the 'hump' current induced by high concentrations change in Estr for a ten fold change of [K+]. was 58 mV, indicating that the cell membrane behaves like a K + electrode in Str the presence of Str. 10-4 M Effects ofextracellular electrolytes on Istr VH (mV) In the presence of intra- and extracellular K+ ([K+]i and -30 [K+]O respectively), Str evoked the outward current at a VH 2 of -50 mV (Figure 6a). The Str-induced outward current was not affected by removal of extracellular Na+, Ca2 , and Cl, but was augmented by the absence of [K+]0, when KCI in the -70 -U) I.. external solution was replaced with equimolar CsCl. In the a) presence of intracellular Cs+, Str induced the inward current a) -110< cr- [Na l r fe fre Control f[KCnoree free free de[Clr-decrease]s 1 nA a 20 s EK VH (mV) I mO m -100 -50 0 10-4 M Str b -

-1 J200 pA 20 s Figure 4 Current-voltage (I-V) relationship of 10-M strychnine Figure 6 Effects of extracellular electrolytes on 10-4M strychnine (Str)-induced response. All responses were normalized to the peak (Str)-induced currents. VH was -50 mV. (a) The cell was perfused with response induced by Str at a VH of -50 mV (see symbol marked with the internal solution containing 120mM K . (b) The cell was perfused asterisk). Each point shows the average of 5 experiments. The inset with the internal solution containing 120mM Cs+ instead of K+. The shows 10 -M Str-induced currents at various VHS in the same cell. horizontal bars indicate the period of application of 10- M Str. STRYCHNINE-INDUCED POTASSIUM CURRENT 495

a Str-induced inward current is completely abolished by removal of extracellular K+; (3) the reversal potential of Str- induced current (Es,,) is quite close to the EK calculated from the Nernst equation.

1 0- M IE=ZI ZZI: 400 pA When suppression of the peak and plateau currents Str 3 mM 10 mM 20 mm 4 appeared with concentrations of Str over 3 x 10-'M, the Ba2+ 4-AP TEA 20s wash-out of Str always induced a transient 'hump' current b (Figure 3a). Such decreases of the peak and plateau currents '1Or may be due to blockade of the K' channel by the Str mol- ecule itself, and the hump on the wash-out of high concentrations of Str is probably due to the activation of undesensitized receptors by Str, released from channels blocked by Str. Such reasoning may also apply to the hump following the wash-out -C, 0.5 of high concentrations of acetylcholine in the voltage-clamped frog endplate (Adams, 1975) and that of pentobarbitone in the frog DRG neurone (Akaike et al., 1987). A variety of voltage-dependent K+ channels have been reported in various invertebrate and vertebrate neurones oL (Adams & Gage, 1978; Adams et al., 1980; Segal & Barker, 1984; Cobbett et al., 1989). At least five distinct K' currents 1 10 100 have been identified as follows: a voltage-activated delayed Concentration (mM) K' current (IV, a transient outward K' current (IA) (Connor Figure 7 Effects of various K+ channel blockers on the strychnine & Stevens, 1971; Neher, 1971; Neher & Lux, 1972), a non- (Str)-induced outward current (Istr). The cells were pretreated with inactivating outward K' current (IM) (Brown & Adams, 1980; standard external solution containing various K+ channel blockers Adams et al., 1982), a Ca2"-activated outward K+ current for 1 min, and successively 10' M Str plus one of the blockers were (IKcE) (Meech & Standen, 1972; Brown & Griffith, 1983; applied simultaneously. VH was -50mV. (a) All recordings were Bourque et al., 1985; Bourque, 1988), and an anomalous recti- obtained from the same cell. Open and closed horizontal bars indicate fier K+ current (IA) which is a voltage- and time-dependent the application of the blockers and Str, respectively. (b) All responses inward current triggered at hyperpolarized potentials induced by a mixture of 10-' M Str plus one of the blockers at various concentrations were normalized to peak current induced by 10-'M (Purpura et al., 1968; Hotson et al., 1979; Halliwell & Adams, Str alone. Abscissa scale shows the concentration of blockers. Each 1982). These have been differentiated on the basis of their point shows the mean of 4 experiments, and vertical bars show + 1 voltage-sensitivity, Ca2+-dependence and pharmacology. In s.e.mean when larger than symbol: (0) Ba2'; (J) 4-aminopyridine molluscan neurones (Thompson, 1977), cultured rat hippo- (4-AP); (0) tetraethylammonium (TEA). campal neurones (Segal et al., 1984), and cultured neurones of the rat supraoptic nucleus area (Cobbett et al., 1989), the extracellular application of TEA and 4-AP mainly attenuated at the same VH of -50mV, and the inward current was com- abolished the of K+ with the 'K and 'A, respectively. The extracellular application of pletely by replacement extracellular Co2+-, Mn2'+ , Cd2'-, La3`-, or organic Ca2'+ blockers Cs+ (Figure 6b). These results also indicate that Ist is carried K+. inhibited the IKc. in Helix aspersa neurones (Meech & by Standen, 1972), molluscan neurones (Thompson, 1977), hippocampal neurones of the guinea-pig (Brown & Griffith, 1983), Effects ofvarious K + channel blockers on Istr and rat supraoptic nucleus neurosecretory neurones (Bourque et al., 1985; Bourque, 1988). The IM was depressed by adding In order to elucidate the pharmacological type of K' channel Ba2+ to the bathing medium (Halliwell & Adams, 1982). The activated by Str, the effects of various K+ channel blockers on IA in hippocampal neurones was affected by the Na'-free the Str-induced outward current were tested at a VH of external solution or by the external solution containing Cs+ -50mV. The neurones were perfused with normal internal (Halliwell & Adams, 1982; Segal & Barker, 1984). In the and external solutions. In these experiments, BaCl2 and 4-AP present preparation, the Ist, was not sensitive to extra- and were directly dissolved in the standard external solution. The intracellular Ca2 + but was suppressed by K + channel external solution containing TEA was made by the replacement of NaCl in the external solution with equimolar TEA-Cl. blockers in the order: Ba2+ > 4-AP > TEA. Moreover, there Ba2 and 4-AP suppressed the Str-induced outward was a linear relationship between the Str-induced K+ current +, TEA, and the holding potential, indicating that ISt, has no voltage- current in a concentration-dependent manner (Figure 7a). The dependence. Interestingly, Str-binding sites exist in the mem- half inhibition doses (IC50) were 3.5 mm for Ba2+, 8 mm for brane fraction associated with the glycine receptor (Young & 4-AP, and 45 mm for TEA (Figure 7b). Snyder, 1974). However, the present preparation has no glycine receptors, although Str increases K + conductance Effects ofinternal perfusion ofBAPTA on IStr which is sensitive to K+ channel blockers. Thus, the results suggest that the Str-induced K+ current is probably due to When the Str-induced outward current was induced at a VH of activation of the Str receptor-K+ channel complex which has -50 mV, intracellular perfusion of BAPTA at concentrations no cooperative interaction with the glycine receptor. of 5- 10 mm had no effects on the amplitude and kinetics of the It is of interest that Str acts not only as an antagonist to Str response in all cells tested (n = 5), indicating that the Str- block the glycine- and GABA-induced Ia (Curtis et al., 1968; induced K+ response is not affected in the presence or the 1971; Ryall et al., 1972; Barron & Guth, 1987; Yakushiji et absence of intracellular Ca2 + al., 1987; Krishtal et al., 1988; Akaike & Kaneda, 1989; Kaneda et al., 1989) but also as an agonist to activate K + Discussion channels. However, at this moment, the functional role of the increase in K+ conductance in DRG cells induced by Str itself The present experiments clearly show that at high concentra- is obscure. Further studies will be necessary to elucidate the tions over 3 x 10-5M Str induces K' currents in dissociated mechanism or functional role of the Str-induced K+ current. rat DRG cells. This K+ current passes through K+ channels as follows: (1) in the presence of intracellular K+, the Str- This study was supported by a Grant-in-Aid for Scientific Research induced outward current is augmented by removal of extra- on Priority Areas (Nos. 02223105 and 02241101) and the Epilepsy cellular K +; (2) in the absence of intracellular K', the Research Foundation to N.A. 496 K. AIBARA et al.

References ADAMS, P.R. (1975). A study of desensitization using voltage clamp. HAMILL, O.P., MARTY, A., NEHER, E., SAKEMANN, B. & SIGWORTH, Pfluegers Arch., 60, 135-144. F.J. (1981). Improved patch-clamp techniques for high resolution ADAMS, P.R., BROWN, D.A. & CONSTANTI, A. (1982). M-currents and current recording from cells and cell-free membrane patches. other potassium currents in bullfrog sympathetic neurones. Pfluegers Arch., 391, 85-100. J. Physiol., 330, 537-572. HOTSON, J.R., PRINCE, D.A. & SCHWARTZKROIN, P.A. (1979). Anom- ADAMS, D.J. & GAGE, P.W. (1978). Ionic currents in response to mem- alous inward rectification in hippocampal neurons. J. Neuro- brane depolarization in an Aplysia neurone. J. Physiol., 289, 115- physiol., 42, 889-895. 142. JOHNSTON, G.A.R. (1978). Neuropharmacology of amino acid inhibi- ADAMS, D.J., SMITH, SJ. & THOMPSON, S.H. (1980). Ionic currents in tory transmitters. Ann. Rev. Pharmacol. Toxicol., 18, 269-289. molluscan soma. Ann. Rev. Neurosci., 3, 141-167. KANEDA, M., WAKAMORI, M. & AKAIKE, N. (1989). GABA-induced AKAIKE, N. & KANEDA, M. (1989). Glycine-gated chloride current in chloride current in rat isolated Purkinje cells. Am. J. Physiol., 256, acutely isolated rat hypothalamic neurons. J. Neurophysiol., 62, C1 153-1159. 1400-1409. KRISHTAL, O.A., OSIPCHUK, YU.V. & VRUBLEVSKY, S.V. (1988). AKAIKE, N., MARUYAMA, T. & TOKUTOMI, N. (1987). Kinetic proper- Properties of glycine activated conductances in rat brain neurones. ties of the pentobarbitone-gated chloride current in frog sensory Neurosci. Lett., 84, 271-276. neurones. J. Physiol., 394, 85-98. KUNO, M. & WEAKLY, J.N. (1972). Quantal components of the inhibi- AKAIKE, N., SHIRASAKI, T. & YAKUSHIJI, T. (1989). Quinolones and tory synaptic potential in spinal motoneurones of the cat. fenbufen interact with GABAA receptor in rat isolated CA1 pyr- J. Physiol., 224, 287-303. amidal neurons. Acta Physiol. Scand., 136, Supplement 582, p21. MEECH, R.W. & STANDEN, N.B. (1972). Potassium activation in Helix BARRON, S.E. & GUTH, P.S. (1987). Uses and limitations of strychnine aspersa neurones under voltage clamp: a component mediation by as a probe in neurotransmission. NIPS, 8, 204-206. calcium influx. J. Physiol., 249, 211-239. BOURQUE, C.W. (1988). Transient calcium-dependent potassium MURASE, K., RYU, P.D. & RANDIC, M. (1989). Excitatory and inhibi- current in magnocellular neurosecretory cells of the rat supraoptic tory amino acids and peptide-induced response in acutely-isolated nucleus. J. Physiol., 397, 331-347. rat spinal dorsal horn neurones. Neurosci. Lett., 103, 56-63. BOURQUE, C.W., RANDLE, J.C.R. & RENAUD, L.P. (1985). Calcium- NEHER, E. (1971). Two fast transient current components during dependent potassium conductance in rat supraoptic nucleus voltage clamp on snail neurons. J. Gen. Physiol., 58, 36-53. neurosecretory neurons. J. Neurophysiol., 54, 1375-1382. NEHER, E. & LUX, H.D. (1972). Differential action of TEA on two K BROWN, D.A. & ADAMS, P.R. (1980). Muscarinic suppression of a current components of a molluscan neurone. Pfluegers Arch., 336, novel voltage-sensitivity K + current in a vertebrate neurone. 87-100. Nature, 183, 673-676. PURPURA, D.P., PRELEVIC, S. & SANTINI, M. (1968). Hyperpolarizing BROWN, D.A. & GRIFFITH, W.H. (1983). Calcium activated outward increase in membrane conductance in hippocampal neurons. Brain current in voltage clamped hippocampal neurones of the guinea- Res., 7, 310-312. pig. J. Physiol., 337, 287-301. RYALL, R.W., PIERCEY, M.F. & POLOSA, C. (1972). Strychnine- COBBETT, P., LEGENDRE, P. & MASON, W.T. (1989). Characterization resistant mutual inhibition of Renshaw cells. Brain Res., 41, 119- of three types of potassium current in cultured neurones of rat 129. supraoptic nucleus area. J. Physiol., 410, 443-462. SEGAL, M. & BARKER, J.L. (1984). Rat hippocampal neurons in CONNOR, J.A. & STEVENS, C.F. (1971). Voltage clamp studies of a culture: potassium conductances. J. Neurophysiol., 51, 1409-1433. transient outward membrane current in gastropod neural somata. THOMPSON, S.H. (1977). Three pharmacologically distinct potassium J. Physiol., 213, 21-30. channels in molluscan neurones. J. Physiol., 265, 465-488. CURTIS, D.R. (1969). The pharmacology of postsynaptic inhibition. YAKUSHIJI, T., TOKUTOMI, N., AKAIKE, N. & CARPENTER, D.O. Prog. Brain Res., 31, 171-189. (1987). Antagonists of GABA responses, studied using internally CURTIS, D.R., DUGGAN, A.W., FELIX, D., JOHNSTON, G.A.R. & perfused frog dorsal root ganglion neurones. Neuroscience, 22, McLENNAN, H. (1971). Antagonism between bicuculline and 1123-1133. GABA in the cat brain. Brain Res., 33, 57-73. YOUNG, A.B. & SNYDER, S.H. (1974). Strychnine binding in rat spinal CURTIS, D.R., HOSLI, L., JOHNSTON, G.A.R. & JOHNSTON, I.H. (1968). cord membranes associated with the synaptic glycine receptor: The hyperpolarization of spinal motoneurones by glycine and cooperativity of glycine interactions. Mol. Pharmacol., 10, 790-809. related amino acids. Exp. Brain Res., 5, 235-258. ZARBIN, M.A., WAMSLEY, J.K. & KUHAR, MJ. (1981). Glycine recep- HALLIWELL, J.V. & ADAMS, P.R. (1982). Voltage-clamp analysis of tor: light microscopic autoradiographic localization with [3H] muscarinic excitation in hippocampal neurons. Brain Res., 250, strychnine. J. Neurosci., 1, 532-547. 71-92. (Received June 18, 1990 Revised September 14, 1990 Accepted September 27, 1990)