during constant current stimulation. within 20 ms, while the slow-transient current took > 10 s to decay. Raised

Transcription

1 Journal of Physiology (1991), 434, pp With 6 figures Printed in Great Britain TWO TRANSIENT POTASSIUM CURRENTS IN LAYER V PYRAMIDAL NEURONES FROM CAT SENSORIMOTOR CORTEX BY W. J. SPAIN, P. C. SCHWINDT AND W. E. CRILL From the Department of Physiology & Biophysics and the Department of Medicine (Division of Neurology), University of Washington School of Medicine, Seattle, WA 98195, USA (Received 19 January 1990) SUMMARY 1. Two transient outward currents were identified in large pyramidal neurones from layer V of cat sensorimotor cortex ('Betz cells') using an in vitro brain slice preparation and single-microelectrode voltage clamp. Properties of the currents deduced from voltage-clamp measurements were reflected in neuronal responses during constant current stimulation. 2. Both transient outward currents rose rapidly after a step depolarization, but their subsequent time course differed greatly. The fast-transient current decayed within 20 ms, while the slow-transient current took > 10 s to decay. Raised extracellular potassium reduced current amplitude. Both currents were present in cadmium-containing or calcium-free perfusate. 3. Tetraethylammonium had little effect on the slow-transient current at a concentration of 1 mm, but the fast-transient current was reduced by 60%. 4- Aminopyridine had little effect on the fast-transient current over the range 20 tm-2 mm, but these concentrations reduced the slow-transient current and altered its time course. 4. Both transient currents were evoked by depolarizations below action potential threshold. The fast-transient current was evoked by a 7 mv smaller depolarization than the slow-transient current, but its chord conductance increased less steeply with depolarization. 5. Voltage-dependent inactivation of the fast-transient was steeper than that of the slow-transient current (4 vs. 7 mv per e-fold change), and half-inactivation occurred at a less negative potential (-59 vs. -65 mv). The activation and inactivation characteristics of each current overlapped, however, implying the existence of a steady 'window current' extending over a range of 14 mv beginning negative to action potential threshold. 6. The fast-transient current displayed a clear voltage dependence of both its activation and inactivation kinetics, whereas the slow-transient current did not. Recovery of either current from inactivation took about 1 s near -70 mv. The recovery of the slow-transient current became faster with hyperpolarization. 7. The contribution of each transient current to repolarization of the action potential was assessed from pharmacological responses. Blockade of calcium influx MS 8203

2 592 W. J. SPAIN, P. C. SCHWINDT AND W. E. CRILL had little or no effect on the rate of action potential repolarization, whereas the selective reduction of either transient current caused significant slowing of repolarization. 8. We conclude that Betz cells possess at least two transient potassium currents, each a member of the rapidly expanding family of voltage-gated potassium currents that have been identified in various cell types. In Betz cells these currents play a major role in repolarization of the action potential and, because of their voltage dependence and kinetics, they can influence the response to stimuli that cause repetitive firing. INTRODUCTION The initial quantitative description of the delayed rectifier current by Hodgkin & Huxley (1952) explained action potential repolarization in axons, but their analysis could not account for the large dynamic range of repetitive firing properties that occur in neurone cell bodies. Subsequently, other investigators showed that a transient, voltage-dependent potassium current (the A-current) was prominent in cell bodies (Hagiwara, Kusano & Saito, 1961; Connor & Stevens, 1971; Neher, 1971). The A-current extended the dynamic firing range of a neurone by allowing repetitive firing to proceed at slow rates (Connor & Stevens, 1971). In recent years numerous additional types of potassium currents have been identified. Among these are potassium currents that differ from the molluscan delayed rectifier or A-current in their kinetics, voltage dependence or pharmacologial properties and which may play different roles in governing cell excitability (reviewed by Rudy, 1988). Large neocortical neurones from layer V of cat sensorimotor cortex ('Betz cells') possess a variety of potassium channel types. We have previously described the functional roles of three apparently different potassium channels of Betz cells that are activated by increases in either intracellular calcium or sodium rather than voltage. These potassium currents have relatively slow kinetics and are modulated by neurotransmitters (Schwindt, Spain, Foehring, Chubb & Crill, 1988a; Foehring, Schwindt & Crill, 1989; Schwindt, Spain & Crill, 1989). Evidence was also presented in brief for the existence of two transient outward currents that contribute to action potential repolarization (Schwindt, Spain, Foehring, Stafstrom, Chubb & Crill, 1988b). In this report we describe in detail the kinetic and pharmacological properties of the two transient potassium currents, and we provide evidence that these voltagegated currents are primarily responsible for action potential repolarization in Betz cells. We find, however, that the activation and inactivation properties of these currents allow them to play a larger role in cell excitability, namely, to affect the transduction of synaptic current into action potential trains. In the following paper (Spain, Schwindt & Crill, 1991) we show how the relative dominance of one of these currents in a certain group of Betz cells affects repetitive firing patterns and thus allows the separation of Betz cells into functional classes. METHODS Large neurones from cortical layer V were studied in brain slices taken from lateral cruciate cortex (area 4y) of pentobarbitone-anaesthetized cats (35 mg kg-', I.P.). After removal of the

3 TRANSIENT POTASSIUM CURRENTS IN BETZ CELLS cortex, the cats were killed by intravenous injection of pentobarbitone (120 mg kg-'). Slices 400,um thick were maintained at C in an interface recording chamber. Full details of animal care, operative procedures, slice preparation and maintenance, identification of cortical layer V and recording methods were described previously (Stafstrom, Schwindt, Flatman & Crill, 1984b; Spain, Schwindt & Crill, 1987). Solutions Normal perfusate, consisting of (in mm): NaCl, 130; KCl, 3; CaCl2, 2; MgCl2, 2; NaH2PO4, 1-25; NaHCO3, 26; dextrose, 10; was bubbled with 95% 02 and 5% CO2 to maintain ph 7-4. Several modified perfusates were employed. Tetrodotoxin (1 #M) was added directly to the perfusate and was present during all voltage clamp experiments. Excess KCl (6 mm), tetraethylammonium chloride (1-50 mm), or 4-aminopyridine (20 /tm-2 mm) were substituted in equimolar amounts for NaCl if their final concentration exceeded 1 mm. Tetraethylammonium chloride was from Eastman-Kodak; all other chemicals were from Sigma Chemical Co. Several procedures were used to block calcium currents. In some experiments, 400,uM-CdCl2 was added to perfusate containing 1-6 mm-cacl2 (cadmium-containing perfusate). To avoid precipitation, NaH2PO4 was omitted, NaHCO3 was reduced to 15 mm, and 10 mm-n-2-hydroxyethylpiperazine-n'-2-ethanesulphonic acid (HEPES: 5-56 mm acid plus 4-46 mm-sodium salt) was added to maintain ph 7-4 during carbogen bubbling at 36 'C. In other experiments, 2 or 3 mm- CoCl2 was substituted for 2 mm-cacl2 in the perfusate with NaH2PO4 omitted to avoid precipitation (cobalt-substituted perfusate). In other experiments, 0 5 mm-ethyleneglycol-bis(f6-aminoethyl ether)n,n',n'-tetraacetic acid (EGTA), titrated to ph 7-4 with NaOH, was added to perfusate in which MgCl2 was raised to 5 mm and CaCl2 was omitted (calcium-free perfusate). Recording Intracellular recording microelectrodes were made from 1-5 mm o.d. thin-wall glass capillary tubing (WPI) and contained 3 M-KCl in most experiments (resistance: 7-14 MQ). In some experiments, the microelectrodes contained 2-5 M-KCl plus 0-1 M-EGTA and 0-1 M-MOPS (3-(Nmorpholino)propanesulphonic acid, ph 7 2). An Axoclamp II amplifier (Axon Instruments) was used in an active bridge mode or a discontinuous mode (DCC) during constant current injection, or in a single-electrode voltage-clamp mode (SEVC). In the DCC and SEVC modes sampling rates of khz were employed at a 30% duty cycle. The head stage output was viewed on a separate oscilloscope, and published procedures (Finkel & Redman, 1985) were followed to set capacitance compensation correctly while maximizing the sampling rate. Following intracellular recording in every experiment, the microelectrodes were tested for polarization artifacts in DCC by using the same switching rate to pass current up to the maximum amplitude used intracellularly. The chord conductances (OF and Gs) associated with the fast- and slow-transient outward currents, respectively, were calculated by dividing the peak fast or slow current measured at each membrane potential by the driving potential for potassium ions assuming potassium equilibrium potential was -100 mv in 3 mm-extracellular potassium. The maximum conductance increases could not be determined because the recording microelectrodes polarized during the passage of large currents at the fast switching rates required for voltage control during large depolarizations. Conductances at fixed membrane potentials could not be compared among cells because a given voltage step evoked larger transient outward currents in cells having larger ohmic 'leakage' conductances. To compare chord conductances among cells we divided peak chord conductance by the ohmic leakage conductance (GL) measured in the same cell. This procedure resulted in plots of peak normalized chord conductance (GF/aL and GS/GL) v8. membrane potential that were similar among the recorded cells. Leakage conductance varied from to 0'159,uS in the cells used for this analysis. Membrane current and potential were recorded on a multichannel FM tape-recorder (frequency response: DC to 5 khz) or on a video cassette-recorder with pulse code modulation (two-channel sampling rate: 44 khz). Recorded data were played back on a storage oscilloscope for analysis and photography or digitized for further analysis by computer. Measurements of resting potentials were made by comparing intracellular with extracellular DC levels recorded on a strip chart-recorder. Linear leakage and capacitative currents were measured during small voltage steps from a holding potential at or near resting potential. In most voltage-clamp records shown here, an analog device was used to scale these currents according to the imposed voltage step and to subtract them from 593

4 594 W. J. SPAIN, P. C SCHWIVDT AlND W. E. CRILL the total membrane current records before photography. Measured parameters are reported as means + S.D. RESUTLTS Data were gathered from 117 neurones from twenty-five cats. Several properties of cells recorded in normal perfusate are listed in Table 1. During constant current stimulation these cells exhibited the responses described previously for large layer V neurones ('Betz cells') from cat sensorimotor cortex in vitro (Stafstrom, Schwindt & Crill, 1984 a: Stafstrom et al. 1984b). As described in the following paper (Spain et al. 1991), intracellular staining identified cells with similar electrical properties as large pyramidal neurones from layer V. Identification of two transient outward currents Figure 1A and B shows typical outward currents evoked by depolarizing voltage steps from -70 mv in the presence of tetrodotoxin (TTX). Two transient outward currents could be distinguished by their strikingly different time course of decay. The fast-transient current peaked and decayed within 20 ms following each voltage step (Fig. 1A). Figure 1B shows the slow-transient current evoked by the same voltage steps. The slow decay of this current is most apparent during the largest two steps. In this cell, the slow-transient current reached a peak (marked by arrow) about 67 ms following a voltage step. At the slow sweep speed of Fig. 1B, the fast-transient current is visible only as the brief initial upward deflection marked by the horizontal arrow-head. The records of Fig. 1 C and D compare voltage-clamp and current-clamp records in order to relate transient current activation to action potential threshold and cell behaviour. The bottom part of each panel shows the membrane response to a constant intracellular current pulse that was injected into the cell before TTX application. This membrane response is superimposed on voltage steps imposed on the same cell after TTX application. The outward currents evoked by the voltage steps are shown in the top part of each panel. Both the fast-transient current (Fig. 1 C) and the slow-transient current (Fig. 1D) were activated by depolarizations subthreshold for action potential initiation. In each cell tested, the fast-transient current was evoked by a smaller depolarization than required to evoke the slowtransient current (7 mv smaller on average). Because the transient currents are active below action potential threshold, they can influence membrane potential behaviour and cell excitability in the subthreshold region. The record of Fig. 1 C, for example, shows a membrane potential trajectory frequently seen in Betz cells during injection of a near-rheobase current pulse. Membrane potential peaks, sags and rises again to trigger the first action potential. The initial rise of membrane potential was terminated at potentials where the fasttransient current was activated during voltage clamp. In the cell of Fig. 1D the rise of membrane potential towards action potential threshold also was interrupted, but this longer-lasting interruption started at the more positive potential where the slowtransient current was first activated. (The fast-transient current cannot be seen at the slow time base used in Fig. 1 D). Currents similar to those in Fig. 1 were seen in other cells examined after calcium currents were blocked in EGTA-containing, calcium-free perfusate alone (n = 8; cf.

5 TRANSIENT POTASSIUM CURRENTS IN BETZ CELLS 595 A 50 mv 2 na B 10 ms 200 ms c D 10 mv 14 na 200 nmns /; v,, ~~~~~~100Ms1 Fig. 1. Two transient outward currents and their relation to membrane potential trajectory during constant current stimulation. A, superimposed traces of fast-transient outward current (upper) evoked by voltage steps (lower) in the presence of tetrodotoxin (TTX). Holding potential -70 mv. Step potentials -50, -46, -41 and -37 mv. B, same records at 10 times slow-sweep speed showing the slow-transient outward current evoked by each voltage step. Arrow-head points to initial fast-transient current; arrow points to peak slow-transient current. C, lower: superimposed records of the membrane potential response evoked by an injected constant current pulse (not shown) before TTX application and voltage steps imposed after TTX application. Part of the voltage steps were removed after photography for better visibility of the membrane potential response. Spikes are truncated. Holding potential -70 mv. Step potentials -54, 49 and -44 mv. C, upper: fast transient currents evoked by the voltage steps after TTX application. D, similar to C but taken from another cell at times slower sweep speed. Only the slowtransient current is visible; it is first evoked at a voltage corresponding to the slow rise of membrane potential to threshold during constant current stimulation. Spikes truncated and retouched. Holding potential -70 mv. Step potentials -55, -50 and -45 mv. Calibration (100 ms) applies to membrane potential response; 200 ms applies to ionic currents and voltage steps. TABLE 1. Representative properties of recorded cells Property Mean + S.D. n Resting potential (mv) Spike height (mv) Spike duration* (ms) Input resistancet (MQ) * Measured at action potential threshold. t Measured at 200 ms during injection of a -1 na current pulse. Fig. 3) or combined with EGTA in the recording microelectrode (n = 1; cf. Fig. 2), or by the addition of 400,tM-cadmium (n = 8), or the substitution of cobalt for calcium (n = 15). Both transient currents deactivated too rapidly for tail currents to be resolved clearly using single-electrode voltage clamp. Thus, we were unable to examine

6 596 W. J. SPAIN, P. C SCHWINDT AND W. E. CRILL reversal potentials directly in either normal or raised extracellular potassium. However, when extracellular potassium was raised from 3 to 9 mm, the amplitudes of both currents were reduced. Current amplitude, voltage dependence and time course were not altered during prolonged impalements with KCl-containing A ^> B 14nA Control - K1 mm-te +10 mm-tea ms 200 ms Fig. 2. Effect of tetraethylammonium (TEA) on transient outward currents. All data from the same cell impaled with an EGTA-containing microelectrode in the presence of tetrodotoxin and calcium-free perfusate. A, fast-transient current (upper) evoked by a voltage step (lower) in calcium-free perfusate (control) and after addition of 1 and 10 mm- TEA. In this and following figures the numbers on the voltage traces indicate membrane potential in mv. B, same records as A at 20 times slower sweep speed showing effect of TEA on the slow-transient current. Fast-transient current is not visible at this sweep speed. microelectrodes, suggesting no chloride dependence, and the currents were reduced by pharmacological agents known to reduce potassium currents as described below. Transient currents differ in sensitivity to tetraethylammonium and 4-aminopyridine These experiments were carried out in solutions expected to block calcium currents (see Methods). Typical results with tetraethylammonium (TEA) are shown in Fig. 2. The records in Fig. 2A show that the fast-transient current was greatly reduced by 1 mm-tea (average 60%, n = 8). This underestimates the true reduction because the fast-transient current peaks during the onset of the slow-transient current, so that some outward current would remain at the time of its peak even if it were blocked completely. A significant decrease of the slow-transient current did not occur until TEA was raised to 10 mm (Fig. 2B, + 10 mm-tea). Among the cells tested, a TEA concentration of 1 mm had little effect on the slow-transient current (n = 5), whereas 5 and 10 mm reduced the current by 37 % (n = 5) and 52 % (n = 2), respectively, and 50 mm resulted in a 77 % reduction (n = 2, data not shown). The reduction of the slow-transient current by TEA was fully reversible, and the time course of the slow-transient current was not altered (data not shown). Typical results with 4-aminopyridine (4-AP) are shown in Fig. 3. The slowtransient current was reduced 38 % by 20,pM-4-AP (n = 3), about 50 % by doses in the range ,aM (n = 8) and 65 % by 2 mm-4-ap (n = 4). The effects of 4-AP were usually irreversible over the time that cell impalement could be maintained. 4- Aminopyridine caused the slow-transient current to decay more rapidly to a steady value (Fig. 3B). In addition, the percentage reduction at large depolarizations was less than at small depolarizations (data not shown), as if the blocking action of 4-AP were partially relieved by depolarization. 4-Aminopyridine had little effect on the fast-transient current at the concentrations employed (20 /tm-2 mm; n = 8).

7 TRANSIENT POTASSIUM CURRENTS IN BETZ CELLS 597 Activation and inactivation of the slow transient current We took advantage of an earlier finding that cobalt slowly blocks the fasttransient current (Schwindt et al b) to study the activation of the slow-transient current in fifteen cells. Figure 4A shows the rapid initial rise of the slow-transient A 0+ Control B mm-4-ap \ _ 4nA 8ms I4nA 2s Control =+0.5 mm-4-ap r_+2 mm-4-ap -39 _ Fig. 3. Effects of 4-aminopyridine on fast- and slow-transient outward currents. A, superimposed records of fast-transient current (upper) evoked by a voltage step (lower) in calcium-free perfusate (control) and after the addition of 0 5 mm-4-aminopyridine (4- AP). B, superimposed records of slow-transient current (upper) evoked by a voltage step (lower) in calcium-free perfusate (control) and after the addition of 0 5 and 2 mm-4-ap. Fast transient current is not visible at this sweep speed. current. It attained 70 % of its peak value at ms (n = 15). This time was not obviously voltage dependent. Figure 4B shows relations between normalized chord conductance (see Methods) and membrane potential derived from nineteen cells in which calcium influx was blocked by various means. The conductance began to activate at about -50 mv and increased steeply between -30 and -20 mv. The limitations of the single-electrode voltage clamp precluded measurements at more depolarized levels. Inactivation of the slow-transient current was incomplete even after 10 s of maintained depolarization (data not shown). The decay of the current was well fitted by two exponential functions of time constants r1 = ms and T2 = s (n = 20). Neither r1 nor r2was obviously voltage dependent over the range -45 to -5 mv. Recovery from inactivation was much quicker than inactivation. In eight cells, inactivation at -40 mv was removed completely during 1 s repolarizing voltage steps negative to -50 mv. About 75% of the inactivation was removed within 250 ms. This early recovery time course was well fitted by one exponential function, and was more rapid at -90 mv than at -50 mv (data not shown). The voltage dependence of inactivation was determined as shown in Fig. 4C. Peak slow-transient current was measured at -40 mv following a series of 1 s hyperpolarizing pre-pulses. A cation current (Ih) was activated during hyperpolarizing pre-pulses (Fig. 4C, trace a), but its deactivation resulted in minimal contamination of the slow-transient current since -40 mv is near Ih reversal potential (Spain et al. 1987). The steady-state inactivation parameter (h.j) for the slow-transient current was calculated by dividing current amplitude following each

8 ~~~~~~~~~~~~ W. J. SPAIN, P. C. SCHWINDT AND W. E. CRILL A B - g/==== =3 t O ~~ na _ ms Membrane potential (mv) C D a, i-o~~~~~1. 12 Peak >e _ ~~~~ ~0.4 -' 50 mv 1 na 4 a 0.2 -s 2-40 mv oo 0 -- ~ 500 ms Membrane potential (mv) Fig. 4. Activation and inactivation of the slow-transient current. Data of A, C and D were obtained after abolition of fast-transient current by substitution of cobalt for calcium. A, superimposed traces showing fast onset of slow-transient current at different membrane potentials. B, relations between normalized peak chord conductance (GS/GL) and membrane potential measured in nineteen cells with calcium influx blocked by various means. C, records showing increased amplitude of slow-transient curreilt (upper) as 1 s hyperpolarizing pre-pulses (lower) were made more negative, indicating removal of the inactivation existing at -40 mv. Current segments labelled 'a' correspond. D, plots of steady-state inactivation (h.*, fitted with sigmoidal curve as explained in the text) and current measured at peak and at 1 s during voltage steps from -70 mv in the same cell. pre-pulse by current amplitude following the most negative pre-pulse. A typical plot of ho, against conditioning pre-pulse potential is shown in Fig. 4D. The data points were fitted by the sigmoidal relation, ha, = [I+exp((V-E)/k)]-', where V is membrane potential, E is the membrane potential where the current is half-inactivated and k is a slope factor. For the slow-transient current, E was mv and k was mv (n = 21). Comparison of the h. relation with the current-voltage relations obtained in the same cell indicated that there was a region of membrane potential (e.g. between -50 and -40 mv in Fig. 4D) where the current would be persistently activated.

9 TRANSIENT POTASSIUM CURRENTS IN BETZ CELLS 599 Activation and inactivation of the fast transient current The fast-transient current peaked at ms following voltage steps to -50 mv and at ms following steps to -35 mv (n = 25). Subtraction of records obtained before and after substitution of cobalt for calcium (not shown) A 5.0 B 4.0 -i ,, 60&A 50 mv 2 na.6._. 1.0 t_#: Membrane potential (mv) 5 ms C S8 00t.a oa Membrane potential (mv) Fig. 5. Activation and inactivation of fast-transient current. A, normalized peak chord conductance (GF/GL) at various membrane potentials in eight cells. B, increase in amplitude of fast-transient current (upper) as 1 s hyperpolarizing pre-pulses (lower) were made more negative, indicating removal of inactivation at -40 mv. C, plots of steadystate inactivation (ho, fitted with sigmoidal curve as explained in the text) and peak current during voltage steps from -70 mv in the same cell. indicated that the rapid advancement of the peak current with depolarization was not a consequence of its superposition on the rising phase of the slow-transient current. Thus, the two transients currents differed both in the time taken to reach peak amplitude and the voltage dependence of this process. Figure 5A shows the relation between normalized chord conductance and membrane potential derived from eight cells. The conductance starts to activate near -60 mv. There is a suggestion that the conductance increase begins to saturate positive to -30 mv. Figure 5B shows how the amplitude of the fast-transient current at -40 mv c C) a.

10 600 W. J. SPAIN, P. C. SCHWINDT AND W. E. CRILL increased as preceding hyperpolarizing pre-pulses were made more negative. Such records were used to determine the voltage dependence of inactivation (Fig. 5C, h.). Half-inactivation occurred at mv with a slope factor of mv (n = 14). Thus, half-inactivation of the fast-transient current occured about 6 mv more A B C 120 mv 02 ms +1 mm-tea 5 mv NL +1 mm-tea 2+ ;\ +0.2 mm-4-ap 0 Ca2+ 5ms Fig. 6. Effect of blocking agents on action potential repolarization. Action potentials in A and C were evoked by 3 ms injected current pulses (not shown). Records labelled 0 Ca2+ were taken min after changing to calcium-free perfusate. Horizontal lines indicate resting potentials. A, superimposed action potentials evoked in normal perfusate (NL), in calcium-free perfusate (0 Ca2+) and after the addition of 1 mm-tea. B, records from another cell showing the abolition by 1 mm-tea of the interruption (at arrow-head) of the rise of membrane potential to action potential threshold (action potentials are truncated). Current pulse (lower) was 2 na. C, superimposed action potentials from another cell evoked in normal and calcium-free perfusate and after the addition of 200 ftm-4- aminopyridine (4-AP). Calibrations in A also apply to C. positive than for the slow-transient current, and inactivation was more steeply dependent on membrane potential. Figure 5 C also shows the current-voltage relation measured for the peak fast-transient current in the same cell. As with the slow-transient current, an overlap of the two curves was seen in each cell tested (n = 14). The voltage range of the window region (about 14 mv) was similar for both transient currents, and the window region of each current extended to potentials below action potential threshold. Experiments employing depolarizing pre-pulses of variable duration showed that the time course of decay of the fast-transient current reflected its inactivation time course (data not shown). Thus, the decay time course was taken as the time course of inactivation, and it could be fitted by a single-exponential function. The relation between inactivation time constant and depolarization was obtained in six cells by subtracting the currents measured after the elimination of the fast-transient current in cobalt-substituted perfusate from control records taken at the same membrane potential. Exponential functions were then fitted to the decay of these difference currents. Inactivation time constants range from ms near -45 mv to ms near -10 mv. Thus, the fast-transient current also differed from the slow-transient current in displaying a clear voltage dependence of its inactivation kinetics. Recovery from inactivation was much slower than inactivation. Between -78 and -40 mv, recovery was described by a single-exponential function having time constants ranging from 203 to 648 ms (n = 4, data not shown).

11 TRANSIENT POTASSIUM CURRENTS IN BETZ CELLS 601 Both transient currents contribute to action potential repolarization We used selective pharmacological blockade of the fast- and slow-transient currents by low concentrations of TEA and 4-AP, respectively, to evaluate the contribution of each current to action potential repolarization. It was important to ensure that the agents (particularly TEA) did not alter action potential repolarization by altering calcium-mediated potassium currents. Blockade of calcium currents using cadmium-containing (n = 6) or calcium-free perfusate (n = 11) was signalled by the abolition of evoked synaptic potentials and calcium-dependent after-hyperpolarizations (data not shown). The effect of calcium current blockade on action potential repolarization in these cells ranged from no measurable effect (Fig. 6 C) to that of Fig. 6A, which was the largest effect observed. These records also illustrate our finding that the effect of calcium current blockade on spike repolarization, though always small, increased in proportion to the duration of the action potential (range: ms) in normal perfusate. After calcium currents were blocked, the effects of 4-AP and TEA were evaluated. The typical action of 1 mm-tea is shown in Fig. 6A. Action potential duration (measured at threshold) increased by an average of 29-5 % in nine cells tested. Above, we attributed the interruption in the rise of membrane potential to action potential threshold to activation of the fast-transient current (cf. Fig. 1 C). Figure 6B shows that this interruption was abolished by 1 mm-tea. Figure 6C shows the typical effect of 4-AP on action potential repolarization. It produced a large effect at 200 JM, but increasing the dose to 2 mm had little additional effect (data not shown). Spike duration increased by an average of 129 % in twelve cells tested using 4-AP concentrations in the range 200 /tm-1 mm. The effects of TEA, but not 4-AP, were fully reversible after min of wash-out (not shown). DISCUSSION We have characterized two voltage-gated potassium currents in Betz cells. Although both currents were transient, their activation and inactivation properties differed both in kinetics and voltage dependence. One of our goals was to discover pharmacological agents and doses that preferentially affect only one of the transient currents so that its functions could be inferred from altered membrane responses during constant current stimulation. The two currents were insensitive to blockade of calcium influx, but they differed in sensitivity to tetraethylammonium, 4- aminopyridine and cobalt. These results suggest that each current arises from a distinctive set of voltage-gated potassium channels. Comparison to other potassium currents The transient currents of Betz cells do not fit neatly into traditional categories of A-current or delayed rectifier, but of the two currents, the slow-transient current most resembled a delayed rectifier. Its inactivation kinetics were similar to those of the delayed rectifier of molluscan neurones (Aldrich, Getting & Thompson, 1979; Ruben & Thompson, 1984) to the extent that inactivation was very slow, while recovery from inactivation was rapid and consisted of two phases. The slow-transient

12 602 W. J. SPAIN, P. C. SCHWINDT AND W. E. CRILL current did not display the cumulative inactivation phenomenon seen in molluscan neurones during repetitive voltage steps (Aldrich et al. 1979). Similar to the slowtransient current of Betz cells, the delayed rectifier current of axons displays a much greater sensitivity to 4-AP than TEA (Ulbricht & Wagner, 1976; Yeh, Oxford, Wu & Narahashi, 1976; Meves & Pichon, 1977). Potassium currents exhibiting rapid onset, slow decay and greater sensitivity to 4- AP than TEA have been identified in a variety of neuronal somata (eg. Gestrelius & Grampp, 1983; Kasai, Kameyama & Fukuda, 1986; Penner, Petersen, Pierau & Dreyer, 1986; Stansfeld, Marsh, Halliwell & Brown, 1986; Dekin & Getting, 1987; Quandt, 1988). These currents appear to differ from each other and from the slowtransient current of Betz cells in the details of their pharmacology and voltage dependence. The slow-transient potassium currents identified in hippocampal pyramidal neurones (Gustafsson, Galvan, Grafe & Wigstrom, 1982; Storm, 1988) display fast onset and slow inactivation, but they also differ from Betz cells in details of voltage dependence and pharmacology. They inactivate completely at -60 mv, they are much more sensitive to 4-AP, and they are insensitive to TEA. Recently, however, oocyte expression of a gene cloned from rat brain resulted in a potassium current that resembled the slow-transient current of Betz cells in time course, voltage dependence and relative sensitivity to 4-AP and TEA (Christie, Adelman, Douglass & North, 1989). This diversity may be explained by the recent findings that transient potassium currents differing in voltage dependence, kinetics and 4-AP sensitivity can arise by three different mechanisms: expression of different genes (Solc, Zagotta & Aldrich, 1987), alternative processing of a gene product (Iverson, Tanouye, Lester, Davidson & Rudy, 1988) and interaction of gene products (Rudy, Hoger, Lester & Davidson, 1988). Thus, a particular type of neurone may be able to express potassium channels that are optimal for its function but differ to some extent from those in other neurones. According to this idea, the slow- and fast-transient currents described here are simply the Betz cell's version of transient potassium currents; they need not correspond precisely to transient currents in other neurones. High concentrations of 4-AP block transient outward currents in neocortical neurones (Zona, Pirrone, Avoli & Dichter, 1988; Woody, Baranyi, Szente, Gruen, Holmes, Nenov & Strecker, 1989). We found, however, that concentrations of 4-AP up to 2 mm had little effect on the fast-transient current, while concentrations above 20 fm caused complex effects on the slow-transient current. 4-Aminopyridine alters inactivation kinetics and causes time- and voltage-dependent channel blockade in both neuronal somata and axons (Ulbricht & Wagner, 1976; Yeh et al. 1976; Meves & Pichon, 1977; Hermann & Gorman, 1981; Thompson, 1982). Thus, 4-AP may have altered the time course of the slow-transient current by these mechanisms. It is also possible that 4-AP preferentially blocked a subset of slowly inactivating channels, revealing that at least two channel types contribute to the slow-transient current. Additional tests are needed to substantiate the latter interpretation. Although potassium currents similar to the slow-transient current of Betz cells have been observed in other cells, the fast-transient current appears to be novel. In its time course, the fast-transient current resembles the fast-transient calciumdependent potassium currents observed in several cells (Siegelbaum & Tsien, 1980;

13 TRANSIENT POTASSIUM CURRENTS IN BETZ CELLS MacDermott & Weight, 1982; Salkoff, 1983; Zbicz & Weight, 1985; Callewaert, Vereecke & Carmeliet, 1986; Bourque, 1988). The fast-transient current of Betz cells, however, was not reduced after the addition of cadmium or in calcium-free perfusate. It is unlikely that its activation is enabled by resting levels of internal calcium as the current was present during long impalement with an EGTA-containing microelectrode. Thus, our observations do not support the idea that the fast-transient current of Betz cells is calcium mediated. The large reduction of the fast-transient current produced by 1 mm-tea and the concomitant effect on action potential repolarization are noteworthy. Up to now, only the fast, calcium-mediated potassium current that flows through largeconductance (BK) channels was thought to be so sensitive to TEA (Blatz & Magleby, 1987). A peculiar property of the fast-transient current is its slow abolition after substitution of cobalt for calcium. The failure of calcium-free perfusate to reproduce this behaviour negates our earlier hypothesis that the fast-transient current channel requires external calcium (Schwindt et al a). Several divalent cations reduce A- current by shifting its activation curve to the right (Galvan & Sedlmeir, 1984; Junge, 1985; Mayer & Sugiyama, 1988; Tsuda, Oyama, Carpenter & Akaike, 1988). This mechanism does not fully explain the reduction of the fast-transient current by cobalt since suface charge effects should occur when the perfusate contacts the membrane rather than tens of minutes later. We think the effect is caused by a slow entry of cobalt into the cell and selective blockade of the fast-transient current from the inside for the following reasons: (i) direct intracellular injection of cobalt into neocortical neurones (Baranyi, Szente & Woody, 1988) caused alteration of the action potential virtually identical to prolonged perfusion with cobalt-substituted perfusate (Schwindt et al a); (ii) recovery was incomplete even after 1 h of washout (W. J. Spain & P. C. Schwindt, unpublished observations), and (iii) reduction of transient potassium currents by internal divalent cations has been observed (Alkon & Shoukimas, 1982; Galvan & Sedlmeir, 1984; Tsuda et al. 1988). We found that larger leakage conductances were associated with larger transient currents. Dividing transient chord conductance by leakage conductance resulted in similar conductance-voltage relations from cell to cell, a result that is understandable if cell size is the common factor underlying the variation of both leakage conductance and transient current amplitude. The ohmic leakage conductance measured during voltage clamp of the soma is an input conductance. Current flows both through open channels in the soma and into the linear electrical network provided by the nonisopotential dendritic tree (Rall, 1977). All else being equal, a larger neurone is expected to have a larger leakage conductance simply because it has a larger membrane area. Membrane time constant (the product of specific membrane resistance and capacitance) is narrowly distributed among Betz cells having a wide range of input conductances (Stafstrom et al. 1984b). Thus, a larger input conductance must result predominantly from a larger membrane area. A correlation between input conductance and size has been observed directly in these cells (Spain et al. 1991). If the potassium channels underlying each transient current have a similar spatial density distribution among cells, a larger cell would contain more channels and generate a larger current. 603

14 604 W. J. SPAIN, P. C. SCHWINDT AND W. E. CRILL Functions of the transient potassium currents Rapid repolarization of the action potential is very important in Betz cell function. Interfering with rapid repolarization can cause epileptiform bursts of action potentials or prolonged depolarizations because inward ionic currents then dominate cell behaviour (Stafstrom, Schwindt, Chubb & Crill, 1985; Schwindt et al. 1988a). Both transient currents seemed likely to be important in action potential repolarization because of their fast onset. Previous pharmacological results supported this idea (Schwindt et al. 1988a), but the differential sensitivity of the transient currents to TEA and 4-AP allowed a more precise evaluation of their contribution. The present results were valuable in demonstrating the specific contribution of the slow-transient current, as our earlier results might have been due predominantly to blockade of the fast-transient current. The concentrations of TEA and 4-AP used should have reduced either transient current to a similar extent. The greater prolongation of the action potential seen with 4-AP suggests that the slow-transient current normally plays a larger role in action potential repolarization. We found that calcium current blockade had little or no effect on the action potential of Betz cells. This finding suggests that calcium-mediated potassium currents of Betz cells turn on too slowly to have much influence on action potential repolarization. Calcium-mediated potassium currents measured during voltage clamp exhibited relatively slow kinetics (Schwindt et al. 1988a, b). These slow currents would be expected to play a greater role the longer membrane potential remained depolarized. Our finding that calcium blockade had more effect on the repolarization of longer duration spikes is consistent with this idea. Similar to our results in Betz cells, no evidence of a fast calcium-mediated potassium current (Ic) was obtained in a recent study of neurones of olfactory cortex (Constanti & Sim, 1987). Evidently, the idea derived from studies of hippocampal neurones (Lancaster & Nicoll, 1987; Storm, 1987) that Ic provides the principle mechanism of action potential repolarization does not apply to all mammalian cortical neurones. Both transient currents were evoked by depolarizations below action potential threshold. In addition, our results suggest that both currents are present as steady 'window' currents below action potential threshold. Our measurements underestimate the window region because our 'activation curve' is based on peak current during which some inactivation has already occurred. Thus, both transient currents can influence the integrative properties of the neurone in the subthreshold region, e.g. by influencing the time course of EPSPs or the rise of membrane potential to action potential threshold. The latter effect was demonstrated directly in this study, but the kinetics and voltage dependence of the two currents, particularly the slowtransient current, suggested that they could play a wider role in governing Betz cell excitability. The influence of the slow-transient current on repetitive firing patterns in Betz cells is described in the following paper (Spain et al. 1991). We thank Sue Blaylock, Karen Chin and Todd Cunnington for assistance with manuscript preparation. Gregg Hinz provided excellent technical support. This work was funded by NINCDS grants NS (W.J.S.), NS and NS (W.E.C. and P.C.S.).

15 TRANASIE.NT POTASSIUM CURRENTS IN BETZ CELLS 605 REFERENCES Al,DRICH, R. W. JR, G(ETTING, P. A. & THOMPSON, S. H. (1979). Inactivation of delayed outward current in molluscan neurone somata. Journal of Physiology 291, ALKON, D. L. & SHOUJKIMAS, J. J. (1982). Calcium-mediated decrease of a voltage-dependent potassium current. Biophysics Journal 40, BARANY, A., SZENTE, M. B. & WOODY, C. D. (1988). Characterization of membrane currents found in electrophysiologically identified neurons in cat motor cortex. Society for Neuroscience Abstracts 14, 298. BLATZ, A. L. & MAGLEBY, K. L. (1987). Calcium-activated potassium channels. Trends in Neurosciences 10, BOURQUE, C. W. (1988). Transient calcium-dependent potassium current in magnocellular neurosecretory cells of the rat supraoptic nucleus. Journal of Physiology 397, CALLEWAERT, G., VEREECKE, J. & CARMELIET, E. (1986). Existence of a calcium-dependent potassium channel in the membrane of cow cardiac Purkinje cells. Pflugers Archiv 406, CHRISTIE, M. J., ADELMAN, J. P., DOUGLASS, J. & NORTH, R. A. (1989). Expression of a cloned rat brain potassium channel in Xenopus oocytes. Science 244, CONNOR, J. A. & STEVENS, C. F. (1971). Voltage-clamp studies of a transient outward membrane current in gastropod neural somata. Journal of Physiology 213, CONSTANTI, A. & SIM, J. A. (1987). Calcium-dependent potassium conductance in guinea-pig olfactory cortex neurones in vitro. Journal of Physiology 387, DEKIN, M. S. & GETTING, P. A. (1987). In vitro characterization of neurons in the ventral part of the nucleus tractus solitarius. II. Ionic mechanisms responsible for repetitive firing activity. Journal of Neurophysiology 58, FINKEL, A. S. & REDMAN, S. J. (1985). Optimal voltage clamping with single microelectrodes. In Voltage and Patch Clamping with Microelectrodes, ed. SMITH, T. G. JR, LECAR, H., REDMOND, S. J. & GAGE, P. W., pp American Physiological Society, Bethesda, MD, USA. FOEHRING, R. C., SCHWINDT, P. C. & CRILL, W. E. (1989). Norepinephrine selectively reduces slow calcium- and sodium-mediated potassium currents in cat neocortical neurons. Journal of Neurophysiology 61, GALVAN, M. & SEDLMEIR, C. (1984). Outward currents in voltage-clamped rat sympathetic neurones. Journal of Physiology 356, GESTRELIUS, S. & GRAMPP, W. (1983). Kinetics of the TEA and 4-AP sensitive potassium current in the slowly adapting lobster stretch receptor neurone. Acta Physiologica Scandinavica 188, GUSTAFSSON, B., GALVAN, M., GRAFE, P. & WIGSTROM, H. (1982). A transient outward current in a mammalian central neurone blocked by 4-aminopyridine. Nature 299, HAGIWARA, S., KUSANO, K. & SAITO, N. (1961). Membrane changes of Onchidium nerve cell in potassium-rich media. Journal of Physiology 155, HERMANN, A. & GORMAN, A. L. F. (1981). Effects of 4-aminopyridine on potassium currents in a molluscan neuron. Journal of General Physiology 78, HODGKIN, A. L. & HUXLEY, A. F. (1952). A quantitative description of membrane current and its application to conduction and excitation in nerve. Journal of Physiology 117, IVERSON, L. E., TANOUYE, M. A., LESTER, H. A., DAVIDSON, N. & RUDY, B. (1988). A-type potassium channels expressed from Shaker locus cdna. Proceedings of the National Academy of Sciences of the USA 85, JUNGE, D. (1985). Calcium dependence of A-currents in perfused Aplysia neurons. Brain Research 346, KASAI, H., KAMEYAMA, K. & FUKUDA, J. (1986). Single transient K channels in mammalian sensory neurons. Biophysics Journal 49, LANCASTER, B. & NICOLL, R. A. (1987). Properties of two calcium-activated hyperpolarizations in rat hippocampal neurones. Journal of Physiology 389, MAcDERMOTT, A. B. & WEIGHT, F. F. (1982). Action potential repolarization may involve a transient, calcium-sensitive outward current in a vertebrate neurone. Nature 300, MAYER, M. L. & SUGIYAMA, K. (1988). A modulatory action of divalent cations on transient outward current in cultured rat sensory neurones. Journal of Physiology 396,

16 606 W. J. SPAIN, P. C. SCHWINDT AND W7. E. CRILL MEVES, H. & PICHON, Y. (1977). The effect of internal and external 4-aminopyridine on potassium currents in intracellularly perfused squid giant axons. Journal of Physiology 268, NEHER, E. (1971). Two fast-transient current components during voltage-clamp on snail neurons. Journal of General Physiology 58, PENNER, R., PETERSEN, M., PIERAU, F.-K. & DREYER, F. (1986). Dendrotoxin: a selective blocker of a non-inactivating potassium current in guinea-pig dorsal root ganglion neurones. IPfiuigers Archiv 407, QUANDT, F. N. (1988). Three kinetically distinct potassium channels in mouse neuroblastoma cells. Journal of Physiology 395, RALL, W. (1977). Core conductor theory and cable properties of neurons. In Handbook of Physiology, vol. 1, section 1, ed. KANDEL, E. R., pp American Physiological Society, Bethesda, MD, USA. RUBEN, P. & THOMPSON, S. (1984). Rapid recovery from K current inactivation on membrane hyperpolarization in molluscan neurons. Journal of General Physiology 84, RUDY, B. (1988). D)iversity and ubiquity of K channels. Neuroscience 25, RUDY, B., HOGER, J. H., LESTER, H. A. & DAVIDSON, N. (1988). At least two mrna species contribute to the properties of rat brain A-type potassium channels expressed in Xenopus oocytes. Neuron 1, SALKOFF, L. (1983). Drosophila mutants reveal two components of fast outward current. Nature 302, SCHWINDT, P. C., SPAIN, W. J. & CRILL, W. E. (1989). Long-lasting reduction of excitability by a sodium-dependent potassium current in cat neocortical neurons. Journal of Neurophysiology 61, SCHWINDT, P. C., SPAIN, W. J., FOEHRING, R. C., CHUBB, M. C. & CRILL, W. E. (1988a). Slow conductances in neurons from cat sensorimotor cortex in vitro and their role in slow excitability changes. Journal of Neurophysiology 59, SCHWINDT, P. C., SPAIN, W. J., FOEHRING, R. C., STAFSTROM, C. E., CHUBB, M. C. & CRILL, W. E. (1988b). Multiple potasssium conductances and their functions in neurons from cat sensorimotor cortex in vitro. Journal of Neurophysiology 59, SIEGELBAUM, S. A. & TSIEN, R. W. (1980). Calcium-activated transient outward current in calf cardiac Purkinje fibres. Journal of Physiology 299, Soi,c, C. K., ZAGOTTA, W. & ALDRICH, R. W. (1987). Single-channel and genetic analyses reveal two distinct A-type potassium channels in Drosophila. Science 236, SPAIN, W. J., SCHWINDT, P. C. & CRILL, W. E. (1987). Anomalous rectification in neurons from cat sensorimotor cortex in vitro. Journal of Neurophysiology 57, SPAIN, W. J., SCIIWINDT, P. C. & CRILL, W. E. (1991). Post-inhibitory excitation and inhibition in layer V pyramidal neurones from cat sensorimotor cortex. Journal of Physiology 434, STAFSTROM, C. E., SCHWINDT, P. C., CHUBB, M. C. & CRILL, W. E. (1985). Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensorimotor cortex in vitro. Journal of Neurophysiology 53, STAFSTROM, C. E., SCHWINDT, P. C. & CRILL, W. E. (1984a). Repetitive firing in layer V neurons from cat neocortex in vitro. Journal of Neurophysiology 52, STAFSTROM, C. E., SCHWINDT, P. C., FLATMAN, J. A. & CRILL, W. E. (1984b). Properties of subthreshold response and action potential recorded in layer V neurons from cat sensorimotor cortex in vitro. Journal of Neurophysiology 52, STANSFELD, C. E., MARSH, S. J., HALLIWELL, J. V. & BROWN, D. A. (1986). 4-Aminopyridine and dendrotoxin induce repetitive firing in rat visceral sensory neurones by blocking a slowly inactivating outward current. Neuroscience Letters 64, STORM, J. F. (1987). Action potential repolarization and a fast after-hyperpolarization in rat hippocampal pyramidal cells. Journal of Physiology 385, STORM, J. F. (1988). Temporal integration by a slowly inactivating potassium current in hippocampal neurons. Nature 336, THOMPSON, S. (1982). Aminopyridine block of transient potassium current. Journal of General Physiology 80, TSUDA, Y., OYAMA, Y., CARPENTER, D. 0. & AKAIKE, N. (1988). Effects of calcium on the transient outward current of single isolated Helix central neurones. British Journal of Pharmacology 95,