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Voltage-clamp analysis of a transient potassium current in rat neostriatal neurons
Brain Research, 473 (1988) 187-192 Elsevier
187
BRE 23191
Voltage-clamp analysis of a transient potassium current in rat neostriatal neurons D. James Surmeier, Jose Bargas and S.T. Kitai Department of Anatomy and Neurobiology, Collegeof Medicine, The Universityof Tennessee, Memphis, TN38163 (U.S.A.) (Accepted 26 July 1988)
Key words: A-current; Neostriatum; Voltage-clamp;Cell culture
Whole cell voltage-clamp recordings were made from cultured rat neostriatal neurons. Depolarizing voltage commands evoked transient and sustained outward K-currents. The transient K-current was activated by depolarizing commands beyond -50 mV; peak current was dependent upon holding potential. Bath application of 4-aminopyridine, but not inorganic calcium channel blockers (Cd, Co, Mn), attenuated the transient current. Reversal was near the K-equilibrium potential. These properties suggest that this transient K-current is similar to the A-current described in a number of other neurons.
Vertebrate neurons are endowed with a rich complement of ionic conductances that regulate their integrative processes. One of the potassium conductances that plays a prominent role in spike repolarization 5'33, patterning u'12'32 and the response to synaptic input 7'9 is the transient A-current. Originally described by Hagiwara et al. 17 and later by Connor and Stevens 1° and Neher 27 in invertebrates, the A-current has been reported to be present in a number of vertebrate n e u r o n s 1,6"13,23,28,30,31,32.37. Based upon current-clamp experiments, Bargas et al. 4 recently have suggested that the A-current is present in rat neostriatal neurons. Aside from its importance to a general model of neostriatal cellular physiology, the existence of an A-current in neostriatal neurons may be crucial to understand the actions of dopamine and other neuromodulators 2,3,8,2°,26. However, current-clamp experiments cannot provide definitive evidence for the presence or absence of this conductance. Our goal was to determine, using whole-cell voltage clamp techniques, whether an A-like conductance is, in fact, expressed by rat neostriatal neurons. Neostriatal cultures were established and main-
tained as described previously34. Briefly, neurons were derived from El7 rat embryos (Sprague-Dawley). Striata were dissected in cold, calcium- and magnesium-free Hank's balanced salt solution (pH 7.4). Dissections were limited to the dorsal portion of the striatal eminence in an attempt to limit cultures to neostriatum; although this procedure does not ensure histological homogeneity, it does guarantee that the contribution of pallidum to our cultures was small. Dissected striata were minced, resuspended in serum-supplemented basal medium, and then dissociated using a graded series of fire-polished pipettes. The suspension was then plated onto poly-cationtreated glass coverslips at a density of 500-2000 viable cells per square mm. The basal medium consisted of a Ham's F12: Dulbecco's modified Eagle's medium mixture (1:1) supplemented with sodium bicarbonate (1.2 g/liter), hydroxyethylpiperazine-n-2-ethane sulfonic acid (HEPES, 3.5745 g/liter), L-glutamine (0.365 g/liter), insulin (5/ag/ml), streptomyocin (50 /ag/ml) and penicillin (50 U/ml). Cells were plated in basal medium supplemented with 10% fetal calf serum. After 4 days in vitro (DIV), cells were treated with cytosine arabinoside (5/aM) to halt glial prolifer-
Correspondence: D.J. Surmeier, Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee, Memphis, TN 38163, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
188 ation and switched to a basal medium supplemented with 5% horse serum, 5% fetal calf serum.
Whole cell recordings from neostriatal neurons were made using conventional techniques TMat room +2,5 mV
.110 mV
+25 mV
-40 mV
B
J -70 mV
50 1
C
f...
D 0.8
::I / -80
-60
-40
-20
0
20
40
VOLTAGE (mV) Fig. 1. Activation characteristics of the transient outward current. A: outward currents evoked by depolarizing voltage commands from -60 to +25 mV when preceded by a 150 ms conditioning pulse of -110 inV. B: currents evoked by the same depolarizing commands when preceded by a conditioning pulse of -40 mV. C: the difference currents resulting from the subtraction of B from A: D: plot of the peak conductances estimated from Fig. 1 as a function of voltage, assuming the current was carried exclusively by potassium. The fit of the Boltzmann equation shown by the smooth line used a least squares criterion (see text). Records were corrected for linear leak currents; DC input resistance (Rn) = 5.3 G ~ , whole cell capacitance (C,) = 9 pF. The cell from which the data were derived was of ovoid type Ia 33 and had been maintained for 7 days in vitro (DIV).
189 temperature (20-23 °C). A low volume (200 /A) recording chamber was mounted on the stage of an inverted microscope fitted with Hoffman optics. The chamber was perfused at approximately 0.2 ml/min in most experiments. The normal bath solution had the following ionic composition (in mM): 120 NaCI, 6 KCI, 2 CaC12, 1 MgCI, 10 N a - H E P E S , 6 glucose; pH was brought to 7.4 with HC1. Na currents were blocked by the inclusion of 1 - 1 0 / ~ M tetrodotoxin (TTX). Electrodes were pulled from Corning 8161 glass, coated with Sylgard (Dow Corning 184) and fire-polished immediately prior to use. The normal ionic composition of the electrode solution was (in mM): 98 K-gluconate, 20 tetraethylammonium-C1
(TEA-CI), 10 H E P E S , 2 MgCI 2, 2 A T P , 0.2 GTP, 5 E G T A , pH = 7.2 (with KOH). T E A was included after preliminary experiments indicated that 20 mM significantly reduced the sustained component of the outward current while leaving the transient component relatively intact 35'36. Electrode D C impedance was normally 2 - 5 MQ. Whole cell recordings were made with an Axopatch (Axon Instruments) electrometer; data acquisition and analysis were performed with the assistance of a microcomputer. Although our electrode impedances and current amplitudes suggested that voltage command errors rarely exceeded a few mV, series resistance compensation (60-80%) was used in most experiments. Electro-
-30 mV
-35 mV
L
-70 mV I
B
1.0
m
0.8
E
o w
\
0.6
o z
I-
o D a z 0 o
0.4
o.2
0.0
\
w
-120
-I00
-80
-60
-40
-20
PRE-PULSE V O L T A G E ( m V )
Fig. 2. Inactivation characteristics of the transient outward current. A: outward currents evoked by a test pulse of-30 mV when preceded by 150 ms conditioning pulses of from - 110 to -35 mV. A subset of the current traces are shown for clarity. B: plot of the peak conductances evoked by the test pulse as a function of conditioning pulse voltage, assuming the current was carried exclusively by potassium. The smooth curve was a least squares fit of the Boltzmann equation described in the text. Records were corrected for linear leak currents. Data were derived from the cell of Fig. 1.
19() tonic analysis of the neuritic tree using an adaptation of the voltage-clamp procedure of Ral129 suggested that, in general, neurons of less than 3 - 4 weeks of age were relatively compact (L <0.4) under our recording conditions. The activation characteristics of the transient outward current were studied by applying depolarizing pulses from -6(1 mV to +30 mV following a 150-300 ms conditioning pulse o f - 1 1 0 inV. The currents generated by this protocol were then subtracted from those produced by the same depolarizing pulses preceded by a conditioning pulse o f - 4 0 mV. This procedure allowed the separation of that portion of the outward current that was inactivated by holding at depolarizing potentials ~'>'23'>'~2"35"36. In Fig. 1 A - C , the whole cell currents evoked in an exemPlary neuron by these two protocols and the difference currents are shown. Conductances estimated from the peak difference currents, assuming a reversal potential equal to that of potassium (see below), were well fit by a Boltzmann equation: G/Gma x = 1/(1 + exp(-(V - Vh)/Vc) (Fig. ID); for the data of Fig. 1C, Vh was -33.3 mV and the slope factor, Vc, was 9.1 mV. Most neurons exhibited Vhs between -45 mV and - 3 0 mV, as in Fig. 1; the remainder of our sample had VhS greater than - 2 0 mV. Inactivation curves for these neurons were commensurately shifted in the depolarizing direction. Slope factors (Vc) for the two subsets were not significantly different (P > 0.05. t-test). A more complete characterization of these differences is underway and will be the subiect of a subsequent report. The steady-state inactivation characteristics of the transient current were determined by measuring the peak current in response to a test pulse o f - 3 0 mV when preceded by 150-300 ms conditioning pulses of - 110 to -35 mV (Fig. 2A). As for the example in Fig. 2B, these data were generally well-fit by a Boltzmann equation: G/Gma x = 1/(1 + e x p ( ( V - Vh)/Vc)); in this example, V h was -80.9 mV a n d Vc was 8.0 inV. A hallmark of the A-current in other neurons is its relative sensitivity to 4-aminopyridine (4-AP) 16'35'36. The transient K current identified by the subtraction procedure outlined above was substantially reduced by bath application of 2 - 5 mM 4-AP, while it was not substantially altered by 20 mM T E A . In Fig. 3, the transient currents derived by the subtraction proce-
A
B
4-AP I
C
I
IAIA
C~LI
III I
I
I
roll
m0oAI
Fig. 3. Effects of 4-AP on the transient outward current. A: difference currents produced by depolarizing voltage commands
from -60 to +10 mV when preceded by -110 and -40 mV conditioning pulses of 150 ms. See Fig. 1 for the voltage protocol. 13: currents produced by the same protocol 2 min after the introduction of 2 mM 4-AP. C: recovery of currents 3 min after the beginning of washing with control medium. Records were corrected for linear leak currents; R, = 2.08 Gf2, C~ = 13 pF. The neuron was of tripolar type I133 and had been maintained for 29 DIV.
dure before (A), during (B), and after washing (C) 4A P application are shown. Qualitatively similar results were obtained in all neurons studied (n = 6). These currents were not blocked by bath application of the inorganic calcium channel blockers C d (up to 500pM; n = 8), Co (3 mM; n = 4), Mn ( 2 m M ; n = 4) or by partial chloride substitution (two thirds with isethionate). To determine the principal ion carrying the transient current, tail currents produced by stepping from a - 3 0 mV test pulse to more negative potentials were studied. Tail currents always reversed within a few millivolts of the computed K-equilibrium potential (n = 6). The reversal potentials for CI, Na and Ca were sufficiently positive that had the channels mediating the current possessed a significant permeability to any of these ions the deviation of the reversal potential from the potassium equilibrium potential should have been apparent. Plots of the tail current amplitude as a function of c o m m a n d potential were invariably well fit by linear regression lines, indicating the channels underlying the transient current were behaving ohmically between -120 and - 3 0 inV. Taken together, these results strongly suggest that the transient current described here can be identified with the A - c u r r e n t 1'6'13'15'16'19'23"25"27'28'3°'31'32'37. Three lines of evidence are consistent with this infer-
191 ence. First, the v o l t a g e d e p e n d e n c e and kinetics of
sensitivity of the c u r r e n t to 4 - A P and its r e l a t i v e re-
the c o n d u c t a n c e s in m o s t n e u r o n s b e a r a s t r o n g re-
sistance to calcium c h a n n e l b l o c k e r s and T E A clearly
s e m b l a n c e to t h o s e of A - c u r r e n t d e s c r i b e d in o t h e r m a m m a l i a n n e u r o n s 6A6"23"25"26"28'31'32'37. G i v e n t h e
d i f f e r e n t i a t e it f r o m t h e C a - d e p e n d e n t o u t w a r d currents6,14,15,16.19.22,24.28,31,35.37,
discharge characteristics of striatal n e u r o n s 21, t h e s e p r o p e r t i e s suggest that it m a y play a role in spike rep o l a r i z a t i o n 5,33, p a t t e r n i n g 11'12'32 and the r e s p o n s e to
W e wish to t h a n k Drs. V. D i o n n e , M. W h i t e and
synaptic input 7'9. S e c o n d , the p r i m a c y of K-ions in
E. M c C l e s k e y for their h e l p in bringing the p a t c h
carrying the c u r r e n t and its o h m i c b e h a v i o r are consistent with p r e v i o u s descriptions 23,32. Lastly, the
clamp t e c h n i q u e to o u r group. This w o r k was sup-
1 Adams, P.R., Brown, D.A. and Constanti, A., M-currents and other potassium currents in bullfrog sympathetic ganglion neurons, J. Physiol. (Lond.), 330 (1982) 537-572. 2 Aghajanian, G.K., Modulation of a transient outward current in serotonergic neurones by alpha-adrenoceptors, Nature (Lond.), 315 (1985) 501-503. 3 Akaike, A., Ohno, Y., Sasa, M. and Takaori, S., Excitatory and inhibitory effects of dopamine on neuronal activity of the caudate neuron in vitro, Brain Research, 418 (1987) 262-272. 4 Bargas, J., Galarraga, E. and Aceves, J., An early outward conductance modulates the firing latency and frequency of neostriatal neurons of the rat brain, Exp. Brain Res., in press. 5 Beluzzi, O. and Sacchi, O., The interactions between potassium and sodium currents in generating action potentials in rat sympathetic neurons, J. Physiol. (Lond.), 397 (1988) 127-147. 6 Beluzzi, O., Sacchi, O. and Wanke, E., A fast transient outward current in the rat sympathetic neuron studied under voltage-clamp conditions, J. Physiol. (Lond.), 358 (1985) 91-108. 7 Byrne, J.H., Quantitative aspects of ionic conductance mechanisms contributing to firing pattern of motor cells mediating inking behavior in Aplysia californica, J. Neurophysiol., 43 (1980) 651-668. 8 Calabresi, P., Mercuri, N., Stanzione, P., Stefani, A. and Bernardi, G., Intracellular studies on the dopamine-induced firing inhibition of neostriatal neurons in vitro: evidence for D1 receptor involvement, Neuroscience, 20 (1987) 757-771. 9 Cassell, J.F. and McLachlan, E.M., The effect of a transient outward current (IA) on synaptic potentials in sympathetic ganglion cells of the guinea-pig, J. Physiol. (Lond.), 374 (1986) 273-288. 10 Connor, J.A. and Stevens, C.F., Voltage-clamp studies of a transient outward membrane current in gastropod neural somata, J. Physiol. (Lond.), 213 (1971) 21-30. 11 Connor, J.A. and Stevens, C.F., Prediction of repetitive firing behavior from voltage-clamp data on an isolated neurone soma, J. Physiol. (Lond.), 213 (1971) 31-53. 12 Connor, J.A., Neural pacemakers and rhythmicity, Annu. Rev. Physiol., 47 (1985) 17-28. 13 Cooper, E. and Shrier, A., Single-channel analysis of fast transient potassium currents from rat nodose neurones, J. Physiol. (Lond.), 369 (1985) 199-208. 14 Galarraga, E., Bargas, J., Sierra, A. and Aceves, J., The role of calcium in the repetitive firing of neostriatal neu-
rons, Exp. Brain Res., in press. 15 Galvan, M. and Sedlmeir, C., Outward currents in voltageclamped rat sympathetic neurones, J. Physiol. (Lond.), 356 (1984) 115-133. 16 Gustafsson, B., Galvan, M., Grafe, P. and Wigstrom, H., A transient outward current in a mammalian central neurone blocked by 4-aminopyridine, Nature (Lond.), 299 (1982) 252-254. 17 Hagiwara, S., Kusano, K. and Saito, N., Membrane changes of Onchidium nerve cell in potassium-rich media, J. Physiol. (Lond.), 155 (1961) 470-489. 18 Hamill, O.P., Many, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 19 Johansen, J. and Kleinhaus, A.L., Transient and delayed potassium currents in the Retzius cell of the leech, Macrobdella decora, J. Neurophysiol., 56 (1986) 812-822. 20 Kaczmarek, L.K. and Strumwasser, F., A voltage-clamp analysis of currents underlying cyclic AMP-induced membrane modulation in isolated peptidergic neurons of Aplysia, J. Neurophysiol., 52 (1984) 340-349. 21 Kita, H., Kita, T. and Kitai, S.T., Active membrane properties of rat neostriatal neurons in an in vitro slice preparation, Exp. Brain Res., 60 (1985) 54-62. 22 Kita, T., Kita, H. and Kitai, S.T., Effects of 4-aminopyridine on rat neostriatal neurons in an in vitro slice preparation, Brain Research, 361 (1985) 10-18. 23 Kostyuk, P.G., Veselovsky, N.S., Fedulova, S.A. and Tsyndrenko, A.Y., Ionic currents in the somatic membrane of rat dorsal root ganglion neurons. III. Potassium currents, Neuroscience, 6 (1981) 2439-2444. 24 MacDermott, A. and Weight, F.F., Action potential repolarization may involve a transient, Ca-sensitive outward current in a vertebrate neuron, Nature (Lond.), 300 (1982) 185-188. 25 Mayer, M.L. and Sugiyama, K., A modulatory action of divalent cations on transient outward current in cultured rat sensory neurones, J. Physiol. (Lond.), 396 (1988) 417-433. 26 Nakajima, Y., Nakajima, S., Leonard, R.J. and Yamaguchi, K., Acetylcholine raises the excitability by inhibiting the fast transient potassium current in cultured hippocampal neurones, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 3022-3026. 27 Neher, E., Two fast transient current components during voltage-clamp of snail neurons, J. Gen. Physiol., 58 (1971) 36-53. 28 Numann, R.E., Wadman, W.J. and Wong, R.K.S., Out-
p o r t e d by U . S . P . H . S . G r a n t s NS20702 and NS23886.
192
29
30 31
32
33
ward currents of single hippocampal cells obtained from the adult guinea pig, J. Physiol. (Lond.), 393 (1987) 331-353. Rail, W., Time constants and electrotonic length of membrane cylinders and neurons, Biophys. J., 9 (1969) 1483- 15118. Rogawski. M.A., The A-current: how ubiquitous a feature of excitable cells is it'?, Trends Neurosci., 5 (1985) 214-219. Segal, M. and Barker, J.L., Rat hippocampal neurons in culture: potassium conductances, J. Neurophysiol., 51 (1984) 1409-1433. Segal, M., Rogawski, M.A. and Barker, J.L., A transient potassium conductance regulates the excitability of cultured hippocampal and spinal neurons, J. Neurosci., 4 (1984) 6(/4-6119. Storm, J., Actio~ potential repolarization and a fast after-
34
35 36
37
hyperpolarization in rat hippocampal pyramidal cells, ./. Physiol. (Lond.), 385 (1987) 733- 759. Surmeier, D.J., Kita, H. and Kitai, S.T., The expression of gamma-aminobutyric acid and leu-enkephalin immunoreactivity in primary monolayer cultures of rat striatum, Dev. Brain Res., 42 (1988) 265-282. Thompson, S.. Aminopyridine block of transient t3otassium current, J. Gen. Physiol., 811 (1982) 1-18. Thompson, S.H., Three pharmacologically distinct potassium channels in molluscan neurones, J. Physiol. (Lond.). 265 (1977) 465-488. Zbicz, K.L. and Weight, F.F., Transient voltage and calcium-dependent outward currents in hippocampal CA3 neurons, J. Neurophysiol., 53 (1985) 1/138-1058.
187
BRE 23191
Voltage-clamp analysis of a transient potassium current in rat neostriatal neurons D. James Surmeier, Jose Bargas and S.T. Kitai Department of Anatomy and Neurobiology, Collegeof Medicine, The Universityof Tennessee, Memphis, TN38163 (U.S.A.) (Accepted 26 July 1988)
Key words: A-current; Neostriatum; Voltage-clamp;Cell culture
Whole cell voltage-clamp recordings were made from cultured rat neostriatal neurons. Depolarizing voltage commands evoked transient and sustained outward K-currents. The transient K-current was activated by depolarizing commands beyond -50 mV; peak current was dependent upon holding potential. Bath application of 4-aminopyridine, but not inorganic calcium channel blockers (Cd, Co, Mn), attenuated the transient current. Reversal was near the K-equilibrium potential. These properties suggest that this transient K-current is similar to the A-current described in a number of other neurons.
Vertebrate neurons are endowed with a rich complement of ionic conductances that regulate their integrative processes. One of the potassium conductances that plays a prominent role in spike repolarization 5'33, patterning u'12'32 and the response to synaptic input 7'9 is the transient A-current. Originally described by Hagiwara et al. 17 and later by Connor and Stevens 1° and Neher 27 in invertebrates, the A-current has been reported to be present in a number of vertebrate n e u r o n s 1,6"13,23,28,30,31,32.37. Based upon current-clamp experiments, Bargas et al. 4 recently have suggested that the A-current is present in rat neostriatal neurons. Aside from its importance to a general model of neostriatal cellular physiology, the existence of an A-current in neostriatal neurons may be crucial to understand the actions of dopamine and other neuromodulators 2,3,8,2°,26. However, current-clamp experiments cannot provide definitive evidence for the presence or absence of this conductance. Our goal was to determine, using whole-cell voltage clamp techniques, whether an A-like conductance is, in fact, expressed by rat neostriatal neurons. Neostriatal cultures were established and main-
tained as described previously34. Briefly, neurons were derived from El7 rat embryos (Sprague-Dawley). Striata were dissected in cold, calcium- and magnesium-free Hank's balanced salt solution (pH 7.4). Dissections were limited to the dorsal portion of the striatal eminence in an attempt to limit cultures to neostriatum; although this procedure does not ensure histological homogeneity, it does guarantee that the contribution of pallidum to our cultures was small. Dissected striata were minced, resuspended in serum-supplemented basal medium, and then dissociated using a graded series of fire-polished pipettes. The suspension was then plated onto poly-cationtreated glass coverslips at a density of 500-2000 viable cells per square mm. The basal medium consisted of a Ham's F12: Dulbecco's modified Eagle's medium mixture (1:1) supplemented with sodium bicarbonate (1.2 g/liter), hydroxyethylpiperazine-n-2-ethane sulfonic acid (HEPES, 3.5745 g/liter), L-glutamine (0.365 g/liter), insulin (5/ag/ml), streptomyocin (50 /ag/ml) and penicillin (50 U/ml). Cells were plated in basal medium supplemented with 10% fetal calf serum. After 4 days in vitro (DIV), cells were treated with cytosine arabinoside (5/aM) to halt glial prolifer-
Correspondence: D.J. Surmeier, Department of Anatomy and Neurobiology, College of Medicine, The University of Tennessee, Memphis, TN 38163, U.S.A. 0006-8993/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
188 ation and switched to a basal medium supplemented with 5% horse serum, 5% fetal calf serum.
Whole cell recordings from neostriatal neurons were made using conventional techniques TMat room +2,5 mV
.110 mV
+25 mV
-40 mV
B
J -70 mV
50 1
C
f...
D 0.8
::I / -80
-60
-40
-20
0
20
40
VOLTAGE (mV) Fig. 1. Activation characteristics of the transient outward current. A: outward currents evoked by depolarizing voltage commands from -60 to +25 mV when preceded by a 150 ms conditioning pulse of -110 inV. B: currents evoked by the same depolarizing commands when preceded by a conditioning pulse of -40 mV. C: the difference currents resulting from the subtraction of B from A: D: plot of the peak conductances estimated from Fig. 1 as a function of voltage, assuming the current was carried exclusively by potassium. The fit of the Boltzmann equation shown by the smooth line used a least squares criterion (see text). Records were corrected for linear leak currents; DC input resistance (Rn) = 5.3 G ~ , whole cell capacitance (C,) = 9 pF. The cell from which the data were derived was of ovoid type Ia 33 and had been maintained for 7 days in vitro (DIV).
189 temperature (20-23 °C). A low volume (200 /A) recording chamber was mounted on the stage of an inverted microscope fitted with Hoffman optics. The chamber was perfused at approximately 0.2 ml/min in most experiments. The normal bath solution had the following ionic composition (in mM): 120 NaCI, 6 KCI, 2 CaC12, 1 MgCI, 10 N a - H E P E S , 6 glucose; pH was brought to 7.4 with HC1. Na currents were blocked by the inclusion of 1 - 1 0 / ~ M tetrodotoxin (TTX). Electrodes were pulled from Corning 8161 glass, coated with Sylgard (Dow Corning 184) and fire-polished immediately prior to use. The normal ionic composition of the electrode solution was (in mM): 98 K-gluconate, 20 tetraethylammonium-C1
(TEA-CI), 10 H E P E S , 2 MgCI 2, 2 A T P , 0.2 GTP, 5 E G T A , pH = 7.2 (with KOH). T E A was included after preliminary experiments indicated that 20 mM significantly reduced the sustained component of the outward current while leaving the transient component relatively intact 35'36. Electrode D C impedance was normally 2 - 5 MQ. Whole cell recordings were made with an Axopatch (Axon Instruments) electrometer; data acquisition and analysis were performed with the assistance of a microcomputer. Although our electrode impedances and current amplitudes suggested that voltage command errors rarely exceeded a few mV, series resistance compensation (60-80%) was used in most experiments. Electro-
-30 mV
-35 mV
L
-70 mV I
B
1.0
m
0.8
E
o w
\
0.6
o z
I-
o D a z 0 o
0.4
o.2
0.0
\
w
-120
-I00
-80
-60
-40
-20
PRE-PULSE V O L T A G E ( m V )
Fig. 2. Inactivation characteristics of the transient outward current. A: outward currents evoked by a test pulse of-30 mV when preceded by 150 ms conditioning pulses of from - 110 to -35 mV. A subset of the current traces are shown for clarity. B: plot of the peak conductances evoked by the test pulse as a function of conditioning pulse voltage, assuming the current was carried exclusively by potassium. The smooth curve was a least squares fit of the Boltzmann equation described in the text. Records were corrected for linear leak currents. Data were derived from the cell of Fig. 1.
19() tonic analysis of the neuritic tree using an adaptation of the voltage-clamp procedure of Ral129 suggested that, in general, neurons of less than 3 - 4 weeks of age were relatively compact (L <0.4) under our recording conditions. The activation characteristics of the transient outward current were studied by applying depolarizing pulses from -6(1 mV to +30 mV following a 150-300 ms conditioning pulse o f - 1 1 0 inV. The currents generated by this protocol were then subtracted from those produced by the same depolarizing pulses preceded by a conditioning pulse o f - 4 0 mV. This procedure allowed the separation of that portion of the outward current that was inactivated by holding at depolarizing potentials ~'>'23'>'~2"35"36. In Fig. 1 A - C , the whole cell currents evoked in an exemPlary neuron by these two protocols and the difference currents are shown. Conductances estimated from the peak difference currents, assuming a reversal potential equal to that of potassium (see below), were well fit by a Boltzmann equation: G/Gma x = 1/(1 + exp(-(V - Vh)/Vc) (Fig. ID); for the data of Fig. 1C, Vh was -33.3 mV and the slope factor, Vc, was 9.1 mV. Most neurons exhibited Vhs between -45 mV and - 3 0 mV, as in Fig. 1; the remainder of our sample had VhS greater than - 2 0 mV. Inactivation curves for these neurons were commensurately shifted in the depolarizing direction. Slope factors (Vc) for the two subsets were not significantly different (P > 0.05. t-test). A more complete characterization of these differences is underway and will be the subiect of a subsequent report. The steady-state inactivation characteristics of the transient current were determined by measuring the peak current in response to a test pulse o f - 3 0 mV when preceded by 150-300 ms conditioning pulses of - 110 to -35 mV (Fig. 2A). As for the example in Fig. 2B, these data were generally well-fit by a Boltzmann equation: G/Gma x = 1/(1 + e x p ( ( V - Vh)/Vc)); in this example, V h was -80.9 mV a n d Vc was 8.0 inV. A hallmark of the A-current in other neurons is its relative sensitivity to 4-aminopyridine (4-AP) 16'35'36. The transient K current identified by the subtraction procedure outlined above was substantially reduced by bath application of 2 - 5 mM 4-AP, while it was not substantially altered by 20 mM T E A . In Fig. 3, the transient currents derived by the subtraction proce-
A
B
4-AP I
C
I
IAIA
C~LI
III I
I
I
roll
m0oAI
Fig. 3. Effects of 4-AP on the transient outward current. A: difference currents produced by depolarizing voltage commands
from -60 to +10 mV when preceded by -110 and -40 mV conditioning pulses of 150 ms. See Fig. 1 for the voltage protocol. 13: currents produced by the same protocol 2 min after the introduction of 2 mM 4-AP. C: recovery of currents 3 min after the beginning of washing with control medium. Records were corrected for linear leak currents; R, = 2.08 Gf2, C~ = 13 pF. The neuron was of tripolar type I133 and had been maintained for 29 DIV.
dure before (A), during (B), and after washing (C) 4A P application are shown. Qualitatively similar results were obtained in all neurons studied (n = 6). These currents were not blocked by bath application of the inorganic calcium channel blockers C d (up to 500pM; n = 8), Co (3 mM; n = 4), Mn ( 2 m M ; n = 4) or by partial chloride substitution (two thirds with isethionate). To determine the principal ion carrying the transient current, tail currents produced by stepping from a - 3 0 mV test pulse to more negative potentials were studied. Tail currents always reversed within a few millivolts of the computed K-equilibrium potential (n = 6). The reversal potentials for CI, Na and Ca were sufficiently positive that had the channels mediating the current possessed a significant permeability to any of these ions the deviation of the reversal potential from the potassium equilibrium potential should have been apparent. Plots of the tail current amplitude as a function of c o m m a n d potential were invariably well fit by linear regression lines, indicating the channels underlying the transient current were behaving ohmically between -120 and - 3 0 inV. Taken together, these results strongly suggest that the transient current described here can be identified with the A - c u r r e n t 1'6'13'15'16'19'23"25"27'28'3°'31'32'37. Three lines of evidence are consistent with this infer-
191 ence. First, the v o l t a g e d e p e n d e n c e and kinetics of
sensitivity of the c u r r e n t to 4 - A P and its r e l a t i v e re-
the c o n d u c t a n c e s in m o s t n e u r o n s b e a r a s t r o n g re-
sistance to calcium c h a n n e l b l o c k e r s and T E A clearly
s e m b l a n c e to t h o s e of A - c u r r e n t d e s c r i b e d in o t h e r m a m m a l i a n n e u r o n s 6A6"23"25"26"28'31'32'37. G i v e n t h e
d i f f e r e n t i a t e it f r o m t h e C a - d e p e n d e n t o u t w a r d currents6,14,15,16.19.22,24.28,31,35.37,
discharge characteristics of striatal n e u r o n s 21, t h e s e p r o p e r t i e s suggest that it m a y play a role in spike rep o l a r i z a t i o n 5,33, p a t t e r n i n g 11'12'32 and the r e s p o n s e to
W e wish to t h a n k Drs. V. D i o n n e , M. W h i t e and
synaptic input 7'9. S e c o n d , the p r i m a c y of K-ions in
E. M c C l e s k e y for their h e l p in bringing the p a t c h
carrying the c u r r e n t and its o h m i c b e h a v i o r are consistent with p r e v i o u s descriptions 23,32. Lastly, the
clamp t e c h n i q u e to o u r group. This w o r k was sup-
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