- Email: [email protected]
Different action of ethosuximide on low- and high-threshold calcium currents in rat sensory neurons
Neurmcience Vol. 51, No. 4, pp. 755 758, 1992
0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd c 1992 1BRO
Printed in Great Britain
Letter to Neuroscience D I F F E R E N T A C T I O N OF E T H O S U X I M I D E ON LOW- A N D H I G H - T H R E S H O L D C A L C I U M C U R R E N T S IN RAT SENSORY N E U R O N S P. G. KOSTYUK, E. A. M O L O K A N O V A , N. F. PRONCHUK,A. N. SAVCHENKO* and A. N. VERKHRATSKY
Department of General Physiology of the Nervous System, A.A. Bogomoletz Institute of Physiology, Bogomoletz str. 4, Kiev-24, GSP 252601, Ukraine
The effects of ethosuximide on calcium channels were studied on dorsal root ganglion neurons from one-dayold rats using the patch-clamp technique. Bath application of ethosuximide induced dose-dependent and reversible suppression of calcium currents without affecting their time-course. Substantial differences between the effects of ethosuximide on the low-threshold and high-threshold (T- and L-) currents were observed. Ethosuximide reduced the T-current with greater potency than the L-current (Kd for T-current is 7 p M vs 15/~M for L-current). This relative specificity of its action still remained if applied at concentrations up to 1 mM. These data support the hypothesis according to which the anti-epileptic action of ethosuximide is related to reduction of the low-threshold calcium currents in sensory neurons. Ethosuximide is a succinimide derivative which has anticonvulsant action ~2and can be used against petit mal epilepsy/ Electrophysiologically, petit mal epilepsy is characterized by 3-Hz spike-wave rhythm in the electroencephalogram, the generation of which may be at least partly due to the activation of low-threshold inactivating calcium current in brain neurons] This hypothesis was confirmed by the results obtained on thalamic neurons,4'5 in which succinimides specifically depressed the low-threshold (T-type) calcium currents. The data obtained seem to be very important, as until now only a few substances were found with specific action on this type of calcium channels. Therefore, the effect of succinimides on different types of calcium channels should be studied in more detail. In the present work the dorsal root ganglion (DRG) neurons were used for
*To whom correspondence should be addressed. Abbreviations: DRG, dorsal root ganglion: DMEM, Dutbecco's modified Eagle's medium; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; HEPES, N-2hydroxyethylpiperazine-N-2-ethanesulphonic acid: L-current, high-threshold current; T-current, lowthreshold current.
this purpose. These cells possess several well-defined types of calcium currents 2'6't° that make them a very convenient object for studying various effects of calcium antagonists. Unidentified neurons from the lumbar DRGs of one-day-old rats were used for preparation of primary culture. Rats were killed by decapitation and DRGs were quickly removed and placed in Dulbecco's modified Eagle's medium (DMEM, Sigma, U.S.A.). After washout of blood the ganglia were transferred into a DMEM medium supplemented with 0.1% protease, type XIV (Sigma, U.S.A.). The ganglia were incubated in this medium at 3YC for 15-20min, and afterwards they were washed and pipetted in DMEM. Cell suspension was plated on flame and ultraviolet presterilized glass coverslips or on sterile plastic Petri dishes (Nunk, Denmark). After 1.5-2 h incubation of cell suspension in 5% CO2 + 95% air gas environment at 35' C, dishes were filled with 2ml of DMEM medium enriched by 10% embryonic calf serum (Vector, Ukraine). Experiments were carried out between day 3 and day 9 of culture. For performing the experiments, plastic dishes containing cultured cells in 1 ml of extracellular solution were mounted on the stage of an inverted phase-contrast microscope. Membrane currents in DRG neurons were studied by whole cell configuration patch-clamp technique. 8 Recordings were performed at room temperature. Patch pipettes were pulled from borosilicate glass (Hilgenberg, Germany) using the conventional two-step procedure. The pipettes had a resistance between 4 and 6 Mf~ when filled with intracellular solution (see below). Current and voltage signals were amplified by EPC-7 amplifier (List Electronic, Germany), filtered at 3 kHz (8 pole Bessel) and sampled at 5-10kHz by Labmaster interface (Axon Instruments, U.S.A.) connected to an AT-compatible computer system. The acquisition and analysis of data were controlled by pClamp software, version 5.5.1 (Axon Instruments,
755
756
P . G . KOSTYUK et al.
A
T type Ill
'c,~'c=r~
-30mV 2 min 90mv~ //,
I~------L__
30
min
0,8 0.6 0.4 0.2
10
20
40
L type
10
10mV .80 mV__F~" ~ 2 . ~ ~
'c./'. . . .
0.8 1 0,6 0.4 0.2 10
20
30
min
Fig. 1. Run-down of low-threshold (A) and high-threshold (B) calcium currents in DRG neurons. Currents were recorded in 2-min intervals after the establishment of the whole cell recording configuration and activated according to stimulation protocol shown in the inset. Peak currents (lcdlc, m~,) were normalized to the current measured after establishment of whole-cell recording configuration and complete washout of potassium currents and plotted as a function of recording time. U.S.A.). The computer system also served as a stimulus generator. To improve the voltage-clamp conditions, all experiments were done with capacitance and series resistance compensation. As a rule, at the beginning of the experiment, immediately after establishing a whole-cell recording configuration and before starting cell dialysis, the resting membrane potential of the neuron was determined in a current-clamp mode. If the resting potential was lower than - 3 0 m V , such a cell was discarded. All solutions were freshly prepared daily. Basic Tyrode solution contained (in mmol/l): NaC1 140; KCI 5.4; CaCI 2 1.8; MgCI 2 1.1; HEPES/NaOH 10; pH 7.4. The sodium-free extracellular solution: CaCI: 1.8; MgCI2 2; N-methyl-D-glucamine chloride 130; tetraethylammonium chloride 10; HEPES/Tris-OH 10; pH 7.4. The intracellular (pipette) solution: Csaspartate 60; CsCI 60; MgCI 2 4; HEPES/CsOH 10; Na2ATP 3; EGTA 10; pH 7.2. Ethosuximide (Sigma, U.S.A.) was bath applied to the cell in a concentration range from 1/z M to 1 mM.
Ethosuximide was readily soluble in the perfusion medium. The duration of application was 15 s. The replacement of solution near the tested cell after ethosuximide application was carried out by means of special suction pipette. Complete replacement with normal solution took 1 s. Previously it has been shown that calcium currents in D R G neurons can be separated into three subtypes differing in potential dependence, kinetic properties, selectivity and pharmacological sensitivity.26,~° On the basis of operational membrane potential range, calcium currents were classified as low- and highthreshold; the latter subsequently were also differentiated according to their behaviour during long-lasting membrane potential displacement--presence or absence of inactivation (T-, N- and L-currents, respectively, according to Tsien and coworkers' nomenclature). 6 In our experiments T-currents were evoked by depolarizing command to - 3 0 mV from a holding potential of - 90 mV. These transient, fully inactivating currents preserved their activity for a long time (Fig. IA). L-currents were evoked by a larger depolarizing command to 10 mV from holding potential - 80 mV; they remained active only for a short time (Fig. IB). N-currents were not specially analysed in the present experiments because of the difficulty of their separation in most of the neurons. We examined the action of ethosuximide on T- and L-currents in 18 neurons. Ethosuximide evoked a reduction of both types of calcium currents in all the neurons studied. However, the threshold concentration of ethosuximide needed for the effects to appear differed for T- and L-currents. The K d values obtained for corresponding Langmuir's isotherms were also different: 7 p M for T- and 15 # M for L-currents. Both currents could be suppressed completely by 1 mM ethosuximide. Lower concentrations of ethosuximide always induced a more substantial reduction of Tthan L-current (Fig. 2).
Iodl ....
1.0
08 06
~
T
~
~
.
04 02 -6 10
I I I ~11111 I -5 -4 10 10 ES concentration(M)
It,ll -3 10
Fig. 2. Dose-dependent effect of ethosuximide on T- (circles) and L- (triangles) calcium currents in DRG neurons (logarithmic scale). Circles: mean value of normalized peak currents obtained from four cells, vertical bars indicate S.E.M. Corresponding K, values are given in text.
757
Effects of ethosuximide on calcium currents ES
in quantitative agreement with those obtained on rat thalamic neurons.4'5 -7O It should be mentioned that effective ethosuximide -60 " -5O concentrations in the present study were even 100 pA I -4o % ~ . , . , ~ , ~ ' ~ * ~ lower than the clinically relevant free serum concentration of ethosuximide (25(~750#M)) 2 This difference can be explained by the fact that in intact brain the access of the drug to the neuronal membrane is difficult due to the blood-brain barrier, 200 P A l Therefore true concentration of ethosuximide near the cell may be lower than the free serum concentration. Ethosuximide application to dissociated cells at clinically relevant concentrations produced a nonspecific suppression of all types of 50 ms 50 ms calcium currents• In thalamic neurons, ethosuximide applied at concentrations of 600-1000yM also B I -II~i. -- 8 ........ evoked more often a reduction of high-threshold currents. 4 Except for the concentration range discussed -300 above, the differences between DRG and thalamic I(pA) neurons concerned the maximal effects of ethosux\ -600 \~ / / imide (40% depression in thalamic neurons against \ '~ Control 100% in the present study) and selectivity ofethosuximide action (ethosuximide reduced T-current, only I0 i i i i i -900 -6 -40 -20 0 20 40 rarely affecting L-current in thalamic neurons)? It is W (rnV) quite possible that the differences may be related to Fig. 3. Ethosuximide (ES) action ([0/~M) on calcium the investigation technique: thalamic neurons were currents in DRG neurons. (A) Representative family of studied in slices, and therefore the differences in calcium current records. Holding potential - 8 0 mV, 200-ms voltage-clamp pulses to the potential indicated near current voltage control between intact, less electrotonically traces• Left, control experiments; right, currents after ethosuximide application. (B) Peak current (Ira) to voltage (Vm} A T type relationship before (open squares) and after (filled squares) application of ethosuximide for a cell illustrated in A.
A
Control
12it/\ ..........
.
Reduction of both calcium currents by ethosuximide was not accompanied by any alteration of their time-courses (Fig. 3A). However, quantitative distinctions could be resolved: the amplitude of Tcurrent was much more affected as compared with that of L-current over the full activation potential range (Fig. 3B). The decrease of T-current amplitude induced by 10 y M ethosuximide was 1.8 2 times larger: the mean reductions of current amplitude were 91.4 + 6.2% and 45.4 + 4.8% (mean + S.E.M., N = 18) for T- and L-currents, respectively (Figs 2, 4). The development of the effects of ethosuximide in time is demonstrated in Fig, 4, The time-course of depression did not show substantial distinctions between T- and E-currents. The action of ethosuximide was fully reversible taking into account the run-down of calcium currents (cf. Fig. 1 without ethosuximide). Thus we found that in D R G neurons ethosuximide reduces T-current with greater potency (Kd for Tcurrent 7 y M vs 15pM for L-current) and higher efficacy than L-current (depression of T-current amplitude was several times higher). A relative specificity of ethosuximide action still remained if applied at concentrations up to l mM. The results about predominant influence of ethosuximide on T-current are
1.0
ES
ES
i1~11~.
i
0.8
I,
0.6
I,
0.4
'1 ~
0.2
i
•\
==
I 10
20
30
40
min
L type Ic,/ic. m~
1,0
.
ES
ES
m
m
0.8 0.6 0.4 0,2
I
10
I
20
i II~
30
min
Fig. 4. Run-down of T- (A) and L- (B) calcium currents in DRG neurons if ethosuximide applied at a concentration 10 #M, application is indicated by bars. Details as described in Fig. 1.
758
P.G. KOSTYUK et al.
c o m p a c t cells in slices a n d enzymatically treated acutely isolated n e u r o n s 3 could occur. Besides, it should be t a k e n into account that the amplitudes of T- a n d L-currents in D R G n e u r o n s were 1.5-2 times a n d five to six times higher, respectively, t h a n those in thalamic neurons. 3'~° Therefore, due to a more considerable suppression o f calcium currents by ethosuximide in D R G neurons, T-currents became equal in amplitude to those in thalamic cells. L-currents, even after some reduction induced by ethosuximide, h a d substantially higher values in D R G neurons t h a n those in thalamic n e u r o n s in control experiments.
We p e r l b r m e d all experiments using one-day-old rats. This age is characterized by specific composition of different types of calcium channels in neuronal m e m b r a n e : larger density of T-channels which later decreases during postnatal development. 9:t It would be o f interest to c o m p a r e the present data a b o u t the effect of ethosuximide on T- and L-currents obtained on n e o n a t a l neurons with its effect on cells from adult animals to find out if the different sensitivity of calcium channels to ethosuximide remains the same.
Acknowledgements--This research was partially supported by Bayer AG research grant to PK and AV. The authors thank L. Grigorovitch for excellent technical assistance.
REFERENCES
1. Browne T. R., Dreiuss F. E., Dyken P. R., Goode D. J., Penry J. K., White B. G. and White P. T. (1975) Ethosuximide in the treatment of absence (petit mal) seizures. Neurology 25, 515-524. 2. Carbone E. and Lux H. D. (1987) Kinetics and selectivity of a low-voltage-activated calcium current in chick and rat sensory neurones. J. Physiol., Lond. 386, 547-570. 3. Coulter D. A., Huguenard J. R. and Prince D. A. (1989) Calcium currents in rat thalamocortical relay neurones: kinetic properties of the transient, low-threshold currents. J. Physiol., Lond. 414, 587~04. 4. Coulter D. A., Huguenard J. R. and Prince D. A. (1989) Characterization of ethosuximide reduction of low-threshold calcium current in thalamic neurones. Ann. Neurol. 25, 582-593. 5. Coulter D. A., Huguenard J. R. and Prince D. A. (1990) Different effects of petit real anticonvulsants and convulsants on thalamic neurons: calcium current reduction. Br. J. Pharmac. 100, 800-806. 6. Fox A. P., Nowycky M. C. and Tsien R. W. (1987) Kinetic and pharmacological properties distinguishing three types of calcium currents in chick sensory neurons. J. Physiol., Lond. 394, 582-593. 7. Gloor P. and Fariello R. G. (1988) Generalized epilepsy: some of its cellular mechanisms differ from those of focal epilepsy. Trends Neurosei. 11, 63~58. 8. Hamill O. P., marty A., Neher E., Sakmann B. and Sigworth F. J. (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfliigers Arch. 391, 85-100. 9. Kostyuk P. G., Fedulova S. A. and Veselovsky N. S. (1986) Changes in ionic mechanisms of electrical excitability of the somatic membrane of rat dorsal root ganglion neurons during ontogenesis. Distribution of ionic channels of inward current. Neurophysiology, Kiev 18, 813-820. 10. Kostyuk P. G., Shuba Ya. M. and Savchenko A. N. (1988) Three types of calcium channels in the membrane of mouse sensory neurons. Pfliigers Arch. 411, 661~569. 11. Kostyuk P., Pronchuk N., Savchenko A. and Verkhratsky A. (1992) Calcium currents in rat aged dorsal root ganglion neurons. J. Physiol., Lond. (in press). 12. MacDonald R. L. and McLean M. J. (1986) Anticonvulsant drugs: mechanisms of action. Achr. Neurol. 44, 713 736.
(Accepted 7 September 1992)
0306-4522/92 $5.00 + 0.00 Pergamon Press Ltd c 1992 1BRO
Printed in Great Britain
Letter to Neuroscience D I F F E R E N T A C T I O N OF E T H O S U X I M I D E ON LOW- A N D H I G H - T H R E S H O L D C A L C I U M C U R R E N T S IN RAT SENSORY N E U R O N S P. G. KOSTYUK, E. A. M O L O K A N O V A , N. F. PRONCHUK,A. N. SAVCHENKO* and A. N. VERKHRATSKY
Department of General Physiology of the Nervous System, A.A. Bogomoletz Institute of Physiology, Bogomoletz str. 4, Kiev-24, GSP 252601, Ukraine
The effects of ethosuximide on calcium channels were studied on dorsal root ganglion neurons from one-dayold rats using the patch-clamp technique. Bath application of ethosuximide induced dose-dependent and reversible suppression of calcium currents without affecting their time-course. Substantial differences between the effects of ethosuximide on the low-threshold and high-threshold (T- and L-) currents were observed. Ethosuximide reduced the T-current with greater potency than the L-current (Kd for T-current is 7 p M vs 15/~M for L-current). This relative specificity of its action still remained if applied at concentrations up to 1 mM. These data support the hypothesis according to which the anti-epileptic action of ethosuximide is related to reduction of the low-threshold calcium currents in sensory neurons. Ethosuximide is a succinimide derivative which has anticonvulsant action ~2and can be used against petit mal epilepsy/ Electrophysiologically, petit mal epilepsy is characterized by 3-Hz spike-wave rhythm in the electroencephalogram, the generation of which may be at least partly due to the activation of low-threshold inactivating calcium current in brain neurons] This hypothesis was confirmed by the results obtained on thalamic neurons,4'5 in which succinimides specifically depressed the low-threshold (T-type) calcium currents. The data obtained seem to be very important, as until now only a few substances were found with specific action on this type of calcium channels. Therefore, the effect of succinimides on different types of calcium channels should be studied in more detail. In the present work the dorsal root ganglion (DRG) neurons were used for
*To whom correspondence should be addressed. Abbreviations: DRG, dorsal root ganglion: DMEM, Dutbecco's modified Eagle's medium; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; HEPES, N-2hydroxyethylpiperazine-N-2-ethanesulphonic acid: L-current, high-threshold current; T-current, lowthreshold current.
this purpose. These cells possess several well-defined types of calcium currents 2'6't° that make them a very convenient object for studying various effects of calcium antagonists. Unidentified neurons from the lumbar DRGs of one-day-old rats were used for preparation of primary culture. Rats were killed by decapitation and DRGs were quickly removed and placed in Dulbecco's modified Eagle's medium (DMEM, Sigma, U.S.A.). After washout of blood the ganglia were transferred into a DMEM medium supplemented with 0.1% protease, type XIV (Sigma, U.S.A.). The ganglia were incubated in this medium at 3YC for 15-20min, and afterwards they were washed and pipetted in DMEM. Cell suspension was plated on flame and ultraviolet presterilized glass coverslips or on sterile plastic Petri dishes (Nunk, Denmark). After 1.5-2 h incubation of cell suspension in 5% CO2 + 95% air gas environment at 35' C, dishes were filled with 2ml of DMEM medium enriched by 10% embryonic calf serum (Vector, Ukraine). Experiments were carried out between day 3 and day 9 of culture. For performing the experiments, plastic dishes containing cultured cells in 1 ml of extracellular solution were mounted on the stage of an inverted phase-contrast microscope. Membrane currents in DRG neurons were studied by whole cell configuration patch-clamp technique. 8 Recordings were performed at room temperature. Patch pipettes were pulled from borosilicate glass (Hilgenberg, Germany) using the conventional two-step procedure. The pipettes had a resistance between 4 and 6 Mf~ when filled with intracellular solution (see below). Current and voltage signals were amplified by EPC-7 amplifier (List Electronic, Germany), filtered at 3 kHz (8 pole Bessel) and sampled at 5-10kHz by Labmaster interface (Axon Instruments, U.S.A.) connected to an AT-compatible computer system. The acquisition and analysis of data were controlled by pClamp software, version 5.5.1 (Axon Instruments,
755
756
P . G . KOSTYUK et al.
A
T type Ill
'c,~'c=r~
-30mV 2 min 90mv~ //,
I~------L__
30
min
0,8 0.6 0.4 0.2
10
20
40
L type
10
10mV .80 mV__F~" ~ 2 . ~ ~
'c./'. . . .
0.8 1 0,6 0.4 0.2 10
20
30
min
Fig. 1. Run-down of low-threshold (A) and high-threshold (B) calcium currents in DRG neurons. Currents were recorded in 2-min intervals after the establishment of the whole cell recording configuration and activated according to stimulation protocol shown in the inset. Peak currents (lcdlc, m~,) were normalized to the current measured after establishment of whole-cell recording configuration and complete washout of potassium currents and plotted as a function of recording time. U.S.A.). The computer system also served as a stimulus generator. To improve the voltage-clamp conditions, all experiments were done with capacitance and series resistance compensation. As a rule, at the beginning of the experiment, immediately after establishing a whole-cell recording configuration and before starting cell dialysis, the resting membrane potential of the neuron was determined in a current-clamp mode. If the resting potential was lower than - 3 0 m V , such a cell was discarded. All solutions were freshly prepared daily. Basic Tyrode solution contained (in mmol/l): NaC1 140; KCI 5.4; CaCI 2 1.8; MgCI 2 1.1; HEPES/NaOH 10; pH 7.4. The sodium-free extracellular solution: CaCI: 1.8; MgCI2 2; N-methyl-D-glucamine chloride 130; tetraethylammonium chloride 10; HEPES/Tris-OH 10; pH 7.4. The intracellular (pipette) solution: Csaspartate 60; CsCI 60; MgCI 2 4; HEPES/CsOH 10; Na2ATP 3; EGTA 10; pH 7.2. Ethosuximide (Sigma, U.S.A.) was bath applied to the cell in a concentration range from 1/z M to 1 mM.
Ethosuximide was readily soluble in the perfusion medium. The duration of application was 15 s. The replacement of solution near the tested cell after ethosuximide application was carried out by means of special suction pipette. Complete replacement with normal solution took 1 s. Previously it has been shown that calcium currents in D R G neurons can be separated into three subtypes differing in potential dependence, kinetic properties, selectivity and pharmacological sensitivity.26,~° On the basis of operational membrane potential range, calcium currents were classified as low- and highthreshold; the latter subsequently were also differentiated according to their behaviour during long-lasting membrane potential displacement--presence or absence of inactivation (T-, N- and L-currents, respectively, according to Tsien and coworkers' nomenclature). 6 In our experiments T-currents were evoked by depolarizing command to - 3 0 mV from a holding potential of - 90 mV. These transient, fully inactivating currents preserved their activity for a long time (Fig. IA). L-currents were evoked by a larger depolarizing command to 10 mV from holding potential - 80 mV; they remained active only for a short time (Fig. IB). N-currents were not specially analysed in the present experiments because of the difficulty of their separation in most of the neurons. We examined the action of ethosuximide on T- and L-currents in 18 neurons. Ethosuximide evoked a reduction of both types of calcium currents in all the neurons studied. However, the threshold concentration of ethosuximide needed for the effects to appear differed for T- and L-currents. The K d values obtained for corresponding Langmuir's isotherms were also different: 7 p M for T- and 15 # M for L-currents. Both currents could be suppressed completely by 1 mM ethosuximide. Lower concentrations of ethosuximide always induced a more substantial reduction of Tthan L-current (Fig. 2).
Iodl ....
1.0
08 06
~
T
~
~
.
04 02 -6 10
I I I ~11111 I -5 -4 10 10 ES concentration(M)
It,ll -3 10
Fig. 2. Dose-dependent effect of ethosuximide on T- (circles) and L- (triangles) calcium currents in DRG neurons (logarithmic scale). Circles: mean value of normalized peak currents obtained from four cells, vertical bars indicate S.E.M. Corresponding K, values are given in text.
757
Effects of ethosuximide on calcium currents ES
in quantitative agreement with those obtained on rat thalamic neurons.4'5 -7O It should be mentioned that effective ethosuximide -60 " -5O concentrations in the present study were even 100 pA I -4o % ~ . , . , ~ , ~ ' ~ * ~ lower than the clinically relevant free serum concentration of ethosuximide (25(~750#M)) 2 This difference can be explained by the fact that in intact brain the access of the drug to the neuronal membrane is difficult due to the blood-brain barrier, 200 P A l Therefore true concentration of ethosuximide near the cell may be lower than the free serum concentration. Ethosuximide application to dissociated cells at clinically relevant concentrations produced a nonspecific suppression of all types of 50 ms 50 ms calcium currents• In thalamic neurons, ethosuximide applied at concentrations of 600-1000yM also B I -II~i. -- 8 ........ evoked more often a reduction of high-threshold currents. 4 Except for the concentration range discussed -300 above, the differences between DRG and thalamic I(pA) neurons concerned the maximal effects of ethosux\ -600 \~ / / imide (40% depression in thalamic neurons against \ '~ Control 100% in the present study) and selectivity ofethosuximide action (ethosuximide reduced T-current, only I0 i i i i i -900 -6 -40 -20 0 20 40 rarely affecting L-current in thalamic neurons)? It is W (rnV) quite possible that the differences may be related to Fig. 3. Ethosuximide (ES) action ([0/~M) on calcium the investigation technique: thalamic neurons were currents in DRG neurons. (A) Representative family of studied in slices, and therefore the differences in calcium current records. Holding potential - 8 0 mV, 200-ms voltage-clamp pulses to the potential indicated near current voltage control between intact, less electrotonically traces• Left, control experiments; right, currents after ethosuximide application. (B) Peak current (Ira) to voltage (Vm} A T type relationship before (open squares) and after (filled squares) application of ethosuximide for a cell illustrated in A.
A
Control
12it/\ ..........
.
Reduction of both calcium currents by ethosuximide was not accompanied by any alteration of their time-courses (Fig. 3A). However, quantitative distinctions could be resolved: the amplitude of Tcurrent was much more affected as compared with that of L-current over the full activation potential range (Fig. 3B). The decrease of T-current amplitude induced by 10 y M ethosuximide was 1.8 2 times larger: the mean reductions of current amplitude were 91.4 + 6.2% and 45.4 + 4.8% (mean + S.E.M., N = 18) for T- and L-currents, respectively (Figs 2, 4). The development of the effects of ethosuximide in time is demonstrated in Fig, 4, The time-course of depression did not show substantial distinctions between T- and E-currents. The action of ethosuximide was fully reversible taking into account the run-down of calcium currents (cf. Fig. 1 without ethosuximide). Thus we found that in D R G neurons ethosuximide reduces T-current with greater potency (Kd for Tcurrent 7 y M vs 15pM for L-current) and higher efficacy than L-current (depression of T-current amplitude was several times higher). A relative specificity of ethosuximide action still remained if applied at concentrations up to l mM. The results about predominant influence of ethosuximide on T-current are
1.0
ES
ES
i1~11~.
i
0.8
I,
0.6
I,
0.4
'1 ~
0.2
i
•\
==
I 10
20
30
40
min
L type Ic,/ic. m~
1,0
.
ES
ES
m
m
0.8 0.6 0.4 0,2
I
10
I
20
i II~
30
min
Fig. 4. Run-down of T- (A) and L- (B) calcium currents in DRG neurons if ethosuximide applied at a concentration 10 #M, application is indicated by bars. Details as described in Fig. 1.
758
P.G. KOSTYUK et al.
c o m p a c t cells in slices a n d enzymatically treated acutely isolated n e u r o n s 3 could occur. Besides, it should be t a k e n into account that the amplitudes of T- a n d L-currents in D R G n e u r o n s were 1.5-2 times a n d five to six times higher, respectively, t h a n those in thalamic neurons. 3'~° Therefore, due to a more considerable suppression o f calcium currents by ethosuximide in D R G neurons, T-currents became equal in amplitude to those in thalamic cells. L-currents, even after some reduction induced by ethosuximide, h a d substantially higher values in D R G neurons t h a n those in thalamic n e u r o n s in control experiments.
We p e r l b r m e d all experiments using one-day-old rats. This age is characterized by specific composition of different types of calcium channels in neuronal m e m b r a n e : larger density of T-channels which later decreases during postnatal development. 9:t It would be o f interest to c o m p a r e the present data a b o u t the effect of ethosuximide on T- and L-currents obtained on n e o n a t a l neurons with its effect on cells from adult animals to find out if the different sensitivity of calcium channels to ethosuximide remains the same.
Acknowledgements--This research was partially supported by Bayer AG research grant to PK and AV. The authors thank L. Grigorovitch for excellent technical assistance.
REFERENCES
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(Accepted 7 September 1992)