Abstract
We examined the effect of the anticonvulsant phenytoin (PT) (20–200 μM) on the persistent Na+ current (INaP), INaP-dependent membrane potential responses and repetitive firing in layer 5 pyramidal neurons in a slice preparation of rat sensorimotor cortex. INaP measured directly with voltage-clamp was reduced in a concentration-dependent manner with an apparent EC50 value of 78 μM. Clear effects on current-evoked membrane potential responses were apparent at 50 μM PT: Subthreshold, depolarizing membrane potential rectification was reduced, rheobase current was increased and the relation between firing rate and injected current was shifted to the right, but action potential amplitude and duration were unaffected. We ascribed these effects of PT largely to the reduction of INaP. A slow decline of firing rate during the injected current pulse also became apparent at moderate PT concentrations. When PT concentration was raised to 150 to 200 μM, this slow adaption was enhanced markedly, and firing ceased during a sufficiently large current pulse. This enhanced slow adaptation and the cessation of firing were associated with a marked decline of spike amplitude and a rise in spike firing level during successive interspike intervals. We ascribe these effects largely to the action of PT on the transient Na+ current. We conclude that the reduction in cortical neuronal excitability by PT depends partly on its reduction of INaP, the effects of INaP blockade are apparent at PT concentrations lower than those required to abolish tonic firing and the cells need not be excessively depolarized for PT to decrease excitability by its effect on INaP.
The anticonvulsant PT is widely used to control generalized and partial seizures (Rogawski and Porter, 1990; Woodbury, 1980). A variety of cellular effects have been observed when PT was applied to different preparations (Rogawski and Porter, 1990; Woodbury, 1980), but the therapeutic effects of PT are thought to derive mainly from its blockade of voltage-gated Na+ channels (MacDonald and Kelley, 1993; MacDonald and McLean, 1986; Rogawski and Porter, 1990). Studies on the fast INa indicate that PT binds tightly but slowly to block the Na+channels in both their open and their inactivated states, whereas PT binds only weakly to the closed (“resting”) channel (Kuo and Bean, 1994; Ragsdale et al., 1991; Willow et al., 1985). The binding of PT to open Na+ channel results in a use-dependent block of INa during brief, repetitive depolarizations (Ragsdale et al., 1991;Schwartz and Grigat, 1989; Willow et al., 1985). The binding of PT to the inactivated channel results in a voltage-dependent blockade of INa (Kuo and Bean, 1994; Ragsdaleet al., 1985; Schwartz and Grigat, 1989; Willow et al., 1985). During a long-lasting depolarization, INa is activated only briefly before becoming inactivated. Thus, binding of PT to the inactivated Na+ channel has been considered the important practical binding mode for PT action on Na+channels.
The properties of PT binding outlined above have led to the idea that PT will preferentially bind to and block Na+channels in neurons that are depolarized tonically and firing action potentials at a high rate. Less effect would be expected on neurons engaged in slow-rate firing because the time-average value of Na+ inactivation (and, therefore, PT binding and INa blockade) is expected to be lower when action potentials are less frequent. Support for this idea has been provided by MacDonald and McLean (1986) and McLean and MacDonald (1983) in experiments on cultured murine neurons. In these experiments, long-lasting, depolarizing, injected current pulses evoked only a few initial action potentials, followed by a cessation of firing in the presence of PT, whereas the same pulses evoked tonic, high-rate firing in control solution.
Many neurons possess a second type of Na+current, a “persistent,” or slowly inactivating, Na+ current (INaP) (Taylor 1993) that is activated at membrane potentials negative to spike threshold (Stafstrom et al., 1982, 1985). Although it is a relatively small current, INaP is an important determinant of excitability near spike threshold because it is largely unopposed by other voltage-gated currents in this subthreshold range of membrane potentials. There is evidence that INaPin cortical pyramidal neurons flows through a fraction of the same Na+ channels that normally give rise to the transient Na+ current but temporarily fail to inactivate for an extended period of time (Alzheimer et al., 1993). This fraction of open Na+ channels would be expected to be susceptible to the open-channel binding of PT, and it has been shown recently that INaP measured in acutely dissociated mammalian central neurons is inhibited by PT in a concentration-dependent manner (Chao and Alzheimer, 1995).
We hypothesized that PT may reduce neuronal excitability by its inhibition of INaP in addition to its well known action on INa. It seemed possible that by its action on INaP, PT may reduce excitability at a lower concentration than needed to block high-rate repetitive firing. Furthermore, the large depolarizations that result in inactivation of INa should not be required for PT to affect INaP because PT can bind to the open Na+ channels that give rise to INaP during small depolarizations. To investigate this question, we examined the action of PT on INaP and INaP-dependent membrane potential responses in layer 5 pyramidal neurons in a slice preparation of rat sensorimotor cortex. Although PT has already been shown to reduce INaP in dissociated cortical neurons (Chao and Alzheimer, 1995), the consequences of INaP reduction on cell excitability have not been examined. The effect of PT on repetitive firing has been examined only in cultured murine neurons bathed in a low Ca++solution (McLean and MacDonald, 1983). Therefore, we also examined the effect of PT on repetitive firing of the layer 5 pyramidal cells to compare, in the same preparation and conditions, the PT concentrations that affected repetitive firing with the concentrations that affected subthreshold, INaP-dependent membrane potential responses and INaP measured directly by voltage-clamp.
Methods
Sprague-Dawley rats (28–35 days postnatal) were anesthetized with ketamine (150 mg/kg) and xylazine (10 mg/kg) and killed by carotid section. A coronal section of cortex 0 to 3 mm posterior to bregma was isolated, and slices 350 μm thick were prepared and maintained as described previously (Schwindt and Crill, 1995). Recorded cells lay 1.0 to 1.3 mm below the pial surface and 2.1 to 3.2 mm from midline, corresponding to layer 5 of areas HL and FL of sensorimotor cortex (Zilles and Wree, 1985).
Intracellular recordings were made with the slice submerged in a chamber of ≈0.25 ml volume maintained at 33° to 34°C and perfused at 1 to 2 ml/min. Slices were perfused with physiological saline consisting of (in mM) 130 NaCl, 3 KCl, 2 CaCl2, 2 MgCl2, 1.25 NaH2PO4 and 10 dextrose saturated with 95% O2/5% CO2, pH 7.4. PT (Sigma Chemical, St. Louis, MO) was prepared as a 100 mM stock solution in DMSO. Aliquots of this stock were added to the perfusate just before intracellular recording to attain final PT concentrations up to 200 μM. Control experiments (n = 3) were performed using the maximal DMSO concentrations (0.1–0.2%) to ensure that the reported effects were not caused by DMSO.
Cells were impaled with sharp microelectrodes made from standard 1.0-mm outer diameter borosilicate tubing and filled with 2.7 M KCl (DC resistance, 30–40 MΩ). An Axoclamp-2A amplifier (Axon Instruments, Foster City, CA) was used to record membrane potential and inject current in active bridge mode or discontinuous current clamp mode or to control somatic membrane potential in single-electrode voltage-clamp using a switching rate of 2.5 to 5.5 kHz (30% duty cycle). Membrane potential and injected current were monitored and filtered at 1 to 10 kHz; membrane current measured in voltage-clamp was filtered at 100 Hz. Signals were amplified and recorded on a multichannel videocassette recorder with pulse code modulation (Neuro-Data, New York, NY; two-channel sampling rate, 44 KHz). Resting potential was taken as the difference between the intracellular and extracellular potentials recorded on a chart recorder. Recorded data were digitized to analyze evoked responses and firing rates using a computer program.
Results
Intracellular recordings were obtained from 26 neurons. The recording microelectrode was placed at a depth below the pial surface to record from layer 5 (see Methods). All recorded neurons had relatively low input resistance (see below), and all exhibited current-evoked repetitive firing responses (burst firing or regular spiking) characteristic of pyramidal neurons (McCormick et al., 1985). Neurons used for this study had a stable resting potential negative to −60 mV and did not fire spontaneously.
Resting potential and input resistance.
The effects of PT on both resting potential and input resistance were examined quantitatively in 11 neurons. PT (25–150 μM) had no effect on resting potential (control, −71.9 ± 5.3 mV; PT, −71.6 ± 5.8 mV; P > .6, paired t test, n = 11). In the same neurons, the voltage responses at the end of a series of 1-sec negative current steps were measured and plotted as function of injected current (data not shown). The slope of a linear fit to this plot (between 0 and −0.5 nA) was taken as input resistance. No significant change of input resistance was found after the application of PT (control, 16.4 ± 4.7 MΩ; PT, 17.5 ± 5.5 MΩ; P > .2, paired t test, n = 11; also see voltage-clamp results below).
Alteration of repetitive firing.
Measurements of repetitive firing properties and their alteration by PT were made in the same 11 neurons described above. Repetitive firing was evoked by 1-sec current steps of increasing amplitude before and after the addition of PT (25–100 μM). Most data were obtained using 50 μM PT. Two aspects of the repetitive response were altered by PT: The firing evoked throughout most of a given current pulse was significantly slower than in control, and a slow decline of firing rate during the pulse became apparent after PT application. Typical results are illustrated in figure 1. Figure 1, A–D, shows how the firing evoked by a current pulse was altered by PT (50 μM). The slower firing rate evoked by this pulse in PT is seen most clearly in figure 1, C and D, which shows the last 600 msec of the response of figure 1, A and B, at a faster time scale. The plots of figure 1, E and F, show instantaneous firing frequency (1/interspike interval) as a function of action potential number during current pulses. The three different curves in each plot correspond to the responses evoked by three different currents. By comparison of the control curves (fig. 1E) with those obtained after application of PT (fig. 1F), it can be seen that firing rate was slower than control even during the first interspike interval at the smallest current pulse (bottom plot) and was slower than control starting from the second interspike interval during the two larger pulses (top two plots). Among the 11 cells examined, PT had no consistent effect on instantaneous firing rate during the first interspike interval (off scale in the plots of fig. 1, E and F) or sometimes during the second interspike interval, when the instantaneous rate during these early intervals (evoked by larger current pulses) was >50 to 100 Hz.
Figure 2 shows how PT altered the relation between firing rate and injected current (F-I relation). These F-I relations were constructed by measuring the average firing rate over the last 600 msec of the response to 1-sec current pulses. The average firing rate evoked by each current in the presence of PT (fig.2A, •) was slower than the control (fig. 2A, ○). This effect of PT was reversible (fig. 2A) and concentration dependent (fig. 2B). The average firing rates obtained after application of PT had a larger variance than in control (see error bars in fig. 2, A and B), but this larger variance resulted from a slow decline of firing rate throughout the current pulse rather than from large fluctuations in the interspike interval (see below). The slope of the F-I relations were measured by fitting a line to the data points between 0 and 0.8 nA (dotted lines in fig. 2). Despite the lower average firing rates obtained in PT, the F-I slopes were essentially unchanged (control slope, 34.2 ± 9.7 Hz/nA; PT slope, 33.4 ± 8.1 Hz/nA, P > .3, pairedt test, n = 11).
This shift of the F-I relation to the right by PT represents decreased excitability. One measure of this decreased excitability is the additional injected current above the control value that was needed to evoke a given firing rate in PT. After application of PT (25–100 μM), current had to be increased by an average of 32 ± 35% (n = 11) to evoke the same steady firing rate. This also applies to the current resulting in the minimum steady firing rate. We measured the minimum current (with a resolution of 0.1 nA) that evoked repetitive firing in control and found that a current pulse ≈30% more than control was needed to initiate steady firing in the presence of PT.
Figure 1, C and D, also illustrates our second finding that a slow decline of firing rate (slow spike frequency adaptation) first appeared or became more prominent in the presence of PT. In the control record (fig. 1C), interspike interval duration remained essentially constant during the last 600 msec of the current pulse, whereas the interspike intervals lengthened progressively in the presence of PT (fig. 1D). The effect of PT (50–100 μM) on slow spike frequency adaptation was examined quantitatively in 4 neurons. For each cell, we fit the adaptation relation from the seventh to the last action potential by a line, as shown in figure 1, E and F. In each cell, the slopes of these lines were independent of injected current strength (P > .2, one-way ANOVA). We calculated the average slope of the line in control solution and after PT application. In the presence of PT, firing rate declined ≈4 times faster than the control (control, −0.035 ± 0.049 Hz/spike; PT, −0.144 ± 0.046 Hz/spike, n = 4).
A higher concentration of PT greatly enhanced this slow decline of firing rate, as illustrated in figure 3. When 200 μM PT was applied to this cell, firing rate declined to zero during injection of a sufficiently large current pulse (fig. 3B, top trace). The neuron did not stop firing during the injection of smaller currents (fig. 3B, bottom trace), but the slow decline of firing rate was much greater than at lower PT concentrations (compare fig. 3B, bottom trace, with fig. 1B). The amplitude of successive action potentials also decreased markedly after PT application in figure 3, whereas they were unaltered at lower PT concentrations (compare fig.3B, bottom trace, with fig. 1B). Both the progressive decline of successive action potentials (at all currents tested) and the cessation of firing (during larger current pulses that evoked tonic firing in control) were observed in each of 4 cells tested when PT concentration was raised to 150 μM (n = 1) or 200 μM (n = 3).
Even from the low-gain records of figure 3, it is clear that the progressive slowing of firing rate and the decline of action potential amplitude are associated with a progressive increase in firing level. By “firing level,” we mean the somatic membrane potential at which the spike starts its rapid rise, and we take this as a measure of spike threshold at that instant in time. This progressive rise in firing level was apparent, although considerably more modest, at lower PT concentrations, as illustrated in figure4. Figure 4A shows superimposed voltage responses evoked by the same current pulse in control (dotted traces) and in the presence of 50 μM PT (solid traces). The epochs marked by the horizontal bars in figure 4A are shown at a faster time scale in figure 4, B and C (corresponding to the left and right bars of fig. 4A, respectively). The superimposed traces of figure 4B show that firing level in PT (marked by the solid horizontal lines in B and C) was higher than that in control by the third spike of the response. Furthermore, firing level in PT rose by an additional 4.2 mV by the end of the pulse, but there was no similar rise of firing level in the control. This progressive rise of firing level in the presence of PT is an expected effect of the slow, voltage- and time-dependent binding of PT to Na+ channels. This rise in firing level may explain why firing rate slowly declined (interspike interval lengthened): It takes a longer time for membrane potential to reach a firing level that rises progressively during successive interspike intervals. The much greater rise in firing level at high PT concentrations (fig. 3B) may explain why the neuron stopped firing: The depolarization provided by the injected current eventually became insufficient to bring membrane potential to firing level.
Alteration of spike threshold and rheobase current.
The effect of PT on spike threshold was investigated further by evoking single spikes with briefer current pulses. In figure5A, the firing level of a single spike, evoked by 100-msec current pulse, was increased by 2.5 mV after the addition of 100 μM PT. Superimposition of the action potentials obtained in each solution (fig. 5B) shows that this concentration of PT had no significant effect on spike amplitude or duration. (The control spike appears be slightly larger in figure 5B because spike threshold was the reference point used for the superposition.) In 4 other cells tested similarly, we found no significant change in the duration or amplitude of single action potentials evoked in the presence of PT concentrations that caused the changes in firing level and repetitive firing described above.
Because a larger injected current was required to evoke steady repetitive firing after PT application, we expected the rheobase current in control solution would fail to evoke an action potential in solution containing PT. As shown in figure 5C (same cell as for fig. 5, A and B), when the current pulse was set to evoke an action potential on every trial in control solution, no action potentials were elicited by the same pulse after PT application. Similar results were obtained in each of 5 cells tested. The failure of membrane potential to reach threshold in PT was associated with the disappearance of the final, linear rise of membrane potential to firing level that was observed in the control (marked by arrow in fig. 5C). Thus, PT affected the subthreshold response of the neurons as well as their repetitive firing properties. The effect of PT was confined to depolarizing responses, however, as illustrated by the records of figure 5D (same cell as for fig. 5, A–C). Subthreshold responses to both negative and positive current pulses are shown. In the presence of 100 μM PT, the response to each current pulse was similar to control except for the most positive current pulse, which evoked a markedly smaller depolarization in the presence of PT. The subthreshold, depolarizing, inward rectification that was abolished by PT is known to be caused by activation of INaP (Stafstrom et al., 1982, 1985). Indeed, the depolarizing rectification observed in this cell after washout of PT was blocked by the subsequent application of TTX (data not shown).
Alteration of persistent Na+ current.
Experiments were conducted on 17 cells to confirm by direct measurement that PT reduced INaP and to determine the effective PT concentrations for comparison with concentrations that affected the near-threshold membrane potential responses and repetitive firing. INaP was recorded in voltage clamp (see Methods) using a voltage command consisting of a slow (1–3-sec duration) ramp depolarization from resting potential. This depolarization is slow enough to inactivate INaand reveal INaP (Alzheimer et al., 1993; Stafstrom et al., 1982, 1985). A typical voltage-clamp response is illustrated in figure 6A. In this figure, the control membrane current evoked by the depolarizing ramp voltage clamp is superimposed on the membrane current measured after application of 50 μM PT and on the current measured after partial washout of PT. Inward rectification is apparent on the control record as the cell is depolarized. PT reduced this inward rectification, and some recovery was observed after 10 min of washout. When membrane potential reached −52 mV, an uncontrolled action current was evoked, as can be seen on both the voltage and current records of all three traces. Because the single-electrode voltage-clamp cannot control the large, fast, inward current associated with an action potential, the ramp depolarization was terminated when somatic membrane potential reached firing level. This limited the depolarization to ≈−50 mV among the cells tested.
The data of figure 6A are replotted as I-V relations (I-V curves) in figure 6B. The initial, linear part of these relations represents a “leakage conductance” that corresponds to the steady-state input conductance measured near resting potential by current-clamp recording. The inward deviation of the I-V relation from this linear portion of the I-V relation signals INaP activation, as demonstrated by the abolition of the inward rectification and the extension of the linear relation by the application of TTX (Stafstromet al., 1982, 1985; see fig.7A). INaP was first activated at ≈−65 mV in the cell of figure 6. PT reduced the inward rectification (i.e., INaP) reversibly, but the leakage conductance was unaffected. The effect of PT on the inward rectification was concentration dependent, as illustrated for another cell in figure 6C. The blockade of INaPin this cell was not quite complete even at 200 μM PT.
We wanted to compare the effects of different PT concentrations on INaP among different recorded cells with different leakage (input) conductances and INaPmagnitudes. In each cell, INaP was isolated by subtracting the linear leakage current from the total current measured during the ramp depolarization. This subtraction was done after extrapolation of the initial, linear portion of the I-V relation to the maximum depolarization attained in the cell. The validity of this procedure is illustrated by the records of figure 7A, which show that the I-V relation became linear after INaP was abolished by TTX, and its slope was similar to the initial linear portion of the control I-V relation. Therefore, the difference between the extrapolated leakage current and the total current gives INaP. Figure 7B shows I-V plots from another cell for INaP isolated as described above. Increasing PT concentration from 50 to 75 μM further reduced INaP, and TTX eliminated it. For further comparison among cells, we divided the I-V relation for INaP measured after applying PT by the control I-V relation for INaP (fig. 7C). The result of this division gives the INaP conductance (GNaP) that remained after PT application as a fraction of the control GNaP (relative GNaP). Over voltages where INaP was small or absent, the division of the two I-V relations resulted in a large scatter of data points (data not shown). Points at which INaP had a sufficiently large amplitude to measure accurately (usually starting at ≈−60 mV) were fit adequately by a horizontal line. This procedure revealed a concentration-dependent reduction of GNaP by PT (fig. 7C).
In each of these 17 cells, we examined the effect of one to three concentrations of PT in the range of 20 to 200 μM. In these cells, the initial, linear portion of the I-V curve did not change significantly after application of PT (P > .4, pairedt test), supporting our finding that input conductance measured in current-clamp was not affected significantly by PT. The average relative GNaP in each cell was based on the I-V relations for INaP between ≈−60 mV and −55 mV. A voltage-dependent reduction of GNaPwas not detected by this procedure, possibly because the reduction was only examined over a 5- to 10-mV range of membrane potential. Values from the 17 cells were pooled and plotted as a function of PT concentration after averaging across cells (fig.8, triangles). The error bars in this plot indicate considerable variability of GNaPreduction at a given PT concentration among the cells tested, a point that also is made by the disparate concentration-response relations shown for 2 individual cells (fig. 8, filled symbols). This variability in PT effectiveness may reflect the existence of cells that differ in their sensitivity to PT. It also could reflect variability in the actual PT concentration attained at the cell membrane in a slice preparation, as mentioned in the Discussion. A fit to this concentration-response curve on the assumption of first-order drug-receptor binding (Kuo and Bean, 1994) resulted in an EC50 value of 78 μM.
Discussion
We found that PT affected INaP, INaP-dependent membrane potential responses and repetitive firing properties in a graded, concentration-dependent manner. The clearest relation between the reduction of INaP and the reduction of excitability was the reduction of the subthreshold, depolarizing rectification that causes membrane potential to rise to firing level (arrow in fig. 5C), which is well documented to depend on INaP activation (Stafstrom et al., 1982, 1985). The failure of membrane potential to reach firing level during injection of a control current pulse necessitated the use of a larger (rheobase) pulse to evoke a spike. Because a larger current was needed to evoke a single spike in the presence of PT, we would expect a larger current also to be needed to evoke steady repetitive firing, as was observed. The requirement for a larger current to evoke repetitive firing in the presence of PT would naturally cause the F-I relation to shift to the right, and we may confidently ascribe part of this rightward shift to the reduction of INaP. INaP is active at membrane potentials traversed in the interspike interval during repetitive firing (Stafstrom et al., 1984). Reduction of INaP would therefore delay the rise of membrane potential to firing level during repetitive firing, resulting in a longer interspike interval and slower firing rate. This is a second way by which the reduction of INaP would contribute to the rightward shift of the F-I curve. These effects were observed with no change in spike amplitude or duration. Therefore, we ascribe these effects principally to the reduction of INaP by low to moderate concentrations of PT. The alteration of these INaP-dependent factors would reduce the excitability of all cells, not just those that are excessively depolarized. Indeed, we observed reduced excitability at rheobase current and at the minimum steady firing rate.
We would expect the reduction of INaP to depend at least partly on the binding of PT to open Na+channels, but our data shed no light on this. At the usual resting potentials encountered in the slice preparation (≈−70 mV), INa is expected to be partly inactivated (Brownet al., 1994; Huguenard et al., 1988). The binding of PT to inactivated channels that might otherwise have returned to the resting state, and even the much weaker binding of PT to channels in the resting state would reduce the total number of Na+ channels available to open during depolarization, including channels that might otherwise have contributed to INaP generation. This mechanism may reduce INaP as effectively as open-channel binding and blockade. One might expect to observe the same type of voltage-dependent block of INaP that is seen for INa. The necessity of evoking an INaP that was sufficiently large to measure accurately at membrane potentials below spike threshold limited our observation to a voltage range (5–10 mV) that was too small to detect a clear voltage dependence of INaP reduction. It should be realized that our methods and preparation were directed toward measuring changes in excitability rather than questions of channel biophysics.
The alteration of repetitive firing was finely graded by PT concentration in our experiments; this allowed us to gain some insight into the factors that result ultimately in the cessation of repetitive firing when PT concentration reached a critical level. PT caused a concentration-dependent increase in slow spike frequency adaptation that was associated with a progressive rise in firing level from interval to interval. Because spike threshold depends on the balance of inward and outward currents and INaP causes a net-inward current to develop in some cells (Stafstrom et al., 1982, 1985), it is possible that the reduction of INaP also contributed to the rise of firing level at moderate PT concentrations at which spike amplitude was unaffected. At high concentrations of PT, the conclusion seems inescapable that the marked rise in firing level and decrease in spike amplitude during the repetitive firing were caused primarily by the progressive reduction of INa as a consequence of the slow binding of PT to inactivated Na+ channels (Kuo and Bean, 1994). The progressive decline of spike amplitude is likely to hasten further the inactivation and blockade of Na+ current. Less-repolarizing K+ current would be activated by a smaller spike, membrane potential during the next interspike interval would become more depolarized (see fig. 3B) and steady-state Na+ inactivation (thus, PT binding) would increase. Thus, we ascribe the rapid decline of firing rate (ultimately to zero) seen at high PT concentrations largely to the reduction of INa by PT.
Our repetitive firing results are consistent in two respects with those of McLean and MacDonald (1983) on cultured murine neurons: We confirmed that PT ultimately can cause the cessation of repetitive firing during a long-lasting depolarization, and the repetitive firing ceased sooner when the initial firing rate was faster. Our results differ in other respects, however. McLean and MacDonald (1983) interpreted their results as indicating that PT preferentially binds to and blocks Na+ channels in neurons that are firing action potentials at a high rate while having little or no effect on those neurons engaged in slow-rate firing. Although we found that PT could block high-rate firing, we found that firing at all rates was slowed by PT. Therefore, the effects of PT appear to be less selective for high-rate firing than previously thought.
A second major difference in our results is the PT concentration that resulted in a cessation of firing. McLean and MacDonald (1983) found this to occur at PT concentrations of 4 to 8 μM, whereas firing ceased at concentrations of 150 to 200 μM in our experiments. This discrepancy may be explained by factors that differed in the two sets of experiments. The average resting potential of the murine neurons was ≈10 mV more depolarized than in this study. To eliminate synaptic potentials, McLean and MacDonald (1983) recorded in a low-Ca++, high-Mg++solution, which was also likely to reduce or block the Ca++-dependent K+ currents that are primarily responsible for repolarizing membrane potential during the interspike interval. The EC50 for INa blockade may decrease ≈100 fold as a neuron is depolarized (Kuo and Bean, 1994). Because of this remarkable dependence of EC50 on membrane potential, differences in resting potential, firing level and membrane potential during the interspike interval could significantly alter the effective concentration at which PT causes firing to cease.
It also is possible that the actual PT concentration at our recorded cells was considerably less than the nominal bath concentration because we recorded in a slice. PT is highly lipophyllic and binds to a variety of proteins (Goldberg, 1980). Our recorded cells invariably were near the center of the slice, and data were collected within 20 min after the solution change to ensure the observed effects were not due to cell deterioration during a long impalement and to allow time for PT washout without excessive cell deterioration. It is possible that the binding of PT to elements in overlying tissue caused the PT concentration at the recorded cell to remain below the nominal bath concentration during the time frame of our recording. Some indication that this might be the case is given by our apparent EC50 value for the reduction of INaP (fig. 8); it was ≈2.5 times greater than that reported by Chao and Alzheimer (1995) for the reduction of INaP in the same type of cells. A possible reason for this discrepancy is that the membrane of the dissociated cells experienced the bath PT concentration, whereas ours did not. Although the use of dissociated cells in the present study would have eliminated this problem, we chose to study the effect of PT on cells with normal structure (e.g., dendrites) and intracellular milieu and at a more physiological temperature, conditions that are difficult to meet using the dissociated cells.
The slow, voltage-dependent binding kinetics of PT makes it difficult to adequately assess the effectiveness of PT in reducing repetitive firing by the use of a 1-sec depolarization. Because we found that the slowing of firing rate continued throughout a 1-sec depolarizing pulse at moderate PT concentrations (e.g., 50 μM), it is possible that firing rate ultimately would have declined to zero had the depolarization been prolonged indefinitely. Our results show, however, that other manifestations of decreased excitability, such as the rightward shift of the F-I relation and raised rheobase current, result from the reduction of INaP as well as INa.
Acknowledgments
We thank Gregg Hinz and Paul Newman for excellent technical assistance.
Footnotes
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Send reprint requests to: Dr. P. C. Schwindt, Department of Physiology and Biophysics, University of Washington School of Medicine, Box 357290, Seattle, WA 98195-7290. E-mail:schwindt{at}u.washington.edu
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↵1 This work was supported by International Human Frontiers Science Program Grant LT-544/94 (I.L.) and National Institutes of Health Grant NS16792 and the Keck Foundation (P.C.S. and W.E.C.).
- Abbreviations:
- PT
- phenytoin sodium
- TTX
- tetrodotoxin
- INaP
- persistent sodium current
- GNaP
- persistent sodium current conductance
- INa
- transient sodium current
- EC50
- concentration causing half-inhibition
- ANOVA
- analysis of variance
- DMSO
- dimethylsulfoxide
- I-V
- current-voltage
- Received May 22, 1997.
- Accepted September 25, 1997.
- The American Society for Pharmacology and Experimental Therapeutics