Biophysical Characterization and Functional Consequences of a Slowly Inactivating Potassium Current in Neostriatal Neurons

Lisa A. Gabel and Eric S. Nisenbaum

Department of Psychology, University of Connecticut, Storrs, Connecticut 06269

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Gabel, Lisa A. and Eric S. Nisenbaum. Biophysical characterization and functional consequences of a slowly inactivating potassium current in neostriatal neurons. J. Neurophysiol. 79: 1989-2002, 1998. Neostriatal spiny projection neurons can display a pronounced delay in their transition to action potential discharge that is mediated by a slowly developing ramp depolarization. The possible contribution of a slowly inactivating A-type K+ current (IAs) to this delayed excitation was investigated by studying the biophysical and functional properties of IAs using whole cell voltage- and current-clamp recording from acutely isolated neostriatal neurons. Isolation of IAs from other voltage-gated, calcium-independent K+ currents was achieved through selective blockade of IAs with low concentrations (10 µM) of the benzazepine derivative,6 - chloro - 7 , 8 - dihydroxy - 3 - allyl - 1 - phenyl - 2 , 3 , 4 , 5 - tetra - hydro1H-3-benzazepine (APB; SKF82958) and subsequent current subtraction. Examination of the voltage dependence of activation showed that IAs began to flow at approximately -60 mV in response to depolarization. The voltage dependence of inactivation revealed that ~50% of IAs channels were available at the normal resting potential (-80 mV) of these cells, but that only 20% of the channels were available at membrane potentials corresponding to spike threshold (about -40 mV). At these depolarized membrane potentials, the rate of activation was moderately rapid (tau  ~ 60 ms), whereas the rate of inactivation was slow (tau  ~ 1.5 s). The time course of removal of inactivation of IAs at -80 mV also was relatively slow (tau  ~ 1.0 s). The subthreshold availability of IAs combined with its rapid activation and slow inactivation rates suggested that this current should be capable of dampening the onset of prolonged depolarizing responses, but over time its efficacy should diminish, slowly permitting the membrane to depolarize toward spike threshold. Voltage recording experiments confirmed this hypothesis by demonstrating that application of APB at a concentration (10 µM) that selectively blocks IAs substantially decreased the latency to discharge and increased the frequency of firing of neostriatal neurons. The properties of IAs suggest that it should play a critical role in placing the voltage limits on the recurring episodes of subthreshold depolarization which are characteristic of spiny neurons recorded in vivo. However, the voltage dependence and recovery kinetics of inactivation of IAs predict that its effectiveness will vary exponentially with the level and duration of hyperpolarization which precedes depolarizing episodes. Thus long periods of hyperpolarization should increase the availability of IAs and dampen succeeding depolarizations; whereas brief epochs of hyperpolarization should not sufficiently remove inactivation of IAs, thereby reducing its ability to limit subsequent depolarizing responses.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

A distinctive electrophysiological property of spiny projection neurons within the neostriatum is their ability to attenuate the amplitude of voltage transients evoked by subthreshold depolarizing current pulses (Bargas et al. 1989; Calabresi et al. 1987b; Galarraga et al. 1994; Jiang and North 1991; Kawaguchi et al. 1989; Kita et al. 1985a,b; Nisenbaum and Wilson 1995; Nisenbaum et al. 1994). This attenuation is manifested as a slowing of the rate of depolarization giving rise to a subthreshold ramp potential that can last for hundreds of milliseconds. When the depolarizing input is maintained, the ramp potential eventually will culminate in action potential discharge with the latency to first spike being prolonged (Bargas et al. 1989; Nisenbaum et al. 1994). Recently, we have studied the voltage dependence, kinetics, and pharmacological properties of this delayed excitation (Nisenbaum et al. 1994). Results showed that the slope of the ramp potential was dependent on the level of depolarization with the ramp becoming evident at membrane potentials near -65 mV. The magnitude of the delayed excitation also was largely dependent on the membrane potential from which it was evoked. More specifically, prior depolarization decreased the slope of the ramp potential and the latency to spike discharge, whereas a conditioning hyperpolarization produced the opposite effect. In addition, the kinetics of recovery from inactivation of the delayed response showed that relatively long periods (2-3 s) of hyperpolarization were required for the ramp depolarization to be developed fully. Pharmacologically, the delayed excitation was reduced by extracellular application of low concentrations (<= 100 µM) of 4-aminopyridine (4-AP), implicating a potassium (K+) current(s) in this response (Bargas et al. 1989; Kita et al. 1985b; Nisenbaum et al. 1994).

A similar 4-AP-sensitive delayed excitation has been described in other cell types, including lateral geniculate nucleus (LGN) relay neurons (McCormick 1991), hippocampal CA1 pyramidal cells (Storm 1988), and prefrontal cortical neurons (Hammond and Crepél 1992). Several lines of evidence from these studies indicate that the delayed excitation depends on a slowly inactivating outward K+ current, termed ID or IAs. These currents become available at subthreshold membrane potentials and have relatively rapid kinetics of activation and slow kinetics of inactivation. These properties of slowly inactivating K+ currents suggest that they should be capable of limiting the initial response to subthreshold depolarizing input but over time should slowly permit the membrane potential to depolarize toward spike threshold. In addition, both the delayed excitation and the slowly inactivating currents can be blocked by low micromolar concentrations of 4-AP, further supporting a role for these currents in the mediating this response (McCormick 1991; Storm 1988).

Voltage-clamp recording experiments from neostriatal spiny neurons have shown that these cells possess at least three types of calcium-independent, depolarization-activated K+ currents. These include 4-AP-sensitive fast (IAf) and slowly (IAs) inactivating A currents and a 4-AP-resistant, persistent K+ current (IKrp) (Nisenbaum et al. 1996; Surmeier et al. 1988, 1991). The hypothesis that, similar to other cell types, IAs underlies the delayed excitation in neostriatal neurons has not been tested because it has not been possible to isolate this current from IAf and IKrp. However, we recently have shown that IAs can be selectively inhibited by low micromolar concentrations of the dopamine D1 receptor agonist, 6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-3-benzazepine (APB) (Nisenbaum et al. 1998). Moreover, the APB-induced reduction in IAs is not mediated by stimulation of D1 receptors but rather by blocking the channels giving rise to this current. Therefore IAs can nowbe isolated from IAf and IKrp by taking advantage of its sensitivity to APB, and a biophysical characterization of this current can be performed.

On the basis of our previous analysis of the delayed excitation in neostriatal spiny neurons (Nisenbaum et al. 1994), we predict that IAs should be endowed with several properties, including a relatively hyperpolarized voltage dependence of activation and inactivation, rapid kinetics of activation, slow kinetics of inactivation, and slow kinetics of recovery from inactivation. In addition, similar to the effects of 4-AP, depression of IAs by APB should reduce the delayed excitatory response of these neurons. These hypotheses were tested in the present experiments using both voltage- and current-clamp recording from acutely isolated neostriatal neurons.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

Acute isolation of neostriatal neurons

Neurons from adult (28-42-days old) rats were acutely isolated from the neostriatum using previously described procedures (Nisenbaum et al. 1996). Male, Sprague-Dawley rats were anesthetized deeply with methoxyflurane and decapitated. Their brains were removed rapidly from the skull and immersed in a cold (~2°C) NaHCO3-buffered saline solution, which contained (in mM) 126.0 NaCl, 2.0 KCl, 2.0 CaCl2, 2.0 MgCl2, 26.0 NaHCO3, 1.25 NaH2PO4, 1.0 pyruvic acid, and 10.0 glucose; pH = 7.4, osmolarity = 300 ± 5 mOsm/l. The brains were blocked, and 400 µm thick coronal sections were cut through the rostrocaudal extent of the neostriatum using a Vibroslice (Campden Instruments, London, UK). Slices then were incubated at room temperature(20-22°C) for 0.5-6.0 h in a holding chamber containing the continuously oxygenated (95% O2-5% CO2) NaHCO3-buffered saline solution. After the incubation period, slices were transferred to a glass petri dish containing a low Ca2+, N-[2-hydroxyethyl]piperanzine-N-[2-ethanesulfonic acid] (HEPES)-buffered saline solution that contained (in mM) 140.0 NaHOCH2CH2SO3 (Na isethionate), 2.0 KCl, 4.0 MgCl2, 0.1 CaCl2, 23.0 glucose, and 15.0 HEPES; pH = 7.4, osmolarity = 300 ± 5 mOsm/l and placed under a dissecting microscope. The dorsal neostriata from each hemisphere was dissected from the surrounding white matter and cortex. The neostriata were placed into a holding chamber containing protease Type XIV (1 mg/ml; Sigma Chemical, St. Louis, MO) dissolved in a HEPES-buffered Hank's balanced salt solution (HBSS 6136; Sigma Chemical) maintained at 37°C and oxygenated (100% O2). After 30-45 min of incubation in the enzyme solution, the neostriata were rinsed three times with the low Ca2+, HEPES-buffered saline solution and triturated using two fire-polished Pasteur pipettes having tips of decreasing diameter. Before whole cell recording, the cell suspension was placed into a 35-mm transparent plastic petri dish that was mounted onto the stage of an inverted microscope (Nikon, Tokyo, Japan). All voltage- and current-clamp recordings were performed at 22°C.

Whole cell recordings

The whole cell variant of the patch-clamp technique (Hamill et al. 1981) was used for recording from acutely isolated neostriatal neurons. Electrodes were pulled from borosilicate capillary tubing (Corning 7052, WPI, Sarasota, FL) using a multistage puller (Sutter Instruments, Novato CA). The electrodes were fire-polished with a microforge before use. The internal electrode filling solution contained (in mM) 65.0-75.0 KF, 2.0 MgCl2, 40.0 HEPES, 3.0bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA),12.0 phosphocreatine, 2.0 Na2ATP, 0.2 guanosine-5'-triphosphate (GTP), and 0.1 leupeptin; pH was adjusted to 7.2 with KOH 1.0 M and osmolarity was adjusted to 270-280 mOsm/l.

The extracellular solution contained (in mM) 140.0 NaCl, 1.0 KCl, 4.0 CaCl2, 1.0 MgCl2, 0.4 CdCl2, 10.0 HEPES, 10.0 glucose, and 0.001 tetrodotoxin (TTX); pH was adjusted to 7.4 with NaOH 1.0 M; osmolarity was adjusted to 300 ± 5 mOsm/l. TTX and CdCl2 were added to block voltage-dependent Na+ and Ca2+ channels, respectively. Application of drugs was accomplished using a five-barrel pipette array made from small diameter (~500 µm) glass capillary tubing. Solutions were contained in 10-ml syringes and positioned ~12 in above the recording chamber. Gravity-induced flow of each solution from the syringe to the corresponding barrel was controlled by electronic valves. The pipette array was positioned 100-200 µm from the cell before seal formation. The isolated cells were bathed continuously in HBSS, which facilitated washing of drug solutions. The solutions from the drug array were rapidly changed (<20 ms) by altering the array position with a DC motorized actuator (Warner Instruments, Hamden, CT).

Upon placing the recording electrode in the bath, offset potentials were corrected and electrode resistances ranged between 2 and 7 MOmega . To reduce the effect of changes in junction potentials associated with alterations in ionic conditions, the bath ground was a 3 M KCl agar bridge to a Ag/AgCl ground well. A small amount of constant positive pressure (2-3 cm H2O) was applied to the electrode as it was advanced through the bath. Once the electrode tip made contact with the cell membrane, negative pressure was applied to the back of the electrode to form a high-resistance seal between the electrode tip and the cell membrane and subsequently to achieve the whole cell configuration. After achieving the whole cell configuration, series resistance was compensated (60-80%) and monitored periodically. Because the whole cell K+ currents recorded typically were <2 nA, errors in voltage resulting from inadequate compensation would not have exceeded a few millivolts. Voltage-clamp recordings were conducted using an Axon Instruments 200A amplifier (Axon Instruments, Foster City, CA). Currents were digitized and monitored with pClamp software (Axon Instruments) running on a PC pentium clone computer.

For some experiments, current-clamp recording of isolated neurons was performed. In these experiments, the whole cell configuration was achieved in voltage-clamp mode using the procedures described above. The I-CLAMP NORMAL mode of the Axopatch 200A amplifier was used during voltage recordings to minimize errors in the response times. At the end of each recording, any pipette offset potentials were measured and used for determining the membrane potential recorded during the experiment.

Pharmacological agents

APB (SKF82958) was obtained from Research Biochemicals International (Natick, MA). A stock solution of 2 mM APB dissolved in a 0.1% ascorbic acid solution was apportioned into 50-µl aliquots and stored a -30°C until the day of recording. Solutions containing APB were kept covered to protect from exposure to light during the experiments. BAPTA, GTP, leupeptin, Na2ATP, and TTX were purchased from Calbiochem (La Jolla, CA). All other chemicals were obtained from Sigma Chemical.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

The results of the present study were collected from recordings of 77 neurons. Acutely isolated neurons selected for recording were all ~10 µm in diameter and had an average whole cell capacitance of 7.2 ± 0.4 pF (mean ± SD; range = 1.4-11.8 pF). Although the morphological identity of these neurons was not determined, several anatomic studies have shown that the spiny projection neurons are of similar size and comprise the vast majority of neurons within the neostriatum (~95%) (Chang et al. 1982; DiFiglia et al. 1976; Kemp and Powell 1971). In addition, previous studies have shown that acutely isolated spiny neurons that were identified after an injection of retrogradely transported fluorescent beads into the substantia nigra have whole cell capacitance values within the range reported here (Surmeier et al. 1992a, 1995). Therefore the population of unidentified neurons recorded here was assumed to be composed primarily of spiny projection cells.

Delayed excitation is present in acutely isolated neostriatal neurons

The acutely isolated cell preparation was chosen for whole cell voltage-clamp recording because the dendritic arbor of these cells is dramatically truncated, permitting complete voltage control of the cell membrane and thus an accurate quantitative description of IAs. However, a critical assumption in these experiments was that IAs and other currents giving rise to the delayed excitation in neostriatal neurons still would be present in this preparation. To test this hypothesis, whole cell current-clamp recordings from acutely isolated neurons initially were performed. Similar to intracellular recordings from spiny neurons in vivo or in neostriatal slice preparations, subthreshold intracellular depolarizing current pulses (500-ms duration) consistently evoked a slowly depolarizing ramp potential in isolated neostriatal neurons (n = 26; Fig. 1A). When threshold depolarizing current pulses were delivered, the ramp depolarization culminated in an action potential discharge at the end of the response (Fig. 1B). Identical current pulses reduced the latency to spike discharge and increased the frequency of firing when the resting membrane potential was more depolarized (Fig. 1C). These results indicated that the currents underlying the ramp depolarization and prolonged latency to discharge are present in the somata of spiny cells and that recruitment of dendritic currents is not required to elicit the delayed excitatory response.


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FIG. 1. Delayed excitation is present in acutely isolated neostriatal neurons. A: voltage deflection produced by a subthreshold intracellular depolarizing current pulse (0.01 nA, 500-ms duration) from relatively hyperpolarized membrane potentials displayed a pronounced ramp potential. B and C: injection of the same amplitude current pulse when the membrane was depolarized evoked action potential discharges with decreasing latencies.

APB selectively blocks IAs

An accurate description of the biophysical properties of IAs required that this current be isolated from the other depolarization-activated K+ currents. Previous studies have isolated slowly inactivating K+ currents by taking advantage of their preferential sensitivity to low micromolar concentrations of 4-AP (e.g., Storm 1988). Although IAs is more sensitive than IAf to 4-AP in neostriatal neurons (Surmeier et al. 1991), in preliminary experiments, we could not identify a concentration of 4-AP that would substantially reduce IAs (yielding a large difference current) without affecting IAf. However, we recently have shown that IAs can be blocked preferentially by low micromolar concentrations of the D1 dopamine receptor agonist, APB (Nisenbaum et al. 1998). The effect of APB is consistently reversible and has rapid onset (tau  = 1.2 s) and recovery (tau  = 1.6 s) kinetics. The actions of APB do not depend on recruitment of GTP-binding protein-dependent signaling pathways nor can the effects of the compound be blocked by D1 receptor antagonists. Although the precise mechanism of action remains to be determined, the evidence suggests that APB exerts its effects on IAs by allosterically regulating or blocking the channels giving rise to this current (Nisenbaum et al. 1998).

On the basis of its preferential sensitivity to blockade by APB, IAs could be isolated by current subtraction procedures. An example of this effect is illustrated in Fig. 2. Verification that the APB-sensitive current and IAs were identical requires an independent method of isolating IAs so that a direct comparison could be made between IAs and the APB-sensitive current. Previous studies have shown that IAs can be separated from IAf and IKrp on the basis of its relatively slow kinetics of recovery (~1 s) from inactivation (Surmeier et al. 1991). By taking advantage of this difference in recovery kinetics, the combined currents, IAf and IKrp, could be evoked by stepping the membrane potential to -40 mV for 5 s and then delivering a brief (25 ms) hyperpolarizing voltage step before a test step to +35 mV (Fig. 2A). Subtraction of these rapidly recovering currents from the total current evoked by the test step alone isolated IAs (Fig. 2B). In the same neuron, application of 10 µM APB reduced the total K+ current evoked by the test step alone, leaving the APB-insensitive current (Fig. 2C). Subsequent subtraction of the APB-insensitive current from the total K+ current isolated the APB-sensitive current (Fig. 2D). Comparison of the APB-sensitive current with IAs showed that their time courses were nearly identical (Fig. 2E). These results confirm that IAs can be isolated on the basis of its preferential sensitivity to APB, and in subsequent experiments, IAs was defined as the 10 µM APB-sensitive current.


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FIG. 2. 6-Chloro-7,8-dihydroxy-3-allyl1 - phenyl - 2 , 3 , 4 , 5 - tetra - hydro - 1H - 3 benzazepine (APB) selectively depresses IAs. A: total K+ current was evoked by a1-s test depolarizing voltage step from -80 to +35 mV. When the test step was preceded by a 5-s conditioning depolarization to -40 mV followed by a 25-ms hyperpolarizing step to -80 mV, the rapidly recovering current was evoked and was composed of IAf and IKrp. B: subtraction of the rapidly recovering (IAf IKrp) current from the total K+ current revealed IAs. C: in the presence of 10 µM APB, the APB-insensitive current evoked by the test step (-80 to +35 mV) alone was kinetically similar to the rapidly recovering current (IAf + IKrp). D: subtraction of the 10 µM APB-insensitive current from the total K+ current yielded the APB-sensitive current. E: kinetics of activation and inactivation of IAs and the APB-sensitive current were nearly identical, indicating that APB selectively block IAs.

IAs becomes available at subthreshold membrane potentials

Evidence from several studies indicates that the ramp depolarization emerges from a competition between depolarizing inward Na+ and Ca2+ currents and polarizing outward K+ currents (Bargas et al. 1989; Kita et al. 1985a; Nisenbaum et al. 1994). If IAs contributes to the outward K+ flux, then this current must flow at subthreshold membrane potentials. This hypothesis was tested by examining the voltage dependence of steady-state activation of IAs. The whole cell K+ current was evoked by 500-ms depolarizing voltage steps from -70 to +35 mV in 15-mV increments (holding potential = -90 mV) during control conditions (Fig. 3A). With the same protocol, K+ currents were elicited in the presence of 10 µM APB (Fig. 3B). Subsequent subtraction of the APB-insensitive currents from the control currents yielded IAs (Fig. 3C). This same subtraction procedure was used to isolate IAs in all of the following figures. Having isolated IAs, the voltage dependence of activation was assessed by first converting the current amplitudes to chord conductances and then normalizing each conductance by the maximum conductance evoked by the voltage step to +35 mV. For all cells tested (n = 15), the normalized conductances at each membrane potential were calculated and a plot of the average normalized conductance as a function of membrane potential was constructed (Fig. 3D). The plotted points were fit with a Boltzmann function of the form
<IT>g</IT>/<IT>g</IT><SUB>max</SUB> = 1/{1 + exp[(<IT>V</IT><SUB>m</SUB><IT> − V</IT><SUB>h</SUB>)/<IT>V</IT><SUB>c</SUB>]}
where g is conductance, gmax is the maximum conductance at +35 mV, Vm is the membrane potential, Vh is the half-activation voltage, and Vc is the slope factor. Examination of the average activation curve shows that IAs becomes available at relatively hyperpolarized membrane potentials near-60 mV. The average Vh and Vc values for IAs were -6.3 ±9.7 mV and 12.1 ± 1.9 mV, respectively. These results confirm the hypothesis that IAs is available at subthreshold membrane potentials (e.g., less than or equal to -40 mV) and thus is capable of contributing to the ramp potential evoked by depolarizing inputs.


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FIG. 3. IAs becomes available at subthreshold membrane potentials. A: voltage dependence of steady-state activation of IAs was assessed by delivering 500-ms depolarizing voltage steps from -70 to +35 mV (15-mV increments; holding potential, -90 mV). During control conditions, the voltage steps evoked IAf, IAs, and IKrp. B: after application of 10 µM APB, the APB-insensitive current was elicited using the same voltage protocol. C: subtraction of the APB-insensitive current from the control current yielded the APB-sensitive current, which was defined as IAs. D: for each cell tested, the peak amplitude of IAs was measured in response to each voltage step. These current values then were converted to conductance and normalized. Normalized conductance values for IAs were averaged and plotted as a function of membrane potential. Normalized data were fit using a Boltzmann function of the form g/gmax = 1/{1 +exp[(Vm - Vh)/Vc]}, where g is conductance, gmax is the maximum conductance at +35 mV, Vm is the membrane potential, Vh is the half-activation voltage, and Vc is the slope factor. As can be seen, IAs became available at hyperpolarized membrane potentials near -60 mV. Note that the Vh and Vc values for IAs were derived from the Boltzmann fit of the average normalized conductance values presented in the plot. These values differ slightly from average Vh and Vc values presented in the text.

Approximately one-half of IAs channels are available at the resting membrane potential

Previous studies have shown that the magnitude of the delayed excitation is directly related to the degree of membrane polarization (Nisenbaum et al. 1994). More specifically, the slope of the ramp potential and the latency to spike discharge increase with membrane hyperpolarization to potentials near rest and decrease with depolarization to potentials near spike threshold. One contributing factor to the voltage dependence of the delayed excitatory response could be that there are fewer IAs channels available at more depolarized membrane potentials. This possibility was tested by measuring the voltage dependence of steady-state inactivation of IAs. The membrane potential was stepped from -80 mV to potentials between -110 and -10 mV for 5 s before delivering a test step to +35 mV for 250 ms. The largest currents were elicited following the most hyperpolarized conditioning potentials (Fig. 4A). For all cells (n = 7), the current amplitudes were normalized relative to the maximum amplitude and the average normalized current was plotted as a function of the conditioning membrane potential (Fig. 4B). The plotted points were fit with a Boltzmann function of the form
<IT>I</IT>/<IT>I</IT><SUB>max</SUB> = 1/{1 + exp[(<IT>V</IT><SUB>m</SUB><IT> − V</IT><SUB>h</SUB>)/<IT>V</IT><SUB>c</SUB>]}
where I is the current evoked by the test step to 0 mV from each conditioning potential, Imax is the current evoked by the test step following the conditioning step to -110 mV, Vm is the membrane potential, Vh is the half-inactivation voltage, and Vc is the slope factor. Inspection of the inactivation curve shows that IAs was slightly inactivated at -100 mV and ~90% inactivated at -50 mV. The average Vh and Vc values of IAs were -78.8 ± 5.1 mV and 10.4 ± 2.2 mV, respectively. These data indicate that ~50% of IAs channels are available at -80 mV, which is near the resting potential of spiny neurons recorded in vivo and in neostriatal slice preparations using similar extracellular concentrations of K+ (Nisenbaum and Wilson 1995; Wilson and Kawaguchi 1996). By contrast, fewer than 10% of IAs channels are available at membrane potentials near spike threshold (about -40 mV). The relatively large percentage of IAs channels inactivated at the resting potential coupled with the moderately steep slope factor indicates that sustained depolarizations from rest will significantly reduce the availability of this current. This reduction in IAs should consequently contribute to the decreased slope of the ramp potential and latency to discharge when evoked from more depolarized membrane potentials (Nisenbaum et al. 1994).


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FIG. 4. Approximately 50% of IAs channels are available at the resting potential of neostriatal neurons. A: voltage dependence of steady-state inactivation of IAs was evaluated by stepping the membrane potential to values between -110 and -10 mV (10-mV increments) for 5 s before delivering a test step to 0 mV. Responses of IAs to the test step are shown with the largest current evoked after the most hyperpolarizing conditioning step (-110 mV) and the smallest current elicited after the most depolarizing conditioning step (-10 mV). B: relationship between the average normalized peak current and conditioning membrane potential are plotted. A Boltzmann fit of the points shows that approximately one-half of the channels are available at membrane potentials corresponding to the resting potential in vivo. Note that the Vh and Vc values for IAs were derived from the Boltzmann fit of the average normalized conductance values presented in the plot. These values differ slightly from average Vh and Vc values presented in the text.

Activation and deactivation kinetics of IAs are voltage dependent

The analysis of the voltage dependence of activation and inactivation of IAs indicated that ~50% of these channels are available at the resting membrane potential and that this current can be recruited by subthreshold depolarizations. The availability of this current supports the hypothesis that it contributes to the subthreshold ramp potential in spiny neurons. However, for IAs to limit the initial response to depolarizing input also requires that its kinetics of activation be relatively rapid at subthreshold membrane potentials. The kinetics of activation of IAs were assessed by delivering 150-ms voltage steps (15-mV increments) from -70 to +35 mV (holding potential, -90 mV; n = 16). Examination of the current traces showed that IAs had a delayed onset that decreased with larger depolarizations (Fig. 5A). Because of this delay, the currents were best fit using a single second-order exponential function
<IT>I</IT><SUB>As</SUB><IT> = I</IT><SUB>max</SUB>[1 − exp(<IT>t</IT>/τ)]<SUP>2</SUP>
where Imax is the maximum amplitude of each current, t is time after the onset of the voltage step, and tau  is the time constant of activation of each current. In Fig. 5A,the exponential fits of the currents are plotted as overlapping lines. The time constants of activation were dependent on the test step potential. For example, the time constants of activation of currents evoked by steps to -25 and +35 mV were73.4 ± 11.9 ms and 7.6 ± 4.2 ms, respectively (Fig. 5C).


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FIG. 5. Activation and deactivation kinetics of IAs are voltage dependent. A: IAs was evoked by 150-ms voltage steps from -70 to +35 mV (15-mV increments, holding potential, -90 mV). Currents exhibited a delayed onset, with the delay becoming shorter with stronger depolarizations. Second-order kinetics were used to fit activation of the IAs. Fitted curves for each trace are superimposed as solid lines. B: kinetics of deactivation of IAs were studied by stepping the membrane potential from -90 to +35 mV for 100 ms before delivery of 150-ms test steps from -25 to -115 mV (15-mV increments). Tail currents elicited upon deactivation were fit using single exponential functions, and the fitted curves are superimposed as solid lines. C: average time constants of activation and deactivation derived from curve fits like those presented in A and B are plotted. Examination of the entire activation/deactivation profile illustrates the voltage dependence of these processes.


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FIG. 6. IAs has slow kinetics of inactivation. A: IAs was evoked by 8-s depolarizing voltage steps from -70 to +35 mV (15-mV increments, holding potential, -90 mV). Decay of IAs was well fit by single exponential functions, which are superimposed over the traces as solid lines. Values adjacent to each curve fit reflect the time constants of inactivation for those currents at the membrane potential indicated. B: relationship between the average time constants of inactivation and membrane potential for IAs are plotted. Average time constants of inactivation varied as a function of step potential, becoming faster at more hyperpolarized membrane potentials. C: for most neurons, the amplitudes of the currents evoked by steps to membrane potentials greater than or equal to -55 mV were relatively small, making it difficult to accurately measure inactivation kinetics. To obtain a more accurate measure of inactivation kinetics at these potentials, currents were elicited by stepping the membrane potential from -90 mV to the desired potential (e.g., -55 mV) for 1 ms and then stepping to +35 mV for 20 ms before delivering a hyperpolarizing step to -55 mV. Successive steps from -55 to +35 mV then were delivered at intervals ranging from 10 ms to 8.3 s. Amplitude of the currents evoked in this manner decreased as a function of time after the initial voltage step to -55 mV. D: when the peak current values were plotted as a function of time after the initial voltage step, the points could be fit with a single exponential function. Responses evoked from -55 mV decayed with an average time constant of ~600 ms. E: with the fitted parameters from the Boltzmann functions describing the voltage dependencies of activation and inactivation of IAs, normalized activation (minfinity ) and inactivation (hinfinity ) curves were generated. Comparison of the activation curve for the noninactivating component of IAs (dashed line) with the minfinity curve shows that their voltage dependencies are very similar. Minimal overlap in the minfinity and hinfinity curves suggests that their is very little window current associated with IAs. F: normalized window conductance was calculated by multiplying the minfinity and hinfinity curves and the graph shows that at approximately -55 mV, the maximal conductance was only ~0.3% of the total conductancefor IAs.

The kinetics of IAs at more hyperpolarized potentials were assessed by examining the deactivation tail currents evoked by 150-ms hyperpolarizing steps (15-mV increments) from -25 to -115 mV after a 100-ms conditioning step from -90 to +35 mV (n = 9; Fig. 5B). The tail currents were well fit using single exponential functions. The time constants of deactivation were longest at the most depolarized voltages and became shorter at more negative voltages (Fig. 5C). An assumption in these experiments is that the activation and deactivation time constants are measuring the kinetics of the same underlying process. This hypothesis is supported by the fact that the time constants of activation (73.4 ± 11.9 ms) and deactivation (76.1 ± 13.5 ms) were similar for test steps to -25 mV. The entire activation/deactivation time constant profile had an inverted-U shape with values increasing from hyperpolarized membrane potentials, reaching a maximum at -25 mV and decreasing at depolarized membrane potentials (Fig. 5C). The average time constants of activation for subthreshold membrane potentials ranged from 23.7 ± 9.2 ms at -85 mV to 59.6 ± 10.5 ms at -40 mV. These kinetics of activation of IAs at subthreshold voltages are relatively rapid and would be expected to be sufficiently fast to attenuate the initial component of a depolarizing voltage transient.

Inactivation kinetics of IAs are slow

Perhaps the most obvious attribute of the delayed excitation is the long latency to spike discharge. Depending on the amplitude of injected current, this delay can easily last for hundreds of milliseconds (Nisenbaum et al. 1994). For IAs to contribute to the prolonged latency to spike discharge, this current must be endowed with relatively slow kinetics of inactivation. The time constants of inactivation of IAs(n = 14) were assessed by delivering 8-s voltage steps from -70 to +35 mV in 15-mV increments (holding potential, -90 mV) and fitting the decay of the evoked currents with a single exponential function of the form
<IT>I = A</IT>[exp(−<IT>t</IT>/τ)] + <IT>c</IT>
where A is the amplitude of the inactivating component of the current, t is time, tau  is the time constant of inactivation, and c is a constant term representing the noninactivating component of the current. It is important to note that for all currents, a component of IAs was noninactivating even after 8-s depolarizations, requiring the added constant term in the exponential equation (see further text). The small amplitude of the currents elicited by voltage steps to potentials more hyperpolarized than -40 mV precluded an accurate measurement of their inactivation kinetics (Fig. 6A). Therefore, only the responses to more depolarized potentials were evaluated in this manner. The time constants of inactivation varied as a function of test step potential. The currents evoked by the most depolarized test steps (+5 to +35 mV) inactivated with time constants of ~2 s. The longest time constants of inactivation were associated with voltage steps to -10 mV (2.9 ± 1.6 s) and -25 mV (2.9 ± 1.7 s). At membrane potentials corresponding to spike threshold (-40 mV), the time constant of inactivation was 1.5 ± 0.6 s (Fig. 6B).

The kinetics of inactivation at more hyperpolarized membrane potentials were evaluated by stepping from -90 to -55 mV for 1 ms and then delivering successive 20-ms steps from -55 to +35 mV at intervals ranging from 10 ms to 8.3 s (n = 6). The currents evoked by the test steps to +35 mV are shown in Fig. 6C. The largest current was elicited after the 1-ms step to -55 mV; the smallest current was evoked after 8.3 s at -55 mV. The amplitude of the currents decreased as a function of time after the initial voltage step to -55 mV. The peak amplitudes of the currents evoked by the successive steps to +35 mV were plotted against the time after the initial test stimulus onset, and the points were fit with a single exponential function (Fig. 6D). With this method, the average time constant of inactivation at -55 mV was 0.6 ± 0.2 s.

As described earlier, a noninactivating component of the current was evident even after 8-s depolarizations. This noninactivating component could arise from several different mechanisms, including a window current predicted by the steady-state voltage dependencies of activation and inactivation, a noninactivating component of IAs, and/or contamination from the persistent K+ current, IKrp, as a result of partial blockade of this current by APB. To distinguish between the first two possibilities, the predicted window current was assessed by examining the degree of overlap of the normalized activation (minfinity ) and inactivation (hinfinity ) curves for IAs. The minfinity and hinfinity curves for voltages ranging from -115 to +35 mV were generated using the fitted parameters from the Boltzmann functions describing the voltage dependencies of activation and inactivation of IAs (Fig. 6E). Inspection of the curves shows that they overlap minimally, suggesting that the predicted window current should be small. Indeed, calculation of the window conductance by multiplying the minfinity and hinfinity curves shows that at approximately -55 mV, the maximal conductance was only ~0.3% of the total conductance for IAs (Fig. 6F). In addition, if the noninactivating component arises from a window conductance, then their voltage-dependent properties should be the same. Conversely, if the noninactivating component represents a true sustained portion of IAs, then its voltage dependence should be similar to the minfinity curve. To test these hypotheses, the activation curve for the noninactivating component was calculated by plotting the normalized conductance values of the constant term, c, derived from the exponential fits of the decay phase of each current by membrane potential, and fitting these points with a Boltzmann function. Comparison of the activation curve for the noninactivating component of IAs (dashed line) with the minfinity curve shows that their voltage dependencies are very similar (Fig. 6E). More importantly, the voltage dependence of the noninactivating component does not parallel that of the window conductance (Fig. 6F). The last possibility that the noninactivating component reflects a contribution from IKrp also seems unlikely because the voltage dependence of activation of the noninactivating component (Vh = -2.0 mV) is ~10 mV more depolarized than that for IKrp (Vh = -13.0 mV) (Nisenbaum et al. 1996). In addition, we previously have shown that IKrp can be isolated in the presence of 10 mM 4-AP (Nisenbaum et al. 1996) and that this current is not affected by APB (Nisenbaum et al. 1998). Collectively, these data indicate that the noninactivating component of IAs reflects a sustained portion of this current.


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FIG. 7. Kinetics of recovery from inactivation of IAs are relatively slow. A: recovery kinetics were assessed by stepping the membrane potential from -80 to -40 mV for 4 s, then delivering a conditioning step to -80 mV for periods of time ranging from 1 ms to 10 s before delivering a 150-ms test step to 0 mV. Largest currents were evoked after the longest conditioning steps. B: for each cell tested, the current amplitudes were normalized relative to the amplitude after a 10-s conditioning step, and the average normalized currents were plotted as a junction of conditioning step duration. Time constant of recovery from inactivation of these averaged normalized values was 1.0 s. Note this value differs slightly from average time constant of recovery from inactivation presented in the text.


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FIG. 8. APB and depolarization decrease the delayed excitation. A: intracellular injection of a 0.01-nA depolarizing current pulse (500-ms duration) evoked a single action potential discharge at the end of the response. B: application of 10 µM APB reduced the latency to discharge and increased the frequency of discharge evoked by the same current pulse. Also note that the spike duration increased. C-E: depolarization of the membrane potential from -97 to -64 mV to inactivate IAs channels reduced the latency and increased the frequency of spike discharge evoked by a constant amplitude depolarizing current pulse (0.006 nA, 500-ms duration).

Kinetics of recovery from inactivation of IAs are slow

Our previous analysis of the delayed excitation in spiny neurons revealed that the prolonged latency to spike discharge evoked by a depolarizing current pulse was highly dependent on the preceding duration of hyperpolarization. That is, a delayed spike discharge was evident only after the membrane potential had been hyperpolarized for several seconds before delivery of the test current pulse (Nisenbaum et al. 1994). To the extent that IAs contributes to this delay, these data suggest that the availability of this current should increase with longer periods of hyperpolarization. This hypothesis was tested by measuring the kinetics of recovery from inactivation of IAs. The voltage protocol involved stepping the membrane potential from -80 to -40 mV for 4 s and subsequently delivering a conditioning step to -80 mV for periods of time ranging from 1 ms to 10 s before delivery of a test step to 0 mV. The currents evoked by the test step are shown in Fig. 7A. The largest currents were evoked after the longest conditioning step durations. For all cells (n = 10) tested, the current amplitudes were normalized relative to the amplitude after a 10-s conditioning step, and the average normalized currents were plotted as a function of conditioning pulse duration. The average time constant of recovery from inactivation of IAs was 1.2 ± 0.4 s (Fig. 7B). The range of recovery kinetics was 0.9-2.2 s. The relatively slow recovery kinetics of IAs are consistent with a contribution of this current to the delayed excitation after prolonged periods of hyperpolarization.

APB-induced decrease in delayed excitation is voltage dependent

The selectivity of 10 µM APB for IAs permitted a direct examination of the contribution of this current to the delayed excitation in spiny neurons. During control conditions, a threshold depolarizing current pulse delivered to a cell resting at a relatively hyperpolarized membrane potential evoked a single spike discharge at the end of the response (Fig. 8A). The ramp potential underlying the prolonged latency to discharge also was evident. Subsequent application of APB (10 µM) decreased the latency to dischargefrom 346.0 ± 148.0 ms during control conditions to 157.8 ±65.9 ms [t(6) = 3.1, P < 0.05] in the presence of the drug. The frequency of firing and the duration of action potential discharge also were increased after blockade of IAs (Fig. 8B). These results support the hypothesis that IAs contributes to the ramp depolarization and delayed spike discharge in spiny neurons.


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FIG. 9. Prior depolarization reduces the effect of APB on spike frequency. A: a suprathreshold depolarizing current pulse (0.02 nA, 500-ms duration) delivered to a cell at -59 mV evoked a high-frequency response. B: subsequent application of APB (10 µM) had little effect on the frequency of discharge when tested at a similar depolarized membrane potential.

The results of the biophysical analysis of IAs showed that this current had a relatively hyperpolarized voltage dependence of inactivation. In particular, ~50% of IAs channels were inactivated at -80 mV and >90% of these channels were unavailable at -40 mV. One predicted consequence of this property of IAs is that sustained depolarization should inactivate these channels and thus diminish the latency to discharge and increase the frequency of discharge in response to a test pulse. This hypothesis was confirmed by recording from cells at several membrane potentials and showing that the latency to discharge decreased and the frequency of firing increased as a function of membrane depolarization (Fig. 8, C-E). An additional implication of the voltage dependence of inactivation of IAs is that blockade of these channels should have little effect on the latency and frequency of discharge when evoked from a depolarized membrane potential due to the large degree of inactivation of this current. To test this prediction, a suprathreshold stimulus was delivered to a neuron at approximately -60 mV during control conditions and elicited a high frequency of discharge having a short delay to onset (Fig. 9A). In contrast to effects observed at more hyperpolarized membrane potentials, subsequent application of APB (10 µM) had little effect on the latency or frequency of the response (Fig. 9B).

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

IAs underlies the delayed excitatory response of spiny neurons

The delayed transition to discharge of neostriatal spiny neurons is an electrophysiological property that distinguishes these cells from other neurons in the neostriatum (Kawaguchi 1992, 1993). A similar delayed excitatory response was described first in hippocampal pyramidal CA1 neurons and was shown to depend on recruitment of a slowly inactivating K+ conductance (Storm 1988). Voltage-clamp recordings from neostriatal neurons also have identified a slowly inactivating A-type K+ current (IAs), raising the possibility that, similar to CA1 neurons, this current is responsible for the delayed excitation in neostriatal cells (Surmeier et al. 1991). In the present experiments, the hypothesis that IAs contributes to the delay in transition to discharge of neostriatal neurons was investigated. Toward this end, a biophysical analysis of IAs initially was performed to determine if the voltage dependence and kinetics of this current were consistent with that of the delayed excitatory response (Nisenbaum et al. 1994). However, a prerequisite for such an analysis was a method for isolating IAs from the other voltage-gated K+ currents in neostriatal neurons. Our recent observation that the dopamine D1 receptor agonist, APB, selectively blocks IAs provided a pharmacological tool by which IAs could be isolated (Nisenbaum et al. 1998). By taking advantage of its preferential sensitivity to APB, IAs could be separated from IAf and IKrp through current subtraction, and a biophysical characterization of this current then could be conducted. In addition, the selectivity of APB for IAs permitted a direct assessment of the contribution of this current to the transition to discharge of neostriatal neurons in current-clamp experiments.

The results from the biophysical experiments strongly support a role for IAs in the delayed excitation of neostriatal neurons. First, the availability of IAs at subthreshold membrane potentials is consistent with the subthreshold nature of the ramp depolarization (Bargas et al. 1989; Kawaguchi et al. 1989; Kita et al. 1985a,b; Nisenbaum et al. 1994). Second, the voltage dependence of inactivation of IAs is similar to that of the delayed spike discharge of spiny neurons. More specifically, a conditioning depolarization to membrane potentials near -60 mV inactivated ~80% of IAs channels and produced a corresponding decrease in the latency to spike discharge. In contrast a conditioning hyperpolarization to approximately -100 mV deinactivated nearly 100% of IAs channels and produced a concomitant increase in the latency to discharge. Third, the kinetics of activation of IAs are relatively rapid at subthreshold membrane potentials, having time constants of activation ranging from 20.2 to 59.6 ms. Assuming a temperature coefficient of 2-3 (Huguenard and Prince 1991), the time constant of IAs activation should be ~15-25 ms at -60 mV, which is consistent with the predicted ability of this current to limit the initial response to depolarizing input. Fourth, the slow time course of inactivation of IAs at subthreshold membrane potentials is consistent with the shallow trajectory of the ramp potential. For example, the time constant of inactivation of IAs at -40 mV (approximately spike threshold) was 1.5 s and should be sufficiently long to permit this current to dampen threshold depolarizing responses and account for the delay to spike discharge, which can last for hundreds of milliseconds (Bargas et al. 1989; Kawaguchi et al. 1989; Nisenbaum et al. 1994). Finally, the moderately slow kinetics of recovery from inactivation of IAs are similar to the slow recovery kinetics of the delayed excitation. Previous results have shown that full development of the ramp potential and delayed spike discharge requires prior hyperpolarization of the membrane for 1-2 s (Nisenbaum et al. 1994; Surmeier et al. 1992b). The time constant of recovery from inactivation of IAs at -80 mV was 1.2 s, which is consistent with this requirement. Collectively, the similarities between the voltage dependence and kinetics of IAs and the delayed excitation support the hypothesis that this current makes a significant contribution to the transition to firing of neostriatal spiny neurons.

A role for IAs in the delayed excitation also was supported by the results of the voltage-recording experiments. The latency to spike discharge was reduced substantially after application of 10 µM APB, which selectively blocks IAs. A similar decrease in spike latency and an increase in frequency of firing was produced by depolarization of the membrane to potentials approaching spike threshold (e.g., -60 mV) before delivery of the test stimulus. At these membrane potentials, only ~20% of IAs channels are available, suggesting that the decreased spike latency and increased frequency of discharge resulted from inactivation of IAs channels. Moreover, subsequent application of APB at these depolarized membrane potentials had little effect on the delayed excitation as would be expected if IAs channels already were inactivated. It should be noted that an additional mechanism underlying the APB-induced decrease in the delayed excitation could have come in part from the dopamine D1 receptor-mediated effects of this compound on inward Na+ and Ca2+ currents. Acutely isolated neostriatal neurons are known to possess D1 receptors (Surmeier et al. 1996), and D1 receptor stimulation has been shown to reduce both of these inward currents in this preparation (Surmeier et al. 1992a, 1995). However, a reduction in Ca2+ currents most likely did not contribute to the decrease in the delayed excitation because the extracellular solution in these experiments contained the Ca2+ channel blocker, CdCl2, at a concentration (400 µM) that previously has been shown to block all Ca2+ currents in these neurons (Bargas et al. 1994; Surmeier et al. 1995). A substantial depression of Na+ currents also seems unlikely because this effect would be expected to further the delay to spike discharge by increasing the level of depolarization required to reach threshold.

Evidence from several studies indicates that the ramp depolarization and delayed spike discharge of spiny neurons emerges from the competing effects of depolarizing inward Na+ and Ca2+ currents and the polarizing outward K+ current, IAs. As such, blockade of inward Na+ and/or Ca2+ currents diminishes the steepness of the trajectory of the ramp potential (Bargas et al. 1989; Calabresi et al. 1987a; Galarraga et al. 1994; Kita et al. 1985a; Nisenbaum et al. 1994). In addition, selective blockade of IAs with either low concentrations of 4-AP (Nisenbaum et al. 1994) or APB (present results) decreases the slope of the ramp depolarization and the latency to spike discharge. However, elimination of either the inward currents or IAs alone does not completely abolish the ramp potential, indicating that none of these currents is solely responsible for the response (Kita et al. 1985a; Nisenbaum et al. 1994). Taken together, these results suggest that subthreshold depolarizing current injection recruits inward Na+ and Ca2+ currents, which act to further depolarize the membrane, and IAs, which tends to slow the rate of this depolarization. However, differences in their voltage dependencies and kinetics suggest that these currents will predominate at different voltages and times during the response to depolarization. Because IAs is available at more hyperpolarized membrane potentials compared to either Na+ or Ca2+ currents (Bargas et al. 1994; Surmeier et al. 1992a), it would be expected to primarily govern the initial portion of the depolarizing response. As the membrane potential becomes more depolarized, inward Na+ and Ca2+ currents will flow more readily and will compete with IAs. As IAs gradually inactivates, the net ionic flow will shift more toward the inward currents and the membrane potential will slowly depolarize toward spike threshold. Thus the interplay between these inward and outward ionic conductances is postulated to give rise to a slowly developing depolarization, which will proceed until termination of the current pulse or the membrane potential reaches spike threshold (Nisenbaum et al. 1994). Moreover, the presence of the delayed excitation in acutely isolated neostriatal neurons indicates that the ionic channels subserving this response are expressed on the perisomatic membrane of these cells.

The possibility that IAf and/or IKrp contribute to the delayed excitation of spiny neurons also should be considered. A role for IAf in the response seems unlikely because it does not become available until the membrane potential reaches spike threshold and its rapid inactivation rate is not consistent with the slow development of the ramp potential (Surmeier et al. 1988). In contrast to IAf, IKrp becomes available at subthreshold membrane potentials and has an inactivation rate which is approximately two times slower than IAs, suggesting that it should be capable of contributing to the total outward K+ flux during the ramp depolarization. However, the relatively slow activation rate of IKrp should limit its contribution to extended depolarizing ramp responses (Nisenbaum et al. 1996).

Comparison of IAs with slowly inactivating K+ currents in other neurons

Since the original description of the slowly inactivating K+ current, ID, in hippocampal pyramidal CA1 cells (Storm 1988), similar currents have been identified in other neurons of the central nervous system, including cortical pyramidal cells (Foehring and Surmeier 1993; Hammond and Crepél 1992; Spain et al. 1991), LGN relay neurons (Budde et al. 1992; McCormick 1991), as well as neostriatal neurons (Surmeier et al. 1991; present results). Comparison of the slowly inactivating K+ currents in these various cell types shows that they share similarities in voltage dependence and pharmacology. Examination of the voltage dependence of activation of these currents reveals that they all become available at subthreshold membrane potentials between -60 to -70 mV (Foehring and Surmeier 1993; McCormick 1991; Storm 1988; present results). As described above, the availability of these currents at hyperpolarized membrane potentials enables them to attenuate subthreshold depolarizing inputs. The voltage dependencies of inactivation of the slowly inactivating K+ currents also are similar such that each of these currents is half-inactivated at membrane potentials between -75 and -90 mV (Hammond and Crepél 1992; McCormick 1991; Storm 1988; present results). This voltage range corresponds to the resting potentials of these cells, indicating that ~50% of slowly inactivating K+ channels normally are inactivated at rest. The slowly inactivating K+ currents also share pharmacological properties. For example, all of the these currents are sensitive to low micromolar concentrations of 4-AP (Foehring and Surmeier 1993; Hammond and Crepél 1992; McCormick 1991; Spain et al. 1991; Storm 1988; Surmeier et al. 1991). In contrast, these currents are relatively resistant to blockade by tetraethylammonium (Hammond and Crepél 1992; McCormick 1991; Storm 1988).

Despite the similarities in voltage dependence and pharmacology, the slowly inactivating K+ currents exhibit differences in their kinetics that most likely contribute to the distinct functional properties of these neurons. For example, the time constant of inactivation of IAs in neostriatal neurons (1.5 s) at membrane potentials corresponding to spike threshold is faster than that of the slow transient current (3.3 s) (Spain et al. 1991) or IKs (3.75 s) (Hammond and Crepél 1992) in cortical neurons, IAs (3.2 s) in LGN relay neurons (McCormick 1991), and ID (~5 s) in hippocampal pyramidal cells (Storm 1988). The longer time constants of inactivation for these other cell types probably account for their ability to dampen threshold depolarizing inputs and delay spike discharge for many seconds (e.g., Storm 1988) compared with hundreds of milliseconds for neostriatal neurons (Nisenbaum et al. 1994). The kinetics of recovery from inactivation of IAs also differ from slowly inactivating currents in other neurons. The time constant of recovery from inactivation of IAs (1.0 s) is intermediate to that for IAs in LGN relay neurons (91 ms) (McCormick 1991) and ID in hippocampal CA1 pyramidal cells (4.7 s) (Storm 1988). In general, slow recovery kinetics combined with slow inactivation rates permit inactivation to accumulate in response to successive depolarizing inputs, thereby enhancing the level of depolarization evoked by later inputs (Nisenbaum et al. 1994; Storm 1988; Surmeier et al. 1992b). The slower recovery kinetics for ID in hippocampal neurons most likely account for the substantially longer periods of time during which hippocampal neurons can integrate depolarizing inputs (Storm 1988). By contrast, the relatively fast recovery kinetics of IAs in LGN relay neurons enable it to contribute to repolarization of low-threshold Ca2+ spikes which underlie the 1- to 4-Hz rhythmic firing of these cells (McCormick 1991; Steriade et al. 1993).

The comparisons made earlier clearly demonstrate that in addition to their similarities, differences exist in the kinetics of the slowly inactivating K+ currents in different cell types. Several types of K+ channel subunits have been identified that give rise to slowly inactivating currents, including Kv2.1, Kv3.1, Kv3.2, and Kv3.3 (Drewe et al. 1992; Perney et al. 1992; Sheng et al. 1992; Tsaur et al. 1992; Weiser et al. 1994). The kinetic differences in the currents described earlier may likely reflect the differential expression of K+ channels subunits in the membranes of these neurons. For example, in situ hybridization studies have shown that high levels of messenger RNA (mRNA) for Kv3.1 and Kv3.2 are expressed in the LGN and CA3 pyramidal cells of the hippocampus, but low or undetectable levels of these mRNAs are present in spiny neurons of the neostriatum (Perney et al. 1992; Weiser et al. 1994). In addition, high levels of Kv2.1 mRNA are present in CA3 pyramidal cells but not in neostriatal neurons (Drewe et al. 1992). However, neostriatal cells do express mRNA for Kv1.4, Kv1.6, and Kv4.2 subunits (Sheng et al. 1992; Surmeier et al. 1994; Tsaur et al. 1992). An additional contributing factor to the functional differences of slowly inactivating K+ currents may arise from the formation of heteromultimeric K+ channels (Ruppersberg et al. 1990; Stühmer et al. 1989). Evidence from in vitro expression studies indicates that subunits from the same K+ channel subfamily can combine to form heteromultimeric channels (Christie et al. 1990; Ruppersberg et al. 1990; Weiser et al. 1994). Moreover, the assembly of these heteromultimeric channels can give rise to currents that have functional properties intermediate to either channel alone (Ruppersberg et al. 1990; Weiser et al. 1994). Therefore, the differences between slowly inactivating K+ currents in different cell types may reflect the differential expression of K+ channel subunits and/or the assembly of distinct heteromultimeric channels.

Role of IAs in the spontaneous activity of spiny neurons

The natural activity of spiny projection neurons recorded intracellularly in vivo is distinguished by recurring episodes of maintained hyperpolarization (about -80 mV) followed by periods of sustained subthreshold depolarization, which can last for hundreds of milliseconds to seconds (Wilson 1993; Wilson and Groves 1981; Wilson and Kawaguchi 1996). The transitions to the depolarized state depend on powerful excitatory input from cortex and thalamus, and are maintained at a relatively constant potential just below the threshold for spike discharge (Wilson 1993, 1994; Wilson et al. 1983; Wilson and Kawaguchi 1996). Once the cell has entered the depolarized state, action potentials may occur and can have long latencies to the discharge and irregular firing patterns. Recent experiments have demonstrated that the limits placed on the amplitude of the depolarized state depend on K+ currents that are recruited by depolarization (Wilson and Kawaguchi 1996). Results from the present experiments would strongly support a role for IAs in limiting the subthreshold depolarizations as well as contributing to the latency to discharge and frequency of firing. The availability of IAs at approximately -60 mV coupled with its relatively rapid rate of activation at subthreshold membrane potentials should enable this current to limit the level of depolarization associated with the onset of the depolarized state. In addition, the slow time course of inactivation of IAs should permit this current to maintain the voltage limits on the depolarized state for moderately long durations. The rapid activation and slow inactivation of IAs suggest that its ability to decrease the likelihood of action potential generation should be greatest as the membrane potential first reaches the depolarized state and should diminish exponentially with time according to its time constants of inactivation. In this manner, IAs is predicted to contribute to the delay to first spike discharge often seen in the depolarized state.

The voltage dependence of inactivation and kinetics of recovery from inactivation suggest that the efficacy of IAs will depend on both the level of polarization associated with the hyperpolarized state as well as the time that the cell previously has spent in this state before receiving an excitatory synaptic barrage. For example, hyperpolarizing episodes lasting for several seconds should substantially remove inactivation of IAs enabling the current to delay spike discharge or even suppress firing entirely. In contrast, brief periods of hyperpolarization should not permit IAs channels to recover from inactivation and would be expected to decrease the efficacy of this current in inhibiting action potential discharge. Thus the voltage history of the neurons should greatly impact on the ability of IAs to shape the subthreshold voltage behavior and discharge characteristics of spiny neurons in response to excitatory synaptic input (Nisenbaum et al. 1994; Wilson 1993).

    ACKNOWLEDGEMENTS

  We thank Dr. Charles Wilson for comments during the preparation of this manuscript.

  This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-34254 to E. S. Nisenbaum.

    FOOTNOTES

  Address reprint requests to E. S. Nisenbaum.

  Received 25 September 1997; accepted in final form 10 December 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/98 $5.00 Copyright ©1998 The American Physiological Society