Muscarinic Receptors Differentially Modulate the Persistent Potassium Current in Striatal Spiny Projection Neurons

Lisa A. Gabel and Eric S. Nisenbaum

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


    ABSTRACT
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Gabel, Lisa A. and Eric S. Nisenbaum. Muscarinic receptors differentially modulate the persistent potassium current in striatal spiny projection neurons. Cholinergic regulation of striatal spiny projection neuron activity is predominantly mediated through muscarinic receptor modulation of several subclasses of ion channels. Because of its critical role in governing the recurring episodes of hyperpolarization and depolarization characteristic of spiny neurons in vivo, the 4-aminopyridine-resistant, persistent potassium (K+) current, IKrp, would be a strategic target for modulation. The present results show that IKrp can be either suppressed or enhanced by muscarinic receptor stimulation. Biophysical analysis demonstrated that the depression of IKrp was associated with a hyperpolarizing shift in the voltage dependence of inactivation and a reduction in maximal conductance. By contrast, the enhancement of IKrp was linked to hyperpolarizing shifts in both activation and inactivation voltage dependencies. Viewed in the context of the natural activity of spiny neurons, muscarinic depression of IKrp should uniformly increase excitability in both hyperpolarized and depolarized states. In the hyperpolarized state, the reduction in maximal conductance should bolster the efficacy of impending excitatory input. Likewise, in the depolarized state, the decreased availability of IKrp produced by the shift in inactivation should enhance ongoing synaptic input. The alterations associated with enhancement of IKrp are predicted to have a more dynamic influence on spiny cell excitability. In the hyperpolarized state, the negative shift in activation should increase the flow of IKrp and attenuate subsequent excitatory synpatic input; whereas once the cell has traversed into the depolarized state, the negative shift in inactivation should reduce the availability of this current and diminish its influence on the existing excitatory barrage.


    INTRODUCTION
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INTRODUCTION
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Clinical evidence indicates that dysfunction of the cholinergic system within the striatum plays a critical role in the manifestation of a variety of neurological disorders such as Parkinson's disease (Barbeau 1962). The striatum has one of the highest concentrations of acetylcholine (ACh) in the mammalian brain (Phelps et al. 1985), and this rich innervation arises from a small population of ACh-containing interneurons (Bolam et al. 1984). One of the primary targets of the cholinergic interneurons is the spiny neurons that give rise to the major striatal projection pathways (Bolam et al. 1984; Wilson et al. 1990). Given the relationship between the activity of spiny neurons and several aspects of motor function (Hikosaka 1994), some of the behavioral consequences of normal and abnormal ACh release in striatum are likely to be mediated by changes in the activity of these spiny cells.

The natural activity of spiny neurons recorded in vivo is characterized by recurring episodes of membrane hyperpolarization (approximately -85 mV) followed by subthreshold depolarization (approximately -45 mV) from which spike discharges can arise (Wilson and Groves 1981). Although the shifts between hyperpolarized and depolarized states require excitatory input from cortico- and thalamostriatal afferents (Wilson et al. 1983), recent evidence indicates that depolarization-activated potassium (K+) currents play a central role in regulating these transitions (Wilson and Kawaguchi 1996). In particular, K+ currents have been shown to shape the transitions to the depolarized state, as well as govern the voltage limits on this state (Wilson and Kawaguchi 1996). Three types of K+ currents have been identified including, 4-aminopyridine-sensitive (4-AP), fast (IAf), and slowly (IAs) inactivating A-type currents and a 4-AP-insensitive, persistent current (IKrp) (Gabel and Nisenbaum 1998; Nisenbaum et al. 1996; Surmeier et al. 1991). Of these K+ currents, IKrp is expected to contribute significantly to limiting the depolarizing periods due to its availability at subthreshold membrane potentials and slow kinetics of inactivation (Nisenbaum and Wilson 1995; Nisenbaum et al. 1996).

Cholinergic modulation of outward K+ currents would be expected to provide an additional layer of regulation of spiny cell activity. In situ hybridization studies indicate that much of the postsynaptic effect of ACh on spiny neurons will be transduced by stimulation of muscarinic receptors (Bernard et al. 1992; Hersch et al. 1994). Indeed, activation of muscarinic receptors has been shown to produce a voltage-dependent modulation IAf in cultured striatal neurons (Akins et al. 1990). Because the whole cell K+ current in cultured neurons is dominated by IAf, modulation of other currents was not readily testable. Given the critical role of IKrp in governing the subthreshold responses of spiny neurons, the possibility that this current may be a target of muscarinic modulation was investigated in acutely isolated cells from adult tissue.


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Neurons from young, adult (28-42 days old) male, Sprague-Dawley rats were acutely isolated from the striatum using previously described procedures (Nisenbaum et al. 1996). Briefly, animals were deeply anesthetized with methoxyflurane and perfused intracardially with a cold (~2°C) NaHCO3-buffered saline solution (in mM): 126 NaCl, 2 KCl, 2 CaCl2, 2 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 1 pyruvic acid, and 10 glucose; pH 7.4; osmolarity, 300 ± 5 mOsm/liter. After the perfusion, their brains were removed rapidly from the skull, and 400-µm-thick coronal sections were cut through the rostrocaudal extent of the striatum. Slices then were incubated at room temperature (~22°C) for 0.5-6.0 h in a continuously oxygenated (95% O2-5% CO2) NaHCO3-buffered saline solution. After the incubation period, the dorsal striata were placed into a HEPES-buffered Hank's balanced salt solution (HBSS) containing protease Type XIV (1 mg/ml; Sigma Chemical, St. Louis, MO) maintained at 37°C and oxygenated (100% O2). After 30-45 min of incubation in HBSS, the striata were triturated using fire-polished Pasteur pipettes, and the cell suspension was placed into a plastic Petri dish mounted onto the stage of an inverted microscope. Whole cell voltage-clamp recordings were performed at 22°C using glass micropipettes containing (in mM) 72 KF, 2 MgCl2, 40 HEPES, 3 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), 12 phosphocreatine, 2 Na2ATP, 0.2 GTP, and 0.1 leupeptin; pH 7.2; osmolarity, 275 mOsm/liter. The extracellular solution contained (in mM) 140 NaHOCH2CH2SO3, 1 KCl, 5 CaCl2, 1 MgCl2, 0.4 CdCl2, 10 HEPES, 10 glucose, and 0.001 tetrodotoxin; pH 7.4; osmolarity, 300 ± 5 mOsm/liter. For a subset of experiments, striatal neurons were retrogradely labeled following bilateral injections of rhodamine latex microspheres (400 µl each hemisphere) into the substantia nigra 2-5 days before electrophysiological recording (Surmeier et al. 1992).


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Carbachol can enhance or depress IKrp in spiny neurons

Consistent with previous reports (Nisenbaum et al. 1996), IKrp could be isolated from the other calcium-independent, depolarization-activated K+ currents in the presence of 10 mM 4-AP. In initial experiments, IKrp was evoked by 500-ms depolarizing voltage steps from -80 mV to +20 mV. Application of carbachol (2 µM) reversibly reduced this current in 31% of cells tested (n = 12 of 39; Fig. 1A). The peak current was depressed by 23% from 1.3 ± 0.7 nA (mean ± SD) in control conditions to 1.0 ± 0.5 nA in the presence of carbachol [t(11) = 4.5; P < 0.001; Fig. 1C]. In contrast, a reversible increase in IKrp was produced by carbachol (2 µM) in 51% of the cells tested (n = 20 of 39; Fig. 1B). For this subset of cells, peak current was enchanced by 30% from 1.0 ± 0.5 nA in control conditions to 1.3 ± 0.5 nA in the presence of carbachol [2 µM; t(19) = 8.9; P < 0.0001; Fig. 1D]. Application of carbachol had no effect on IKrp in 18% of the total number of neurons tested (n = 7 of 39). Although not systematically quantified, ~3-8 s of carbachol application was required for the depression of IKrp, whereas the enhancement of the current was fully developed after ~10-15 s of drug application.



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Fig. 1. Muscarinic receptor stimulation can produce either a reduction or an enhancement in IKrp. A and B: IKrp was isolated in 10 mM 4-aminopyridine (4-AP) and evoked by 500-ms depolarizing voltage steps from -80 to +35 mV. Carbachol (2 µM) either reduced or enhanced IKrp. C and D: histograms of the average (± SD) carbachol-induced decrease or increase in peak IKrp. E: plot of peak IKrp as a function of time and extracellular solution. Application of atropine (2 µM) alone did not affect IKrp nor did coapplication of carbachol (2 µM). However, carbachol alone produced a reversible enhancement in the current. F: atropine also blocked the carbacol-induced depression of IKrp.

Because the vast majority (>95%) of neurons within the striatum belong to the class of spiny projection neurons (Kemp and Powell 1971), the responses of the population of unidentified neurons were assumed to primarily have been recorded from spiny cells. To verify this assumption we conducted additional recordings from identified spiny projection neurons that were retrogradely labeled following injections of rhodamine microspheres into the substantia nigra. The effects of carbachol on IKrp recorded from these identified striatonigral spiny cells showed that the agonist produced either a depression (41%, 9 of 22 cells), an enhancement (45%, 10 of 22 cells), or no change in (14%, 3 of 22 cells) in IKrp.

The muscarinic receptor specificity of the carbachol-induced enhancement and depression of IKrp was tested using the specific muscarinic antagonist, atropine. IKrp was evoked by voltage steps from -80 to +20 mV delivered every 7 s, and the peak amplitude of IKrp was measured as a function of time and extracellular solution. Application of atropine (2 µM) alone had no effect on IKrp nor did subsequent addition of carbachol (2 µM; Fig. 1E). However, the carbachol-induced enhancement of IKrp was evident following removal of atropine. The peak amplitude of IKrp returned to control levels during the wash period. Atropine also blocked the carbachol-induced depression of IKrp (Fig. 1F). The potential contribution of nicotinic receptors to these responses was tested using the antagonist, mecamylamine (10-20 µM). Neither the carbachol-induced enhancement nor depression of IKrp was affected by nicotinic receptor blockade. Collectively, these results demonstrate that the modulatory effects of carbachol on IKrp depend on stimulation of muscarinic receptors.

Carbachol differentially affects the voltage-dependence of IKrp

The biophysical mechanisms through which carbachol exerted its effects on IKrp also were investigated. The differential effects of carbachol could have been produced by either shifts in the voltage dependencies of activation, inactivation, and/or changes in maximal conductance. Possible alterations in the voltage dependencies of activation were tested by stepping the membrane potential from -90 mV to potentials between -70 and +35 mV. The normalized conductances were plotted as a function of step potential and fit with a Boltzmann function. The carbachol-induced decrease in IKrp was not associated with an alteration in the voltage dependence of the current. The average half-activation voltages (Vhact) were not significantly different (control Vhact = 2.4 ± 5.3 mV; carbachol Vhact = 0.1 ± 5.2 mV, n = 16), nor were the slope factors (Vcact; control Vcact = 11.6 ± 1.1 mV; carbachol Vcact = 11.3 ± 1.5 mV). However, carbachol produced a consistent decrease in maximal conductance (gmax) of IKrp from 14.5 ± 6.9 nS in control conditions to 11.6 ± 6.3 nS in the presence of the agonist [t(15) = 2.9; P < 0.0001, n = 16; Fig. 2, A-C]. In contrast to the depressive effects of carbachol, the enhancement of IKrp was associated with a hyperpolarizing shift in the voltage dependence of activation (Fig. 2, D-F). The magnitude of the current was considerably larger, particularly at hyperpolarized membrane potentials. The average Vhact values shifted toward more hyperpolarized potentials from 6.5 ± 1.8 mV during control conditions to -0.9 ± 2.0 mV in the presence of carbachol [t(6) = 13.4; P < 0.0001, n = 7]. No change in the slope factor (control Vcact = 11.5 ± 1.0 mV; carbachol Vcact = 9.8 ± 4.4 mV, n = 7) or maximal conductance was observed (control gmax = 18.6 ± 4.9 nS; carbachol gmax = 18.9 ± 6.3 nS, n = 7).



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Fig. 2. Muscarinic-induced reduction and enhancement of IKrp is marked by differential effects on the voltage dependence of activation. A, B, D, and E: voltage dependence of steady-state activation of IKrp was assessed by delivering 500-ms depolarizing voltage steps from -70 to +35 mV (15-mV increments; holding potential, -90 mV) during control and carbachol conditions. C and F: peak amplitude of IKrp was measured in response to each voltage step and converted to conductance. The conductance (C) or normalized conductance (F) values for IKrp were averaged and plotted as a function of membrane potential. The data were fit using a Boltzmann function of the form g or 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. The reduction in IKrp was associated with a decrease in gmax, but no change in Vh or Vc. The enhancement of IKrp was associated with a negative shift in Vh.

Possible shifts in the voltage dependencies of inactivation of IKrp were assessed by stepping the membrane potential from -80 mV to potentials between -110 and -10 mV for 5 s before delivering a test step to +35 mV for 250 ms (Fig. 3). The current amplitudes were normalized, plotted as a function of the conditioning membrane potential and were fit with a Boltzmann function (Fig. 3, C and F). For cells (n = 12) in which carbachol decreased IKrp, the voltage dependence of inactivation of the current was shifted toward hyperpolarized membrane potentials. The average half-inactivation voltage (Vhinact) shifted from -49.0 ± 4.6 mV during control conditions to -59.6 ± 5.1 mV in the presence of carbachol [t(11) = 9.0; P < 0.0001]. No change in the slope factor (Vcinact) of the current-voltage relationship was observed (control Vcinact = 14.0 ± 0.9 mV; carbachol Vcinact = 14.6 ± 1.0 mV). This considerable shift in voltage dependence may have accounted for some of the apparent decrease in maximal conductance given the holding potential of -90 mV. A similar hyperpolarizing shift in the voltage dependence of inactivation of IKrp was associated with the enhancement of this current (n = 8). The average Vhinact values were -37.7 ± 3.9 mV and -45.9 ± 3.1 mV for control and carbachol conditions [t(7) = 5.6; P < 0.001], respectively. This hyperpolarizing shift in voltage dependence also was accompanied by a change in the steepness of the conductance-voltage relationship {control Vcinact = 10.7 ± 1.0 mV; carbachol Vcinact 12.7 ± 1.6 mV; [t(7) = 5.7; P < 0.001]}. An unexpected observation was that the control Vhinact values for cells in which IKrp was reduced (-49.0 ± 4.5 mV) or enhanced (-37.7 ± 3.9 mV) were significantly different ([t(7) = 5.1; P < 0.005), suggesting that two subpopulations of spiny neurons are present. Indeed, the voltage dependence of inactivation for a given cell could be used to predict its response (i.e., increase or decrease) to muscarinic receptor stimulation. No differences in Vhact values were found for the two groups of neurons.



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Fig. 3. Muscarinic modulation of IKrp is associated with hyperpolarizing shifts in the voltage dependencies of inactivation. A, B, D, and E: voltage dependence of steady-state inactivation of IKrp 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. C and F: relationship between the average normalized peak current and conditioning membrane potential is shown and is fit with a Boltzmann function of the form I/Imax = 1/{1 + exp[(Vm - Vh)/Vc+]}, where I is current, Imax is the current evoked by the test step from -110 mV, Vm is the membrane potential, Vh is the half-inactivation voltage, and Vc is the slope factor. C: both the reduction and enhancement of IKrp were associated with negative shifts in the voltage dependence of inactivation. G: carbachol-induced increase in IKrp was evident when the current was evoked from relatively hyperpolarized potentials (-80 mV). In contrast, the same neuron displays a reduction in IKrp when evoked from depolarized potentials (-40 mV).


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The present results demonstrate that IKrp can be depressed or enhanced by muscarinic receptor stimulation in striatal spiny neurons and that the biophysical nature of these effects are considerably different. The depression of IKrp is characterized by a hyperpolarizing shift in the voltage dependence of inactivation and a reduction in gmax. In contrast, the enhancement of IKrp is associated with hyperpolarizing shifts in both the voltage dependence of activation and inactivation. The functional consequences of these two effects on the responses of spiny neurons to excitatory input in the hyperpolarized and depolarized states should be markedly different. As described above, the membrane potential of spiny neurons resides at potentials near -85 mV in the hyperpolarized state and -45 mV in the depolarized state (Stern 1998; Wilson and Kawaguchi 1996). On the basis of its voltage dependence and kinetic characteristics, IKrp has been postulated to contribute significantly to limiting the level of depolarization associated with the depolarized state (Nisenbaum et al. 1996). As such, muscarinic stimulation in the hyperpolarized state should decrease the subsequent availability of IKrp through a reduction in maximal conductance and thereby enhance the level of depolarization evoked by impending excitatory input once the cell makes the transition into the depolarized state. Likewise, in the depolarized state, the decreased availability of IKrp produced by the hyperpolarizing shift in inactivation voltage dependence should bolster the efficacy of ongoing synaptic input. Thus muscarinic depression of IKrp should uniformly augment spiny cell excitability.

The hyperpolarizing shifts in activation and inactivation voltage dependence associated with the muscarinic receptor-mediated enhancement of IKrp are similar to those previously described for IAf in striatal neurons (Akins et al. 1990). These alterations are predicted to have a more dynamic influence on spiny cell excitability such that in the hyperpolarized state the negative shift in activation should increase the flow of IKrp and attenuate subsequent excitatory synpatic input, thereby maintaining the neuron in this state. In contrast, once the cell has traversed into the depolarized state, the negative shift in inactivation of IKrp should reduce the availability of this current, diminishing its influence on the existing excitatory barrage leading to greater depolarization. These hypotheses were tested within the context of the present experiments. Results showed that when IKrp was evoked from negative potentials (e.g., -80 mV) corresponding to the hyperpolarized state, muscarinic stimulation enhanced the current. However, when evoked from potentials corresponding to the depolarized state (e.g., -40 mV), IKrp was reduced (Fig. 3G). Thus these muscarinic effects are postulated to confer a stabilizing effect on the prevailing state (i.e., hyperpolarized or depolarized) of spiny neurons (Akins et al. 1990).

Although the muscarinic receptor subtypes mediating the effects on IKrp were not investigated, of the five receptors that have been cloned, m1 and m4 are preferentially expressed on spiny neurons (Bernard et al. 1992; Hersch et al. 1994). Therefore one explanation for the differential modulation of IKrp is that the nature of the effect depends on selective stimulation of m1 or m4 receptors. However, this hypothesis is difficult to reconcile with the extensive colocalization (~60%) of m1 and m4 receptors on spiny neurons (Bernard et al. 1992). Nonetheless, previous studies in spiny neurons have demonstrated distinct m1 and m4 receptor modulation of calcium (Ca2+) currents. The m1 modulation is dependent on intracellular Ca2+ concentration [Ca2+]i and insensitive to pertussis toxin (PTX) and targets L-type Ca2+ channels, whereas the m4 modulation is independent of [Ca2+]i and PTX-sensitive and targets N-and P-type Ca2+ channels (Howe and Surmeier 1995). In light of these findings, it is possible that both m1 and m4 modulation of IKrp could be revealed in spiny cells by varying [Ca2+]i and/or inhibition of PTX-sensitive G-proteins respectively (Howe and Surmeier 1995). Alternatively, the differential modulation of IKrp may indicate that the specific K+ channels subtypes that give rise to IKrp differ between spiny neurons, a possibility supported by the differences in inactivation voltage dependence of the current. Further studies will be required to distinguish between these possibilities.


    ACKNOWLEDGMENTS

This research was supported by a grant from the National Institute of Neurological Disorders and Stroke (NS-34254) to E. S. Nisenbaum.


    FOOTNOTES

Present address and address for reprint requests: E. S. Nisenbaum, Lilly Research Laboratories, LCC Drop Code 0510, Eli Lilly and Company, Indianapolis, IN 46285.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 11 September 1998; accepted in final form 8 November 1998.


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