Department of Psychology, University of Connecticut, Storrs, Connecticut 06269
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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.
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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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METHODS |
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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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RESULTS |
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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGMENTS |
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This research was supported by a grant from the National Institute of Neurological Disorders and Stroke (NS-34254) to E. S. Nisenbaum.
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FOOTNOTES |
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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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REFERENCES |
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