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The Journal of Neuroscience, May 15, 1998, 18(10):3537-3547
The weaver Mutation Causes a Loss of Inward Rectifier
Current Regulation in Premigratory Granule Cells of the Mouse
Cerebellum
Paola
Rossi,
Giovanna
De
Filippi,
Simona
Armano,
Vanni
Taglietti, and
Egidio
D'Angelo
Istituto di Fisiologia Generale, I-27100, Pavia, Italy, and
Istituto Nazionale per la Fisica della Materia, Pavia Unit, Italy
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ABSTRACT |
Considerable interest has recently focused on the
weaver mutation, which causes inward rectifier channel
alterations leading to profound impairment of neuronal differentiation
and to severe motor dysfunction in mice (Hess, 1996 ). The principal
targets of mutation are cerebellar granule cells, most of which fail to differentiate and degenerate in a premigratory position (Rakic and
Sidman, 1973a,b). Two hypotheses have been put forward to explain the
pathogenetic role of mutant inward rectifier channels: namely that
inward rectifier channel activity is either lacking (Surmeier et al.,
1996) or altered (Kofuji et al., 1996; Silverman et al., 1996;
Slesinger et al., 1996). We have examined this question by recording
inward rectifier currents from cerebellar granule cells in
situ at different developmental stages in wild-type and weaver
mutant mice. In wild-type mice, the inward rectifier current changed
from a G-protein-dependent activation to a constitutive activation as
granule cells developed from premigratory to postmigratory stages. In
weaver mutant mice, G-protein-dependent inward rectifier currents were
absent in premigratory granule cells. A population of putative granule
cells in the postmigratory position expressed a constitutive inward
rectifier current with properties compatible with mutated GIRK2
channels expressed in heterologous systems. Because granule cells
degenerate at the premigratory stage (Smeyne and Goldowitz, 1989), the
loss of inward rectifier current and its regulation of membrane
potential are likely to play a key role in the pathogenesis of weaver
neuronal degeneration.
Key words:
cerebellum; granule cell; channellopathy; weaver; inward rectifier; mouse
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INTRODUCTION |
Ionic channels in neurons have the
dual role of controlling membrane potential, thus allowing information
coding that is essential for nervous system computation, and of
regulating basal cellular processes such as growth, survival, and death
(Spitzer, 1991). K+ channels play an important role
in membrane potential regulation. In the weaver mutation,
which causes neuronal degeneration and severe motor dysfunction in
mice, an inward rectifier K+ channel is altered
(Patil et al., 1995), and neuronal degeneration is observed in the
cerebellum, substantia nigra, and hippocampus. In particular, most
cerebellar granule cells fail to differentiate and die in ectopic
position, causing severe disruption of the cerebellar network (Rakic
and Sidman, 1973a,b; Sotelo and Changeux, 1974). Although the weaver
mutation has been identified, its implications in terms of the
pathogenetic mechanism remain largely unknown (Hess, 1996).
It has been shown that the weaver mutant channel expressed in oocytes
has lower K+ selectivity than the wild-type and has
lost the modulatory G-protein control, thus becoming constitutively
active (Slesinger et al., 1996). An anomalous Na+
influx would cause membrane depolarization, possibly facilitating neuronal degeneration through secondary
Ca2+-dependent processes (Kofuji et al., 1996). The
absence of inward rectifier currents in granule cells in culture has
suggested, however, that a loss of function was the main effect of the
mutation (Surmeier et al., 1996). Here too, uncontrolled membrane
depolarization would link the mutation to its pathological
consequences. To be realistic, these hypotheses need to be based on a
knowledge of the native properties of the inward rectifier current and
its effect on membrane potential. This is particularly relevant to granule cells, whose electroresponsiveness follows a specific developmental pattern in situ in terms of both inward
rectification and action potential generation (D'Angelo et al., 1997).
We therefore have compared inward rectifier currents in wild-type and
weaver granule cells, as well as their G-protein modulation and effect on membrane potential, at different developmental stages using whole-cell patch-clamp recordings in acute cerebellar slices.
Here we show that in wild-type mice an inward rectifier current was
elicited by G-protein activation in premigratory granule cells. The
inward rectifier current changed voltage dependence and kinetics in
postmigratory granule cells, thus becoming constitutively active. In
weaver mutant mice, neither constitutive nor
G-protein-dependent inward rectifier currents were observed in
premigratory granule cells, providing compelling evidence that a loss
of inward rectifier function occurs at the time of granule cell
degeneration (Smeyne and Goldowitz, 1989; Surmeier et al., 1996). Some
neurons in a deep position that expressed a constitutive inward
rectifier current with properties compatible with those reported in
reconstituted systems (Kofuji et al., 1996; Silverman et al., 1996;
Slesinger et al., 1996) apparently corresponded to granule cells that
had migrated and developed synaptic connections (Sotelo and Changeux, 1974). A loss of inward rectifier current at the premigratory stage and
its regulation of membrane potential are therefore likely to play the
key role in the pathogenesis of weaver neuronal degeneration.
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MATERIALS AND METHODS |
Patch-clamp whole-cell recordings were performed in granule
cells in 250-µm-thick mouse cerebellar slices (Edwards et al., 1989).
Either wild-type (Swiss strain) or homozygous weaver mutant mice
(B6CBACa-Aw-J/A-wv) were used. Homozygous
weaver mice were obtained from intercross mating of
heterozygous weaver mice (Jackson) and amounted to ~25% of littermates, consistent with simple mendelian segregation. Homozygous weaver mice were identified on the basis of their evident motor dysfunction, their dramatic cerebellar atrophy, and the abnormal
morphology of cerebellar tissue during microscopic observation of the
slices (Rakic and Sidman, 1973a,b). The slices were obtained on
postnatal days 9-15 (day of birth = P1) from both wild-type and
weaver mice. Cerebellar slices were prepared as reported previously (D'Angelo et al., 1995, 1997). Briefly, the mice were anesthetized with halothane (Aldrich, Milwaukee, WI) before they were decapitated. Krebs' solution for slice cutting and recovery contained (in
mM): NaCl 120, KCl 2, MgSO4 1.2, NaHCO3 26, KH2PO4 1.2, CaCl2 2, glucose 11. This solution was equilibrated with
95% O2 and 5% CO2, pH 7.4. Slices were
maintained at room temperature before being transferred to the
recording chamber (1.5 ml) mounted on the stage of an upright microscope (Zeiss Standard-16). The preparations were superfused at a
rate of 5-10 ml/min with Krebs' solution and maintained at 30°C
with a feed-back Peltier device (HCC-100A; Dagan Corporation, Minneapolis, MN).
Data recording and analysis. The excitable
responses and membrane currents of the granule cells were recorded
using an Axopatch 200-A amplifier. The data were sampled with a TL-1
DMA Interface (sampling time = 250 µsec for current-clamp
recordings, 10 µsec for voltage-clamp recordings) and analyzed with
P-CLAMP software (Axon Instruments, Foster City, CA). Mossy fiber
stimulation was performed with a bipolar tungsten electrode (Clark
Instruments, Pangbourne, UK) via a stimulus isolation unit. The
stimulating electrode was placed over the mossy fiber bundle, and
stimuli were applied at a frequency of 0.1 Hz. Control and test
solutions were applied locally through a multi-barrel pipette.
Perfusion of control solution was commenced before seal formation and
was maintained until we switched to test solutions. QX-314 was obtained from Alomone (Tel-Aviv, Israel), and BAPTA tetrapotassium salt was
obtained from Molecular Probes (Eugene, OR).
Patch pipettes were pulled from borosilicate glass capillaries
(Hingelberg, Malsfeld, Germany) and had 8-12 M resistance before a
seal was formed with a filling solution containing (in mM):
potassium gluconate 126, NaCl 4, MgSO4 1, CaCl2
0.02, BAPTA 0.1, glucose 15, ATP 3, HEPES 5 (pH was adjusted to 7.2 with KOH), and either GTP 0.1 or GTP- -S 0.1. This solution allowed
current-clamp recordings of the excitable response as well as
voltage-clamp recordings of the inward rectifier current. Considering
that this solution was diluted by 10% before use, and assuming a 0.75 activity coefficient for K+ salts (Pitzer and
Mayorga, 1973), the nernstian reversal potential for
K+ ions was  85.7 mV. Intracellular
Ca2+ was buffered at 100 nM, similar to
the resting Ca2+ concentration measured in granule
cells (Marchetti et al., 1995).
After a gigaseal was formed (seal resistance was usually >20 G),
electrode capacitance was carefully cancelled before the patch was
ruptured to allow for the electronic compensation of pipette charging
during subsequent current-clamp recordings (fast current-clamp mode). Depending on the high input-to-series resistance ratio, bridge balancing in current-clamp recordings proved of little
effect and was not used routinely (D'Angelo et al., 1995). Membrane
potential was measured relative to an agar-bridge reference electrode.
Reported membrane potential values have been adjusted off-line for
liquid-junction potentials (usually <5 mV). Voltage-clamp recordings
were used to record the inward rectifier current (see below) and to
estimate the passive granule cell membrane properties. For this
purpose, current transients were elicited by 10 mV hyperpolarizing pulses from a holding potential of 70 mV. The transients showed a
monoexponential relaxation (time constant = 81 ± 27 µsec;
n = 40) and were used to estimate series resistance
(21.1 ± 8.7 M; n = 40) and input resistance
and capacitance.
The experimental tracings were analyzed using P-Clamp (Axon
Instruments) and Origin (Microcal Software, Northampton, MA) software. Data are reported as mean ± SD, and statistical comparisons were performed using Student's t test.
Recording and analysis of inward rectifier current. The
inward rectifier current was activated with hyperpolarizing voltage pulses from a holding membrane potential of 40 mV. Alternatively, I-V relationships were constructed using voltage
ramps. Transient current and leakage subtraction were performed either
by using tracings recorded before GTP--S action or tracings in which
the inward rectifier current was blocked pharmacologically, as
explained in Results. The I-V relationships were
fitted with a modified Boltzmann equation (Hille and Schwarz, 1978) as
follows:
![I(V)=[G<SUB><UP>max</UP></SUB>(V−V<SUB><UP>rev</UP></SUB>)]/[1+<UP>exp</UP>((V−V<SUB>1/2</SUB>)/k)]](/content/vol18/issue10/fulltext/3537/img001.gif)
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(1)
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where Gmax is the maximal conductance,
Vrev is the reversal potential,
V1/2 is the potential at which the current is
half-activated, and k is the voltage dependence of
activation (V 1). Once
Vrev and Gmax were known,
and because G(V) = I(V)/(V Vrev), activation curves of the
form:
![G/G<SUB><UP>max</UP></SUB>=1/[1+<UP>exp</UP>((V−V<SUB>1/2</SUB>)/k)]](/content/vol18/issue10/fulltext/3537/img002.gif)
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(2)
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were plotted and used to compare inward rectifier voltage
dependencies.
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RESULTS |
We investigated inward rectifier currents and their relationship
with granule cell electroresponsiveness in wild-type and weaver mutant
granule cells in acute mice cerebellar slices. Recordings were
performed at different depths in the cerebellar cortex, allowing the
electrophysiological properties to be correlated with neuronal developmental stage. Unless stated otherwise, the granule cells were
recorded at P10-P15.
Electroresponsiveness in wild-type granule cells
In wild-type mice, at P10-P15 the germinal layer and the
premigratory zone, constituting the external granular layer (EGL) of
the cerebellar cortex, are clearly recognizable (Fig.
1A) (Rakic and Sidman,
1973a). After migration, the granule cells populate the internal
granular layer (IGL), become synaptically connected with mossy fibers,
and complete their maturation. The electrophysiological properties of
granule cells recorded at different depths are shown in Figure
1B and Table 1.
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Figure 1.
Electroresponsiveness in developing wild-type
granule cells. A, Schematic view of a cerebellar slice
at P15 (after Rakic and Sidman, 1973a,b; Sotelo and Changeux, 1974).
The indices a through c correspond to
different positions in the cortex from which granule cells were
recorded [a = germinal layer,
b = premigratory zone, c1 = postmigratory zone (immature cells), c2 = postmigratory zone (mature cells)]. B, Positive and
negative current pulses have been injected into granule cells recorded
using patch pipettes containing GTP and maintained at the indicated
resting potential with constant current injection. No inward
rectification can be observed in granule cells
a-c1, whereas a marked nonsagging
inward rectification (arrowhead) is expressed in the
granule cell c2 (also see D'Angelo et al., 1995, 1997).
This granule cell is synaptically connected, as demonstrated by the
EPSP generated by mossy fiber stimulation (inset:  mossy fiber stimulation. Calibration: 10 mV, 100 msec). The
current-clamp protocol is illustrated at the bottom. C,
Voltage-current plot for the cells in B. Membrane
potential has been averaged over the last 500 msec of the tracings.
D, Voltage recordings from a granule cell of the
premigratory zone using a patch pipette containing GTP--S. Although
the cell shows no inward rectification at the beginning of recording
(t0), inward rectification developed
after 5 min (t5). To compare the
effect of injected current pulses, tracings have been recorded from an
arbitrary membrane potential (70 mV) maintained with constant current
injection. Therefore the 14 mV hyperpolarization occurring with
GTP--S action is not seen in the figure. The bottom voltage-current
plot reports membrane potentials averaged over the last 500 msec of the
tracings, illustrating the induction of inward rectification by
GTP--S.
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In an initial set of experiments we used patch pipettes containing 100 µM GTP (Fig. 1B). The granule cells in the
germinal layer (a) and premigratory zone (b) of
the EGL showed a rather high resting membrane potential (a,
24 ± 1.4 mV, n = 8; b, 22.9 ± 3, n = 11). These cells did not generate any action
potential when they were injected with depolarizing currents, although
the membrane had previously been hyperpolarized with a constant
negative current. The injection of hyperpolarizing pulses did not
reveal any inward rectification. The granule cells in the IGL showed either immature (c1) or mature (c2) electrical
responses, which were distinguished on the basis of the nature of
action potentials and the presence of inward rectification (D'Angelo
et al., 1997). Immature responses consisted of slow spikes, absence of
inward rectification, and high resting potential (c1,
36.3 ± 9.1 mV; n = 8). Mature responses
consisted of fast spikes, inward rectification, and a negative resting
membrane potential (c2 57.5 ± 10.6 mV; n = 6). Similar granule cells were most commonly
observed after P21 (seven of eight; data not shown), when migration is
concluded and the EGL is no longer evident. The
V-I relationships shown in Figure 1C
compare voltage response elicited by current injection into the granule
cells, evidencing constitutive inward rectification in the granule cell
that generates fast spikes.
When the nonhydrolyzable G-protein activator GTP--S was included in
the patch pipette, inward rectification could be induced in postmitotic
granule cells that did not show it constitutively (Fig.
1D). In these cells GTP--S induction of inward
rectification was associated with a significant lowering in resting
membrane potential (b, 13 ± 3.7 mV,
n = 9; c1, 16.1 ± 4.3 mV,
n = 8; p < 0.02 in both cases). Inward
rectification was manifest as a decrease in input resistance in
V-I plots. Inward rectification extended well
over the K+ reversal potential (85.6 mV) into the
resting membrane potential region, as would be expected from current
voltage dependence measured in voltage-clamp experiments (see below).
No significant inward rectification or membrane potential changes were
observed in granule cells of the germinal layer [a, 4 ± 3.2 mV, n = 8; not significant (NS)], or in
fast spiking granule cells of IGL (c2, 1.1 ± 3.2 mV,
n = 7; NS). It should be noted that both the
constitutive and GTP--S dependent inward rectification did
not sag, indicating that the underlying current had
activation kinetics that were faster than passive membrane
charging.
G-protein modulation of the inward rectifier current in
wild-type granule cells
The nature of the current generating inward rectification, and its
modulation by G-proteins (Wickman and Clapham, 1995), has been
investigated (Fig. 2A),
and the time course of GTP--S action has been compared with that of
GTP (Fig. 2B).
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Figure 2.
GTP--S modulation of the inward rectifier
current in wild-type cerebellar granule cells. A, The
recordings were performed with patch pipettes containing GTP--S.
Tracings were taken at the beginning
(t0) and 6 min after the beginning of
recordings (t6). The
GTP--S-sensitive current is shown in the subtraction tracings
(t6-t0).
Note that an inward rectifier current develops in granule cells of the
premigratory zone (b; P12) and in immature postmigratory
granule cells (c1; P12), but not in granule cells in the
germinal layer (a; P13). Mature postmigratory granule
cells showed a constitutive inward rectifier current, which slightly
decreased with time (c2; P22). Note that an outward tail
current usually developed on returning to 40 mV, probably reflecting
I-A rebound activation. The voltage-clamp protocol is illustrated at
the bottom. B, The plots report the time course of
inward rectifier modulation with pipettes containing GTP--S ( )
compared with pipettes containing GTP ( ). The recordings commenced
at t = 0. The current is reported relative to
control for membrane pulses from 40 mV to 120 mV. The data are
reported as mean ± SD (n = 6-9 for each
plot). Note that GTP--S elicited an inward rectifier current in
b and c1 but not in a. A
slight decrease in the constitutive current was observed in
c2 with both GTP--S and GTP. Labels
a-c have the same meaning as in Figure
1A.
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(a) In the germinal layer, no inward rectifier current could be
demonstrated, despite the presence of GTP--S in the patch pipette (8 of 8).
(b) In the premigratory zone, the granule cells showed inward rectifier
current expression during GTP--S internal perfusion (G-IIR; 9 of 10). No inward
rectifier current was observed at the beginning of the recording or
when patch pipettes containing GTP were used (9 of 9), indicating the
absence of constitutive activation. The time course of GTP--S action
was similar to that reported previously in culture (Surmeier et al.,
1996).
(c1) In the IGL, some of the granule cells (7 of 12) showed inward
rectifier current modulation during GTP--S internal perfusion (G-IIR), but no inward
rectifier current was measured at the beginning of the recording.
Inward rectifier current modulation in these granule cells was
therefore similar to that in cells in the premigratory zone.
Accordingly, when we used patch pipettes containing GTP, no inward
rectifier current was observed in the majority of granule cells (7 of
10), whereas a constitutive inward rectifier current was observed in
the remaining granule cells (3 of 10; see point c2).
(c2) In the IGL, some of the granule cells showed a constitutive inward
rectifier current (C-IIR). In
addition to being observed at the beginning of the recordings with
pipettes containing GTP--S (5 of 12),
C-IIR was also observed when patch
pipettes containing GTP were used (3 of 10). At a more advanced
developmental stage (P21-P22),
C-IIR was recorded in the majority
of granule cells (seven of nine; data not shown).
These results indicate that the inward rectifier current follows a
precise developmental time course in situ, leading from G-protein-dependent to constitutive activation.
Biophysical and pharmacological properties of the inward rectifier
current in wild-type mice
G-IIR and C-IIR
displayed similar pharmacological sensitivity (Fig.
3A). These currents were
completely blocked by 1 mM Ba2+
(97.2 ± 6.1% G-IIR block,
n = 5; 98.6 ± 4.3% C-IIR
block, n = 4) but were only partially blocked by 5 mM Cs+ (68.4 ± 25.4%
G-IIR block, n = 8; 58.6 ± 41.6% C-IIR block, n = 4).
Moreover, these currents were insensitive to 100 µM
QX-314 (3.5 ± 2.2% G-IIR block,
n = 4; 16.6 ± 19.3%
C-IIR block, n = 4),
a cationic channel blocker known to affect Na+
channels as well as inward rectifier channels with partial
Na+ permeability (Perkins and Wong, 1995; Kofuji et
al., 1996; Slesinger et al., 1996). G-IIR and
C-IIR were analyzed using tracings taken after
pharmacological block with 1 mM Ba2+ for
leakage and transient current subtraction (Fig. 3A).
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Figure 3.
Biophysical and pharmacological properties of the
wild-type inward rectifier current. A1, A2, Both the
current generated by GTP--S (A1,
G-IIR) and that constitutively
expressed (A2, C-IIR)
in granule cells were blocked by 1 mM
Ba2+. The Ba2+-sensitive currents
have been obtained by subtraction (control Ba). The
voltage-clamp protocol is illustrated at the bottom. The
inset illustrates the insensitivity of
G-IIR to 100 µM QX-314 and
partial block by 1 mM Cs+ in two
different cells (the tracings are leakage-subtracted, demonstrating the
extent of block). B1, B2, Tracings obtained using the
subtraction protocol shown in A have been used for
kinetic analysis. G-IIR (B1)
was fitted with a double exponential function of the form
i(t) = A1 × exp(t/ 1) + A2 × exp(t/2) + C. The
individual exponential components as well as the biexponential function
are superimposed on the experimental tracing (A1 = 40.2 pA; 1 = 1.1 msec; A2 = 16.1 pA;
2 = 18.5 msec; C = 42).
C-IIR (B2) was fitted with a
single exponential function of the form
i(t) = A1 × exp(t/1) + C. The
monoexponential function is superimposed on the experimental tracing
(A1 = 31.6 pA; 1 = 2.7 msec;
C = 31.6 pA). The tracings in B1
and B2 were generated by a voltage pulse from 40 mV to
120 mV. C1, C2, The voltage dependence of the fast
inward rectifier current components of
G-IIR (C1) or
C-IIR (C2) was obtained
using a voltage-ramp protocol. The experimental points have been fitted
to Equation 1 to yield the following parameters: (C1)
Gmax = 1.17 nS;
Vrev = 79.66 mV;
V1/2 = 73.33 mV; k = 24.4 mV1; (C2)
Gmax= 2.78 nS;
Vrev = 84.8 mV;
V1/2 = 82.4 mV; k = 8.94 mV1. The voltage-ramp protocol is indicated
at the bottom.
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G-IIR did not show inactivation (Fig.
3A1). G-IIR activation comprised a
fast and a slow component and could be fitted with a biexponential
function (Fig. 3B1). The fast component had almost voltage-independent time constants in the submillisecond range and
accounted for 84% of G-IIR amplitude at 120
mV. The slow component had time constants in the 100 msec range and was
better resolved at potentials lower than 100 mV. The fast component therefore accounted for most of the G-IIR in
the physiological range of membrane potentials and should correspond to
the "instantaneous" rectification reported in other papers (Kofuji
et al., 1996; Surmeier et al., 1996). The slow component may itself be
composite (Doupnik et al., 1995), although no different subcomponents
were detectable in the time scale used here. The voltage dependence of
the fast G-IIR component was investigated using
a voltage-ramp protocol (Fig. 3C1). The
I-V relationship was fitted with Equation 1,
yielding the Boltzmann parameters V1/2 and
k as well as maximum conductance (Gmax) and reversal potential
(Vrev) (Table
2). Because of its (1) reversal potential
close to that of K+ ions, (2) fast kinetics, (3)
high sensitivity to Ba2+ block, and (4) modulation
by G-proteins, G-IIR appeared as the native
counterpart of GIRK2-containing channels measured in reconstituted systems (Duprat et al., 1995; Kofuji et al., 1995; Krapivinsky et al.,
1995; Silverman et al., 1996; Slesinger et al., 1996). A similar
current has been reported in mice granule cells in culture (Surmeier et
al., 1996).
C-IIR is shown in Figure 3A2.
C-IIR activated monoexponentially with time
constants in the millisecond range, whereas no slow components could be
detected (Fig. 3B2). The voltage dependence of
C-IIR was investigated using a voltage-ramp
protocol (Fig. 3C2). The results of an analysis similar to
that used for G-IIR are reported in Table 2. No
such constitutive fast inward rectifier current has been reported
previously in granule cells in culture, although its existence was
suggested by voltage recordings in slice preparations (D'Angelo et
al., 1995, 1997).
Table 2 and Figure 4 condensed data on
the biophysical properties of G-IIR and
C-IIR. C-IIR had
steeper voltage dependence than G-IIR,
and an activation time constant that was slower than the fast component
of G-IIR. Together with constitutive activation and the absence of a slow component, these were the major properties of
C-IIR that distinguished it from
G-IIR.
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Figure 4.
Average kinetics of wild-type inward rectifier
currents. A, Average I-V
plots of the fast inward rectifier current components of
G-IIR and C-IIR
have been reconstructed using Equation 1 and the average values
reported in Table 2. B, Normalized activation curves of
G-IIR and C-IIR
have been reconstructed using Equation 2. C, Voltage
dependence of the fast activation time constant of
G-IIR and of the activation time constant
of C-IIR. A-C show that
activation kinetics are faster but less voltage dependent in
C-IIR than in
G-IIR.
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Electroresponsiveness in weaver granule cells
In weaver mutant mice, cerebellar architecture is
profoundly disrupted (Fig. 5A)
(Rakic and Sidman, 1973a; Sotelo and Changeux, 1974). Below the
germinal layer, large neurons probably corresponding to ectopic
Purkinje cells were surrounded by groups of granule cells, and no IGL
could be identified. The small neurons beside or below Purkinje cells
in a deep position should correspond to granule cells "which have
achieved a complete migration reaching their normal position and
developing their normal connections" (Sotelo and Changeux, 1974),
although no direct differentiation vis-à-vis other neuronal types
(stellate or basket cells) could be achieved in the present
experiments.
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Figure 5.
Electroresponsiveness in developing weaver granule
cells. A, Schematic view of the homozygous weaver
cerebellum (after Rakic and Sidman, 1973a,b; Sotelo and Changeux,
1974). The indices
awv-cwv
correspond to different positions in the cortex, as illustrated in the
top inset (awv = germinal
layer, bwv = premigratory zone,
cwv = deep layer). B,
Positive and negative current pulses have been injected in granule
cells recorded in P10-P15 acute slices of the cerebellar vermis using
patch pipettes containing GTP, and maintained at the indicated resting
potential with constant current injection. Note that no inward
rectification can be observed in the granule cells
awv-bwv,
whereas marked sagging inward rectification (arrowhead)
is expressed in the granule cell cwv (also
see D'Angelo et al., 1995, 1997). This granule cell is synaptically
connected, as demonstrated by the EPSP generated by mossy fiber
stimulation (inset: mossy fiber stimulation.
Calibration: 10 mV, 100 msec). The current-clamp protocol is
illustrated at the bottom. C, Voltage-current plot for
the cells in B. Membrane potential has been averaged
over the last 500 msec of the tracings. D, Voltage
recordings from a granule cell of the premigratory zone using a patch
pipette containing GTP--S. Unlike wild-type granule cells, no inward
rectification developed after 5 min of GTP--S internal perfusion
(t6 as compared with
t0), nor was membrane
hyperpolarization measured (the tracings have been recorded from an
arbitrary membrane potential of 70 mV). The voltage-current plot at
the bottom reports membrane potentials averaged over the
last 500 msec of the tracings, illustrating the absence of inward
rectification induction.
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The granule cells displayed different electrophysiological behavior
depending on their location, as shown in Figure 5B and reported in Table 1 for slices obtained at P10-P15. When patch pipettes containing GTP were used, the granule cells in the germinal layer (awv) and in the premigratory zone
(bwv, recorded at 50-100 µm from the
pial surface) appeared similar to those of the wild type. They had a
rather high resting membrane potential
(awv, 22 ± 7.2 mV,
n = 8; bwv, 32 ± 2.7, n = 10) and did not generate either action
potentials or inward rectification during current injection. Some of
the granular neurons in a deeper position
(cwv, recorded at 100-200 µm from the
pial surface), however, had a more negative resting membrane potential
(57.6 ± 5.4 mV, n = 6), generated action
potentials (in three neurons a solitary fast spike was measured,
whereas the other three neurons generated a fast repetitive spike
discharge), and showed sagging inward rectification. These
neurons therefore exhibited a resting membrane potential and spike
discharge comparable to those of wild-type granule cells at an advanced
developmental stage in the IGL, although the nature of the inward
rectification was different (compare Fig. 1A, panel
C2). These neurons could be activated by afferent fiber
stimulation with a time delay of 1.5 msec for EPSP activation (Fig.
5B, inset). These neurons therefore are putative
postmigratory granule cells that developed until they formed functional
synapses. The V-I relationships in Figure
5B show that subthreshold voltage responses lacked
inward rectification in premigratory weaver granule cells, whereas
a constitutive inward rectification was evident in putative
postmigratory granule cells.
A major difference between weaver and wild-type granule cells was
revealed using patch pipettes containing GTP--S. Despite the
apparent similarity of their electrical response to the wild-type cells, the weaver granule cells showed neither induction of inward rectification (Fig. 5D) nor hyperpolarization from rest
during GTP--S perfusion (awv 5.1 ± 4.9 mV, n = 8; bwv 0 ± 3.3 mV, n = 10; cwv 5.6 ± 3.2 mV, n = 6; NS in all cases).
G-protein modulation of the inward rectifier current in weaver
granule cells
As in wild-type granule cells, the nature of the inward rectifier
current in weaver mutant mice, and its G-protein
sensitivity, were investigated (Fig.
6A), and the time
course of GTP--S action was compared with that of GTP (Fig.
6B).
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Figure 6.
GTP--S modulation of the inward rectifier
current in weaver cerebellar granule cells. A, The
recordings were performed with patch pipettes containing GTP--S.
Tracings were taken at the beginning
(t0) and 6 min after the beginning of
recordings (t6). The
GTP--S-sensitive current is shown in the subtraction tracings
(t6-t0).
Note that no inward rectifier current develops in either granule cells
in the premigratory zone (bwv) or
those in the germinal layer (awv).
Granule cells in a deep position showed a constitutive inward rectifier
current, which slightly decreased with time
(cwv; note the different scale than
in awv,
bwv). The voltage-clamp protocol is
illustrated at the bottom. B, The plots
report the time course of inward rectifier modulation with
GTP--S-containing pipettes () compared with GTP-containing
pipettes (). The recordings commenced at t = 0. The current is reported relative to control for membrane pulses from
40 mV to 120 mV. The data are reported as mean ± SD
(n = 5-10 for each plot). Note that neither
GTP--S nor GTP elicited any inward rectifier current in
awv or bwv. A
slight decrease in the constitutive current was observed in
cwv with either GTP--S or GTP. Labels
awv-cwv
have the same meanings as in Figure 5A.
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(awv) As in wild-type mice, granule cells
in the germinal layer did not show any inward rectifier currents
despite the presence of GTP--S in the patch pipette (10 of 10).
(bwv) Unlike in wild-type mice, granule
cells in the premigratory zone did not show any inward rectifier
currents, despite the presence of GTP--S in the patch pipette (14 of
14). These granule cells were interesting, because they may be cells in
which mutation has caused a loss of G-protein sensitivity in the inward rectifier current (Surmeier et al., 1996). The weaver currents therefore differed from wild-type inward rectifier currents in their
G-protein-dependent modulation.
(cwv) Putative postmigratory granule
cells showed a constitutive inward rectifier current with anomalous
properties, wv-IIR (6 of 7) (Fig.
6C). In addition to being observed at the beginning of the
recordings in the presence of GTP--S,
wv-IIR was observed also when patch
pipettes containing GTP were used (7 of 7).
Biophysical and pharmacological properties of the inward rectifier
current in putative weaver granule cells
The weaver inward rectifier current,
wv-IIR, showed distinctive
pharmacological properties. Unlike the wild-type inward rectifiers, wv-IIR was inhibited by 100 µM QX-314 (60.6 ± 19.3% block; n = 5) (Fig. 7A). Sensitivity to
external QX-314 at low concentrations is typical of the homomultimeric
wv-GIRK2 channels expressed in oocytes (Kofuji et al.,
1996; Slesinger et al., 1996). The residual current was then blocked by
the addition of 0.5 mM Cs+ (98 ± 6% block; n = 5) (Fig. 7A). The other major
inward rectifier channel sensitive to submillimolar
Cs+, Ih, has never
been reported to be sensitive to submillimolar external QX-314
(although it can be blocked by 10 mM internal QX-314)
(Perkins and Wong, 1995). An additional four neurons that expressed an
inward rectifier current that was insensitive to QX-314 but was readily
blocked by 0.5 mM Cs+ have not been
considered as wv-IIR and have been
discarded from the present analysis. These neurons had a significantly
higher membrane capacitance than
wv-IIR-expressing neurons (15.4 ± 3.2 pF, n = 4, vs 7.9 ± 2.5 pF,
n = 6; p < 0.01), were autorhythmic, and generated an initial burst followed by a steady discharge during
current injection and were probably Purkinje cells.
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Figure 7.
Biophysical and pharmacological properties of the
weaver inward rectifier current. A,
wv-IIR was inhibited by 100 µM QX-314 and completely blocked by subsequent addition
of 0.5 mM Cs+. The QX-314- and QX-314
Cs+-sensitive currents have been obtained by
subtraction (control QX-314, control QX-314/Cs+). The voltage-clamp protocol is
illustrated at the bottom. B, QX-314
Cs+-sensitive
wv-IIR has been used for
kinetic analysis. wv-IIR was
fitted to a single exponential function of the form
i(t) = A1 × exp(t/1) + C. The
monoexponential function is superimposed on the experimental tracing
(A1 =184 pA; 1 =41.84 msec;
C = 203.6 pA). The tracing was generated by a
voltage pulse from 40 mV to 120 mV. C, Because of
its slow kinetics, the voltage dependence of
wv-IIR was obtained using
steady-state measurements from the
QX-314-Cs+-sensitive current (see
A). Experimental points have been fitted to Equation 1
to yield the following parameters: Gmax = 6.88 nS; Vrev = 61 mV;
V1/2 = 79.3 mV; k = 9.5 mV1.
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wv-IIR was analyzed using tracings
obtained after pharmacological block with Cs+ for
leakage and transient current subtraction after QX-314 sensitivity had
been verified (n = 6) (compare Fig. 7A).
wv-IIR had slow kinetics, activating
with time constants of tens to hundreds of milliseconds (Fig.
7B), and no fast components could be detected. Voltage
dependence was analyzed using Equation 1 (Fig.
8), and average parameter values are
reported in Table 2. The wv-IIR
reversal potential was ~20 mV more positive (64.1 ± 6.3 mV;
n = 4) than C-IIR or G-IIR, consistent with the reduced
selectivity of wv-GIRK2 homomultimers for
K+ ions (Navarro et al., 1996; Kofuji et al., 1996;
Slesinger et al., 1996). wv-IIR is unlikely to
contribute significantly to depolarizing putative granule cells,
however, because its activation voltage dependence was steep and
confined to negative membrane potentials. Moreover, unlike expression
systems, there was no net increase in leakage current in putative
weaver granule cells compared with wild-type granule cells (Table
1).
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Figure 8.
Average kinetics of the weaver inward rectifier
currents. A, Average I-V
plots of wv-IIR have been
reconstructed using Equation 1 and the average values reported in Table
2. B, A normalized activation curve of
wv-IIR was reconstructed
using Equation 2. The activation curves of
C-IIR and G-IIR
are replotted for comparison (dotted lines).
C, Voltage dependence of the fast activation time
constant of wv-IIR. Note
that the activation time constants are one to two orders of magnitude
larger than those of C-IIR and
G-IIR (compare Fig. 4C).
A-C show that
wv-IIR activation kinetics
differs from those of C-IIR and
G-IIR.
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DISCUSSION |
It has been proposed that cerebellar granule cell degeneration in
the weaver mutation is induced by either the absence of G-protein modulation or altered biophysical properties of an inward rectifier current (Kofuji et al., 1996; Silverman et al., 1996; Slesinger et al., 1996; Surmeier et al., 1996). In this paper we report
that the expression of inward rectifier currents and their G-protein
modulation in granule cells of the mice cerebellum are developmentally
regulated in situ. In the weaver mutation, the
absence of G-protein sensitivity at the premigratory stage appears to
be the primary process in the pathogenesis of the mutated phenotype.
The possibility that abnormal channel properties are manifest at more
advanced developmental stages in surviving granule cells is
discussed.
Development of inward rectifier currents in wild-type mice
We found the earliest functional evidence for inward rectifier
current expression in postmitotic premigratory granule cells. In these
cells the inward rectifier current was strictly G-protein-dependent (G-IIR). G-IIR
was also measured in postmigratory granule cells in the first 3 postnatal weeks. In these cells, however, other ionic currents also
differed from those in premigratory granule cells (D'Angelo et al.,
1994, 1997), allowing action potentials to be generated. In mature
granule cells, which generate fast action potentials, a constitutive
inward rectifier current was found
(C-IIR). This may indicate a change in
either channel properties or G-protein modulation. On the one hand, the
higher voltage sensitivity and the lack of a slow current component in
C-IIR compared with G-IIR suggest a change in channel properties.
GIRK channel properties arise from the assembly of different subunits
(Duprat et al., 1995; Kofuji et al., 1995; Krapivinsky et al., 1995),
and the expression of slow kinetics depends on the insertion of GIRK1 subunits into GIRK2-containing channels (Slesinger et al., 1996). Therefore a developmental regulation of channel subunit expression may
contribute to transforming G-IIR into
C-IIR. On the other hand, it seems unlikely
that constitutive C-IIR activation results from
a G  -independent channel regulation by intracellular Na+ (Silverman et al., 1996), because the
pipette solution contained a low intracellular
Na+ concentration (4 mM) in granule
cells expressing both G-IIR and C-IIR. However, we cannot rule out that changes
in G-protein-coupled receptor activation regulate inward rectifier
channels (Wickman and Clapham, 1995). Among several
neurotransmitters and modulators, glutamate has been shown to regulate
granule cell GIRK channels by activating metabotropic receptors
(Surmeier et al., 1996).
G-IIR and C-IIR were
probably both involved in setting the granule cell membrane potential,
although in quite different ways. G-IIR
activation extended into the high-voltage range, being well suited to
hyperpolarizing the membrane of immature granule cells from its
quiescent state at around 30 mV in response to exogenous modulatory
actions. Conversely, C-IIR was constitutively
active in a narrow negative voltage range, tending to clamp the
membrane within a negative potential region. In addition to changes in the inward rectifiers, other factors may contribute to stabilizing the
resting membrane potential at more negative values in mature rather
than immature granule cells, including a lowering in the GABA reversal
potential (Brickley et al., 1996) and increased activation of
K+ currents under muscarinic receptor control
(Watkins and Mathie, 1996).
Inward rectifier currents in weaver mice
Unlike the wild-type cells, premigratory weaver granule cells did
not show any inward rectifier currents induced by G-protein stimulation. This probably indicates a loss of G-protein modulation rather than a lack of inward rectifier channel expression, as suggested
by the observation that GIRK1, which is expressed in the immature
cerebellum, inhibits wv-GIRK2-containing channels (Navarro
et al., 1996; Slesinger et al., 1996). A loss of G-protein-dependent inward rectifier currents has also been reported in weaver granule cell
cultures by Surmeier et al. (1996). It should be noted that no increase
in the leakage current was detectable in our experiments (Table 1), as
in those of Surmeier et al. (1996) but unlike those of Kofuji et al.
(1996).
By recording from putative granule cells that escaped degeneration at
the premigratory stage (Sotelo and Changeux, 1974), we found an inward
rectifier current with slow kinetics dubbed wv-IIR that showed constitutive
activation and a marked reduction after the extracellular
application of low concentrations of QX-314. As well as being sensitive
to QX-314, wv-IIR showed a positive shift in its ionic reversal potential as compared with wild-type currents, consistent with reduced selectivity for K+
in favor of Na+ and Ca2+ ions.
These properties are compatible with the formation of
wv-GIRK2 homomultimers (Navarro et al., 1996; Kofuji et
al., 1996; Slesinger et al., 1996). However, the identification of
wv-IIR as the native counterpart of
wv-GIRK2-containing channels is not definitive. First,
unlike wv-GIRK2 homomultimers, the neurons expressing
wv-IIR did not show any net increase
in leakage current (Table 1). Second, wv-IIR shares with
Ih submillimolar Cs+
sensitivity and slow kinetics associated with sagging voltage responses
to step current injection. Although the
wv-IIR reversal potential was not as
high as expected from Ih, the distinction based on QX-314 sensitivity cannot be considered as absolute, because a
high intracellular QX-314 concentration blocked neuronal Ih (Perkins and Wong, 1995). Finally, although
neurons expressing wv-IIR did not
show intrinsic firing discharge or complex spikes, as occurs in
Ih-expressing Purkinje neurons (Crepel and
Penit-Soria, 1986), it is possible that neurons like stellate or basket
cells, which have unknown inward rectifier currents, could also have been recorded. Therefore, a direct identification of cell types associated with an extended analysis of inward rectifier currents in
cerebellar neurons is needed to confirm the suggestion that wv-IIR is the native counterpart of
mutated wv-GIRK2-containing channels.
Pathogenetic implications
The most important finding in seeking to identify the pathogenetic
role of the GIRK mutation (Patil et al., 1995) is that no
inward rectifier currents could be induced by G-protein stimulation in
weaver premigratory granule cells. These cells cannot use their inward
rectifier current (G-IIR), which is
presumably under the control of specific neuromodulators (Surmeier et
al., 1996), to hyperpolarize the membrane from rest. A failure to
regulate membrane potential may cause uncontrolled activation of
voltage-sensitive N-type Ca2+ channels and NMDA
receptors, which in normal conditions drive granule cell motility and
differentiation (Komuro and Rakic, 1993a,b), leading to
Ca2+ accumulation, neurotoxicity, and death
(Garthwaite, 1994; Choi, 1995). The role of NMDA receptors is
particularly interesting, because they are stimulated by ambient
glutamate in the EGL (Slater and Rossi, 1996) and membrane
depolarization unblocks the channel, causing Ca2+
permeation. The absence of G-IIR activation
seems likely to be the major pathogenetic mechanism, because most
granule cells degenerate when they are postmitotic in the premigratory
region of the EGL (Smeyne and Goldowitz, 1989; Surmeier et al.,
1996).
The few granule cells that pass the premigratory stage (Rakic and
Sidman, 1973b; Sotelo and Changeux, 1974) might suffer further neurodegenerative damage. To the extent that
wv-IIR is the native counterpart of
the mutated GIRK-2 channel expressed in oocytes, it might promote
further neurodegeneration through an anomalous Na+
and Ca2+ influx (Navarro et al., 1996; Silverman et
al., 1996). We noted, however, that the reversal potential remained
rather negative and the voltage dependence remained steep, so that
wv-IIR activation was confined to
low potentials. Consistently, wv-IIR
was not associated with any marked resting depolarization. We would
also point out that constitutive
wv-IIR activation was not
specifically related to the pathogenetic process, because a similar
property was typical of IGL wild-type granule cells (also see D'Angelo
et al., 1995, 1997).
Conclusions
The inward rectifier in cerebellar granule cells is
developmentally regulated, changing from G-protein-dependent to
constitutive activation as the granule cells develop from premigratory
to postmigratory stages. The G-protein-dependent form probably serves
to hyperpolarize the granule cell membrane under the control of
specific neurotransmitters and hormones (Kofuji et al., 1996; Surmeier
et al., 1996). The failure of inward rectifier G-protein activation
identifies an important correlate of the weaver mutation, localizing
its functional expression at the premigratory stage. This is probably
just the first step in a more complex neurodegenerative process (Hess, 1996), because granule cell development involves cell-cell
interactions and the weaver mutation acts nonautonomously (Gao et al.,
1992; Gao and Hatten, 1993; Hatten and Heintz, 1995). An altered
constitutive inward rectifier current may be expressed in weaver
granule cells that develop mature excitable properties (Kofuji et al.,
1996; Silverman et al., 1996; Slesinger et al., 1996), causing further cellular damage in a two-stage pathogenetic process. Our results support the concept that developmental regulation of GIRK channel subunit expression, and modulation of inward rectifier channel activation, may provide important signals for neuronal survival and
differentiation.
| |
FOOTNOTES |
Received Dec. 8, 1997; revised Feb. 26, 1998; accepted Feb. 27, 1998.
Correspondence should be addressed to Dr. Egidio D'Angelo, Istituto di
Fisiologia Generale, Via Forlanini 6, I-27100, Pavia, Italy.
This project was supported by Telethon Grant E.464 and by Istituto
Nazionale per la Fisica della Materia.
| |
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Top
Abstract
Introduction
