 |
 Previous Article | Next Article 
The Journal of Neuroscience, August 1, 2001, 21(15):5813-5823
Expression of Protein Kinase C Inhibitor Blocks Cerebellar
Long-Term Depression without Affecting Purkinje Cell
Excitability in Alert Mice
Jeroen
Goossens1, 2,
Hervé
Daniel3,
Armelle
Rancillac3,
Johannes
van der Steen1,
John
Oberdick4,
Francis
Crépel3,
Christiaan I.
De
Zeeuw5, and
Maarten A.
Frens1
1 Department of Physiology, Neuroscience Institute,
Erasmus University Rotterdam, 3000 DR Rotterdam, The Netherlands,
2 Department of Medical Physics and Biophysics, University
of Nijmegen, 6500 HB Nijmegen, The Netherlands,
3 Laboratory of Neurobiology, Institute of Neuroscience,
University of Paris IV, 75252 Paris, France, 4 Department
of Cell Biology, Ohio State University, Columbus, Ohio 43210, and
5 Department of Anatomy, Neuroscience Institute, Erasmus
University Rotterdam, 3000 DR Rotterdam, The Netherlands
 |
ABSTRACT |
A longstanding but still controversial hypothesis is that long-term
depression (LTD) of parallel fiber-Purkinje cell synapses in the
cerebellum embodies part of the neuronal information storage required
for associative motor learning. Transgenic mice in which LTD is blocked
by Purkinje cell-specific inhibition of protein kinase C (PKC) (L7-PKCI
mutants) do indeed show impaired adaptation of their vestibulo-ocular
reflex, whereas the dynamics of their eye movement performance are
unaffected. However, because L7-PKCI mutants have a persistent multiple
climbing fiber innervation at least until 35 d of age and because
the baseline discharge of the Purkinje cells in the L7-PKCI mutants is
unknown, factors other than a blockage of LTD induction itself may
underlie their impaired motor learning. We therefore investigated the
spontaneous discharge of Purkinje cells in alert adult L7-PKCI mice as
well as their multiple climbing fiber innervation beyond the age of 3 months. We found that the simple spike and complex spike-firing properties (such as mean firing rate, interspike interval, and spike
count variability), oscillations, and climbing fiber pause in the
L7-PKCI mutants were indistinguishable from those in their wild-type
littermates. In addition, we found that multiple climbing fiber
innervation does not occur in cerebellar slices obtained from 3- to
6-month-old mutants. These data indicate (1) that neither PKC
inhibition nor the subsequent blockage of LTD induction disturbs the
spontaneous discharge of Purkinje cells in alert mice, (2) that
Purkinje cell-specific inhibition of PKC detains rather than prevents
the developmental conversion from multiple to mono-innervation of
Purkinje cells by climbing fibers, and (3) that as a consequence the
impaired motor learning as observed in older adult L7-PKCI mutants
cannot be attributable either to a disturbance in the baseline simple
spike and complex spike activities of their Purkinje cells or to a
persistent multiple climbing fiber innervation. We conclude that
cerebellar LTD is probably one of the major mechanisms underlying motor
learning, but that deficits in LTD induction and motor learning as
observed in the L7-PKCI mutants may only be reflected in differences of
the Purkinje cell signals during and/or directly after training.
Key words:
heterosynaptic plasticity; motor learning; multiple
climbing fiber innervation; cerebellar and vestibular nuclei; genetic
manipulation; phosphorylation
| |
INTRODUCTION |
A challenge faced by neuroscience is
to understand how model systems of information storage in the brain,
such as long-term potentiation and long-term depression (LTD), function
in neural circuits that control behavioral learning. Cerebellar LTD is
an attenuation of the granule cell axon-Purkinje cell synapse that occurs after conjunctive stimulation of the granule cell axons and
climbing fiber inputs (Ito et al., 1982 ; Linden
and Connor, 1995). It has been suggested that LTD underlies
several forms of motor learning, including adaptation of the
vestibulo-ocular reflex (VOR) and eye blink conditioning (Ito,
1989).
Several studies using knock-out mice have supported this claim by
showing correlations between deficits in LTD and behavioral learning
(Aiba et al., 1994; Conquet et al., 1994;
Funabiki et al., 1995; Kashiwabuchi et al.,
1995; Shibuki et al., 1996). However, the lack
of anatomical specificity and functional compensation via similar gene
family members has complicated the analyses of knock-out mice. For
example, global knock-outs of the type I metabotropic glutamate
receptor (mGluR1) show a blockade of LTD induction and impaired eye
blink conditioning (Aiba et al., 1994; Conquet et al., 1994), but because this receptor is expressed at multiple sites in the brain, it is difficult to correlate the physiological and
behavioral phenotype of the cell (cf. Ichise et al.,
2000). To overcome these limitations, we have created a
transgenic mouse in which a protein kinase C (PKC) inhibitory peptide,
PKC[19-31], is selectively expressed in Purkinje cells (De
Zeeuw et al., 1998). Cultured Purkinje cells from these L7-PKCI
mice show a complete blockade of LTD induction, and behavioral analysis
indicates a loss of VOR adaptation, whereas the default eye movement
performance is unaffected. These data support the idea that PKC
activation is necessary for LTD induction and that cerebellar LTD is
required for plasticity of the VOR.
However, it is currently unknown whether PKC inhibition inside Purkinje
cells directly corrupts their signals, which in turn could disturb the
learning process. If so, the impaired motor learning in L7-PKCI mutants
may not necessarily reflect a lack of LTD. Perhaps LTD itself is
essential for a normal operation of the cerebellar circuitry without
being involved in memory formation at all. As proposed by De
Schutter (1995), LTD might instead normalize Purkinje cell
excitation as a protective mechanism.
It is not unlikely that L7-PKCI Purkinje cells show abnormal firing
properties, because some of their kinase-regulated ion channels may
control interspike intervals (ISIs) (Nelson et al., 1996) and some of their kinase-regulated proteins involved in calcium metabolism, such as calretinin, can influence both simple spike
and complex spike firing (Schiffmann et al., 1999).
Moreover, inhibition of PKC could result in multiple climbing fiber
innervation, which has been observed in adult global PKC knock-outs
(Kano et al., 1995) and in L7-PKCI transgenics up to
35 d of age (De Zeeuw et al., 1998).
Thus, if the baseline discharge properties of Purkinje cells are
abnormal, there are ample reasons to believe that the impaired adaptation of L7-PKCI mutants results from side effects rather than
from a lack of LTD as a mechanism of plasticity (see also Lisberger, 1998). We therefore examined the spontaneous
activity of Purkinje cells in alert L7-PKCI mice as well as the
persistence of their multiple climbing fiber innervation beyond the age
of 3 months.
| |
MATERIALS AND METHODS |
Mice
We used heterozygous transgenic L7-PKCI mice and their wild-type
littermates. In the L7-PKCI mouse, the pseudosubstrate PKC inhibitor,
PKC[19-31], is selectively expressed in Purkinje cells under the
control of the pcp-2(L7) gene promotor (De Zeeuw et al.,
1998). All animals were bred in a C57BL/6 mouse strain
background. Wild-type and mutant mice were treated equally as far as
housing and experimental conditions were concerned.
Electrophysiology in alert animals
Surgery. Mice, 3- to 12-months old, were prepared for
chronic neurophysiological experiments under inhalant anesthesia with N2O/O2 and halothane (1.5-2%). First, a head
holder was tightly fixed to the skull by embedding two nuts and four
bone screws in dental cement, which allowed for rigid head restraint
during the experiments. Second, a small hole ( 3 mm) was drilled in the left occipital bone, and the exposed dura was partly removed with
great care. Subsequently, dental cement was molded around the hole to
form a recording chamber, which was then closed using bone wax. After
surgery, the animals were allowed to recover for 1-2 d before the
recording sessions.
In vivo recordings. Extracellular activity was recorded in
the cerebellar vermis and paramedian lobule using glass micropipettes filled with 2 M NaCl (tip diameter ~2-5 µm; impedance
~2.5 M at 1 kHz). The electrode tip was positioned on the
cerebellar surface under visual guidance (Olympus VS-IV; Olympus
Optical, Tokyo, Japan) using a micromanipulator (David Kopf
Instruments, Tujunga, CA) and moved downward by a hydraulic
microdrive (Trent Wells) equipped with a stepping motor (TL Elektronik
SMS 87). The electrode signal was amplified and filtered (bandwidth
10-6000 Hz; Dagan 2400; Dagan, Minneapolis, MN) and was sampled
at 12.5 kHz (CED 1401plus, Spike2, Cambridge, UK). Single-unit Purkinje cells were identified on-line by the presence of a brief pause in
simple spikes after the complex spike (see Fig.
1 for representative examples). In the
off-line analysis, simple spikes and complex spikes were detected and
discriminated using custom software implemented in Matlab (version
5.2-5.3; Mathworks, Natick, MA), which clustered groups of
spikes by amplitude, duration, and shape using a linear discriminant
analysis on the first four principal components of the spike wave forms
(Eggermont, 1990).
View larger version (42K):
[in this window]
[in a new window]
|
Figure 1.
Representative extracellular single-unit
recordings from two Purkinje cells in the alert mouse. Top
traces in A and B show 1 sec of raw
electrode signal, sampled at 12.5 kHz after amplification and band-pass
filtering (see Materials and Methods). Bottom traces depict
the simple spike (SS) and complex spike (CS)
waveforms at higher temporal resolutions (25 events with the mean
superimposed). Note that the complex spikes in A are easily
identified in the raw signal by their large positive component, but in
B, the complex spike (arrow) cannot be
discriminated from the simple spikes by polarity. However, the waveform
analysis procedure (see Materials and Methods) enabled reliable
discrimination of simple spikes and complex spikes in both examples as
well as in all other, more intermediate cases.
|
|
Spike train analysis. ISI histograms,
I(t), were derived from 2- to 5-min recording epochs of
spontaneous simple spike and complex spike activity (bin widths of
 t = 0.5 and t = 50 msec, respectively). The mean and variance (µt and
  ) of the simple spike and complex spike
ISIs as well as mean firing rates (f = 1/µt) and coefficients of variation
(CV = t/µt) were computed using
standard methods (Gabbiani and Koch, 1998). The most
likely ISI ( t) was derived from the peak of
the ISI histogram.
The long-term dynamics (seconds to minutes domain) of both simple spike
and complex spike firing were evaluated using the index of dispersion
or Fano factor, F(T), which is the ratio of the spike count
variance, V(T), to the mean spike count, N(T), in
a certain counting window T [F(T) = V(T)/N(T)]
(Gabbiani and Koch, 1998). Estimates of N(T)
and V(T) were obtained by subdividing a spike train into
partly overlapping intervals of duration T, and the counting
time was systematically varied. F(T) was evaluated by
plotting V(T) versus N(T) in double-log
coordinates, and linear regression lines, X =  Y +  [with X = log N(T) and
Y = log V(T)], were fitted to the
resulting curves for N(T) >1. If spiking can be described
as a renewal process (that is, if the ISIs are independently and
identically distributed), the data would fall on a straight line with
slope = 1 and bias = 2 log CV
because under these conditions
V(T) CV2·N(T)
[except for short T because F(T) converges to 1 for T  0] (Cox, 1962).
Simple spike activity was inspected for the presence of oscillations
using autocorrelation histograms [r( ) in spikes per second; bin width t = 1.0 msec; lag time
 300  300 msec]. The degree of simple spike
rhythmicity was quantified by a rhythm index (RI),
calculated in a manner similar to Lang et al.
(1997):
in which c(p,i) and
c(v,j) are the autocorrelation
coefficients at lag times p,i and
v,j corresponding to the ith peak (i = 1,2, ··· ,np) and jth
valley (j = 1,2, ··· ,nv),
respectively. Autocorrelation coefficients were calculated from the
conditional firing rate, r(), using the following
equation:
in which b is the baseline firing rate given by
b = Ntot/Trec
(Ntot is the total number of spikes and
Trec is the total recording time) and
t is the bin width of the histogram. Only successive peaks and valleys that deviated more than ±2 SD from the baseline were
included. The SD of activity about the baseline was measured at time
lags of 250-300 msec, which modulation of the conditional firing rate
was absent. The oscillation period, Tosc, was
calculated from the following equation:
Oscillatory frequency was given by fosc = 1/Tosc. To examine whether the oscillatory
patterns could be understood from the regularity of the ISIs (see
Results), renewal density histograms
[R+(); lag time, 0 < 300 msec] were computed from the (first order) ISI histograms,
I(), according to the following equation:
in which I(k)() denotes the
result of a k-fold convolution of I(), thus
representing the second- and higher-order ISI distributions, Ik+1 (), formed by joining adjacent ISIs
(Eggermont, 1990). t is the bin width of
I(). An alternative procedure for obtaining these
histograms is to compute averaged autocorrelation histograms from spike
trains with randomly shuffled ISIs (Mountcastle et al.,
1969).
Cross-correlation histograms of simple spikes and complex spikes (in
spikes per second; bin width, t = 1.0 msec;
lag time, 300 300 msec) were used to quantify the
climbing fiber pause (CFP) (Simpson et al., 1996). In
addition, ISI histograms of the interval between a complex spike and
the first following simple spike were made (bin width, t = 1.0 msec) to evaluate the complex spike-simple spike interval
(CSI) statistics.
Electrophysiological and fluorescence measurements in
cerebellar slices
Slice preparation. Sagittal slices (250-300 µm
thick) were prepared from the cerebellar vermis as described previously
(Conquet et al., 1994). Cerebellar LTD and the climbing
fiber innervation of Purkinje cells were tested in slices prepared from
mice of 3 weeks to 6 months of age. Slices were incubated at room
temperature in saline solution gassed with 95%
O2/5% CO2 for at least 2 h before
recording. During experiments, the recording chamber was continuously
perfused at a rate of 2 ml/min with oxygenated saline solution of the
following composition (in mM): 124 NaCl, 3 KCl, 24 NaHCO3, 1.15 KH2PO4,
1.15 MgSO4, 2 CaCl2, 10 glucose,
and the GABAA antagonist bicuculline methiodide (10 µM; Sigma, St. Louis, MO), final pH of 7.35 at 28°C,
330 mOsm/l. Recordings of Purkinje cells under visual control
were performed at the somatic level, using the whole-cell patch-clamp
technique with an Axopatch 1D or an Axopatch 200 amplifier (Axon
Instruments, Foster City, CA). For pairing experiments, synaptic
activation of metabotropic glutamate receptor experiments, and kinetic
measurements in climbing fiber innervation experiments (see Results),
access resistance was partially (50-70%) compensated according to the
procedure described by Llano et al. (1991). For
analysis, electrophysiological data were filtered at 2 kHz and
digitized at 20 kHz. Climbing fiber and parallel fiber-mediated
responses were analyzed on-line and off-line using the Acquis1 computer
program (Biologic, Grenoble, France).
Pairing experiments. In pairing experiments, the patch
pipettes (2-3.5 M) were filled with an internal solution containing (in mM): 140 KCl, 8 NaCl, 10 HEPES, 2 ATP-Mg, 0.75 EGTA,
final pH of 7.3 with KOH, 300 mOsmol/l. Cells were maintained at
a holding potential of 70 mV and, as reported previously
(Crépel and Jaillard, 1991), parallel fibers were
stimulated at 0.33 Hz through a monopolar electrode placed at the
surface of the slice, in the lower half of the molecular layer at the
level of the proximal dendrites of the recorded cells. Throughout the
recording, parallel fiber-mediated EPSCs were elicited on a 10 mV hyperpolarizing voltage step that allowed testing of the passive
properties of the recorded cells as well as the stability of access
resistances. In these experiments, parallel fiber-mediated EPSCs were
first evoked in Purkinje cells during a control period of at least 5 min to obtain baseline data. Next, two successive pairing protocols
separated by 5 min were performed in an attempt to saturate LTD. For
each of these pairings, the recording mode was changed to current clamp
for 1 min, and parallel fiber-mediated EPSPs were evoked at 1 Hz during
this period, in conjunction with Ca2+ spikes
elicited in Purkinje cells by both a steady depolarizing current passed
through the recording electrode and depolarizing steps timed to
coincide with parallel fiber stimulation. The voltage-clamp mode and
the initial frequency of stimulation were resumed just after the end of
the pairing protocol.
Synaptic activation of metabotropic glutamate receptor
experiments. In these experiments, the patch pipettes (2-3 M)
were filled with a solution containing (in mM): 140 CsCl, 8 NaCl, 10 HEPES, 2 ATP-Mg, 0.5 EGTA, pH of 7.3 with CsOH, 300 mOsm/l. To obtain synaptically induced metabotropic glutamate
receptor responses, Purkinje cells were voltage clamped at 70 mV and
recorded in the presence of bicuculline methiodide (10 µM; Sigma) to block GABAA receptors and in
the presence of AP-5, (50 µM; Tocris) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 µM;
Tocris Cookson, Bristol, UK) to block ionotropic glutamate
receptors (Batchelor and Garthwaite, 1997; Tempia
et al., 1998). As established previously (Tempia et al.,
1998), a brief train of stimulation pulses was delivered at 100 Hz through a monopolar electrode in the molecular-to-parallel fibers
layer to induce postsynaptic currents mediated by metabotropic glutamate receptors.
Climbing fiber innervation experiments. In the
climbing fiber innervation experiments, the EGTA concentration was
raised to 5 mM, and 0.5 mM CaCl2
was added to the internal solution that was used in pairing
experiments. Moreover, 2 mM
N-2,6-dimethylphenylcarbamoylmethyl triethylammonium bromide
(Alomone Labs, Jerusalem, Israel) was added to block sodium
spikes. Purkinje cells were maintained at 80 mV. Climbing fibers were
stimulated at 0.33 Hz through a monopolar electrode placed at the
surface of the slice, near the soma of the recorded cell. For each
cell, several locations of the stimulating electrode in the internal
granular layer and in the lower half of the molecular layer were tested
for their ability to evoke potential multistep climbing fiber-mediated
EPSCs. Throughout the recording, climbing fiber-mediated EPSCs were
elicited on a 10 mV hyperpolarizing voltage step that allowed
monitoring of the passive electrical properties of the recorded cell.
Fluorescence measurements. As described previously
(Daniel et al, 1999), fluorescence measurements with the
dye fluo3, a high-affinity Ca2+ indicator, were
performed to detect changes in intracellular free calcium into the
Purkinje cell dendrites. Fluo3-free acid (100 µM) added
to the internal medium was introduced into the cell via the patch
pipette. The recording session started 30-45 min after whole-cell
"break in," to allow dye diffusion in the dendrites and to wash out
the background fluorescence in surrounding tissue that occurs because
of leakage of the dye from the pipette just before the formation of the
seal. The Ca2+-sensitive dye was excited by light
from a 100 W xenon lamp, and the epifluorescence excitation wavelength
was at 485 ± 22 nm. The emitted light was collected by a
photometer through a barrier filter at 530 ± 30 nm, from an area
(15 × 15 µm) centered on proximal dendrites. Because this
single wavelength method does not determine absolute free
Ca2+ levels, the fluorescence changes in fluo3 were
expressed as a ratio with respect to the initial background-corrected
resting fluorescence. Fluorescence data were analyzed on-line and
off-line by using Acquis 1 computer software (Biologic).
Statistics
Statistical evaluation of the data included Student's
t tests, Kolmogorov-Smirnov tests (KS tests), and a
 2 test (Press et al., 1992).
Significance levels are indicated in Results.
| |
RESULTS |
Spontaneous Purkinje cell activity in alert L7-PKCI mutants
We recorded spontaneous Purkinje cell activity in the cerebellar
vermis and paramedian lobule of alert LTD-deficient L7-PKCI+/ mice
(n = 10) and their wild-type littermates (L7-PKCI/;
n = 12). Cells were selected for recording once they
were well isolated (Fig. 1). Histograms
of simple spike activity triggered on the occurrence of a complex spike
were made to verify that each Purkinje cell showed a clean climbing
fiber pause (Simpson et al., 1996) (see below for
quantitative data). In this manner, simultaneous records of simple
spikes and complex spikes were obtained from 95 single-unit Purkinje
cells (44 wild types and 51 mutants).
Interspike intervals
To examine whether inhibition of various PKC isoforms and/or the
consequent blockage of LTD induction affected the short-term stochastics of Purkinje cell firing, we plotted ISI histograms. Figure
2 shows the simple spike and complex
spike ISI histograms obtained from a wild-type (L7-PKCI/) (Fig. 2
A1, A2) and a mutant Purkinje cell (L7-PKCI+/) (Fig. 2 B1,
B2). The mean firing rate, f, and the
coefficient of variation, CV, of the simple
spike and complex spike discharge were quantified for each cell. Figure 2, C and D, shows comparisons of the population
averages of the mean simple spike and mean complex spike rates for the
two genotypes. Note that the average values of the simple spike rates
in L7-PKCI+/ and L7-PKCI/ mice are not significantly different
( 60 spikes/sec for both
genotypes; Student's t test; p = 0.8) (Fig.
2C). The average values of the complex spike rates are also very similar for the two genotypes (
1.0 spikes/sec in both cases; Student's t test;
p = 0.3) (Fig. 2D). It should be
noted, however, that there is considerable cell-to-cell variability in
the mean firing rates and that this variability rather than the
population average might be different in the two genotypes. To overcome
this potential pitfall, we also compared the distributions of mean
firing rates. It turned out, however, that the distribution of both
simple spike rates and complex spike rates is indistinguishable (KS
test; p > 0.6). Even when the mean complex spike rate
of individual Purkinje cells is plotted against their mean simple spike
rate (Fig. 2E, scatter plot), it is observed that the
two-dimensional (2D) distribution of the PKCI/ data (Fig.
2E,  ) overlaps the distribution of the
PKCI+/ data (Fig. 2E,  ), indicating that there is no significant difference (2D KS test; p = 0.7). Similar results were obtained with regard to the temporal jitter
in simple spikes and complex spikes, as quantified by the
CV. This is shown in the scatter plot
of Figure 2F, in which the complex spike coefficient of variation is plotted versus the simple spike coefficient of variation for wild-type (Fig. 2F, ) and
mutant (Fig. 2F, ) Purkinje cells. Note
that the simple spike coefficient of variation was <1 for the vast
majority of cells, indicating that the simple spike ISI distributions
are typically more regular than the distributions associated with a
pure Poisson process (for which CV equals 1). The complex spike coefficients of variation were also <1 for most wild-type and mutant neurons, but the average values were closer to
unity than the ones obtained for simple spike firing
( 0.9 versus
 0.7 in both genotypes). Only
a limited fraction of the cells showed a burst-like simple spike and/or complex spike discharge, resulting in coefficients of variation substantially greater than 1. The distribution of complex spike versus
simple spike coefficients of variation was not significantly different
for the two genotypes (2D KS test; p = 0.4).
View larger version (29K):
[in this window]
[in a new window]
|
Figure 2.
Purkinje cells in alert wild-type and
L7-PKCI mutant mice show similar mean firing rates and coefficients of
variation. A, Interspike interval histograms of simple spike
(SS) and complex spike (CS) activity of a
wild-type (L7-PKCI/) Purkinje cell. Mean firing rates,
fss = 78 spikes/sec and
fcs = 1.2 spikes/sec. Coefficients of
variation, CVss = 0.39 and
CVcs = 0.81. Bin width is 0.5 msec in
A1 and 50 msec in A2.
B, Interspike interval histograms of a mutant (L7-PKCI+/)
Purkinje cell. Mean firing rates, fss = 58
spikes/sec and fcs = 1.2 spikes/sec.
Coefficients of variation, CVss = 0.64 and
CVcs = 1.09. C, D, Population
average of the mean simple spike rates (C) and the
mean complex spike rates (D) in wild-type and mutant
Purkinje cells. Error bars indicate 1 SD. Mean ± SD in wild types
(n = 44) (fss = 59 ± 26 spikes/sec and fcs = 0.9 ± 0.4 spikes/sec) and in mutants (n = 51)
(fss = 58 ± 21 spikes/sec and
fcs = 1.0 ± 0.4 spikes/sec). Neither
simple spike nor complex spike firing rates are significantly
different. E, Scatter plot of simple spike versus complex
spike mean firing rates for wild-type and mutant Purkinje cells. Note
the considerable yet very similar cell-to-cell variability in wild-type
() and mutant () mice. F, Scatter plot of simple
spike versus complex spike coefficients of variation for both cell
populations. Typically, CVss and
CVcs are <1, and the distributions largely
overlap. Mean ± SD in wild types, CVss = 0.7 ± 0.4 and CVcs = 0.9 ± 0.2; in mutants, CVss = 0.7 ± 0.3 and CVcs = 0.9 ± 0.2.
|
|
Spike count variability
In subsequent analyses, we evaluated spike count variability
[quantified by the Fano factor, F(T)] to examine possible
differences in the long-term discharge dynamics (see Materials and
Methods). For counting windows on the order of one to five times the
mean ISI, corresponding to mean spike counts of approximately one to five spikes, F(T) was typically approximately equal to the
corresponding CV2, as
expected for a renewal process. For longer integration times, however,
the Fano factor evolved differently for both simple spike and complex
spike firing. This is illustrated in Figure
3, A and B, which
depicts the variance versus mean spike count curves in double-log
coordinates for simple spike (Fig. 3A) and complex spike
(Fig. 3B) firing of a wild-type and a mutant Purkinje cell. Note that the curves are approximately linear for mean spike counts >1, but the variance in the number of simple spike events grows faster
than the mean (Fig. 3A), indicating long-term correlations in the simple spike data. Temporal order in the simple spike sequences was also evident from plots of serial correlation coefficients, which
showed positive correlations between contiguous and noncontiguous ISIs
(data not shown). In contrast, the curves for complex spike variability
exhibit slopes that are less than unity (Fig. 3B), suggesting that some regularity is imposed. The scatter
plots in Figure 3, C and D, summarize the
results of this analysis for all cells. Note that for simple spike
dispersion, the slopes of the regression lines exceed unity in nearly
all Purkinje cells (range 0.96-1.84). Thus, regardless of the
coefficient of variation, a substantial proportion of the simple spike
variability is not explained by interspike interval variability. For
complex spike dispersion, slopes ranged between 0.22 and 1.61, and they
depended on the complex spike coefficient of variation. There were no
significant differences between the two cell populations (KS test;
p > 0.2), indicating that the long-term dynamics of
simple spike and complex spike firings are very similar in wild-type
and mutant mice.
View larger version (36K):
[in this window]
[in a new window]
|
Figure 3.
Spike count variability is similar in Purkinje
cells of alert wild-type and L7-PKCI mutant mice. A, B,
Spike count variance as a function of the mean for simple spike
(SS) (A) and complex spike (CS)
(B) activity of a wild-type (L7-PKCI/) and a
mutant (L7-PKCI+/) Purkinje cell. Note that the curves are
approximately linear for N(T) > 1 (corresponding to
counting times that exceed the mean ISI duration). Slopes of the linear
regression lines fitted to these data were ~1.3 in A and
~0.8 in B. The identity line corresponds to the Fano
factor for a Poisson process (for which F(T) = 1). Note
the scaling differences between A and B. C,
D, Scatter plots of the regression-line slope versus the
coefficient of variation for simple spike (C) and
complex spike (D) activity in wild-type () and
mutant () Purkinje cells. Slopes >1 imply long-term correlations in
the data. Slopes obtained for simple spike activity exceed unity in
virtually all Purkinje cells (mean ± SD,
ss = 1.4 ± 0.2 in both genotypes;
Pearson's correlation coefficients, 0.988-0.999). Slopes obtained for
complex spike activity are related to the complex spike coefficient of
variation and range between 0.22 and 0.61 variation (mean ± SD,
cs = 0.8 ± 0.3 in wild types and
cs = 0.9 ± 0.3 in mutants;
Pearson's correlation coefficients, 0.819-0.999).
|
|
Simple spike oscillations
Autocorrelation histograms indicated the presence of
high-frequency oscillations in the simple spike discharge of most
Purkinje cells (>95%). Figure 4,
A and B, shows the autocorrelation histogram of
simple spike activity in two wild-type and two mutant Purkinje cells,
respectively. Note that the conditional firing rates in Figure 4,
A1 and B1, peak at
approximately ±11 msec, whereas peaks occur at approximately ±14,
±28, and ±42 msec in Figure 4, A2 and
B2. For the vast majority of wild-type and
mutant Purkinje cells, these findings reflected the regularity of the
ISI distribution (compare Fig. 2, A1 and
B1, for the ISI histograms of the two cells in
Fig. 4, A1 and B1). This
is illustrated in Figure 4, A1,2 and
B1,2, by superimposing the renewal density
histograms, R+() (solid
curves), computed from the corresponding ISI histograms (see
Materials and Methods). Note that the predicted oscillatory patterns
correspond quite well to the observed ones. As may be inferred from the
R+() equation (see Materials and
Methods), these results suggest that the oscillation period,
Tosc, will approximately equal the duration of
the most likely simple spike ISI, t. After all, for a unimodal, regular ISI distribution, the contribution of the
first (dominant) term, I(), tends to produce a peak in R+() at t, whereas the contribution of each higher-order
joint ISI distribution, Ik + 1(), tends
to produce an additional peak at (k + 1)·t. The tendency for I() and
Ik + 1() to yield significant peaks
depends of course on the degree of regularity of I() and
declines as a function of k. Figure 4C shows that
for the vast majority of cells, the oscillation period does indeed
match the duration of the most likely simple spike ISI very closely in
both genotypes (Pearson's correlation coefficients, >0.9). However,
as expected from the presence of serial correlations in the ISIs (Fig.
3), the amplitudes of peaks and valleys in the renewal density
functions were typically reduced compared with those in the
autocorrelation histograms. Figure 4 D-G summarizes the
observed oscillatory patterns. Approximately 50% of the cells showed
two or more significant peaks in both cell populations (Fig. 4
D, E), and there was no significant difference
between the frequency distribution for the two genotypes
(2 test; p = 0.2). A rhythm index,
RI, (see Materials and Methods) was used to further quantify
the strength of the simple spike oscillations. For cells showing at
least one peak, rhythm indices ranged between 0.005 and 0.192 in
wild-type mice and between 0.004 and 0.399 in mutant mice (Fig. 4,
F and G, respectively), but their distributions
were not significantly different (KS test; p = 0.2).
View larger version (32K):
[in this window]
[in a new window]
|
Figure 4.
Purkinje cells in alert
wild-type and L7-PKCI mutants show comparable simple spike oscillation
patterns. A, B, Simple spike autocorrelation
histograms from two wild-type (L7-PKCI/) and two mutant
(L7-PKCI+/) Purkinje cells. Bin width, 1 msec. For the two wild-type
cells in A, fosc = 90 Hz,
RI = 0.066 (A1) and
fosc = 69 Hz, RI = 0.121
(A2). For the two mutant cells in B:
fosc = 81 Hz, RI = 0.052
(B1) and fosc = 73
Hz, RI = 0.103 (B2).
Superimposed are renewal density histograms (solid curves),
calculated from the simple spike ISI histograms (see Materials and
Methods). Note that these curves resemble the data remarkably well in
all four cases. The ISI histograms used to compute the curves in
A1 and B1 are plotted in
Figure 2, A1, and B1,
respectively (same cell recordings). C, Simple spike
oscillation period versus the most likely simple spike ISI for
wild-type () and mutant () Purkinje cells. Note that the data lie
close to the identity line (dotted line). Pearson's
correlation coefficients are 0.99 and 0.92 for the L7-PKCI/ and
L7-PKCI+/ population, respectively. D, E, Frequency
distribution of the number of significant peaks in the autocorrelation
histograms for the L7-PKCI/ and L7-PKCI+/ population
(n = 44 and n = 51, respectively). Note
that ~50% of the cells in both genotypes showed two or more
significant peaks. F, G, Frequency distribution of rhythm
indices for both genotypes. Average RI values for the
L7-PKCI/ and L7-PKCI+/ cell population were 0.06 ± 0.04 and
0.09 ± 0.1 (mean ± SD), respectively.
|
|
Climbing fiber pause
As illustrated by the examples in Figure
5, A1 and
B1, cross-correlation histograms of simple spike
and complex spike activity indicated a transient pause (complete
suppression) of simple spike activity for all isolated Purkinje cells.
After this pause (referred to as climbing fiber pause in the
literature; Armstrong, 1974), the simple spike activity
showed one of three general patterns (McDevitt et al.,
1982; Sato et al., 1992; Simpson et al.,
1996): a return to baseline within a few milliseconds (pure
pause), a gradual return to baseline over several tens of milliseconds
(pause-reduction), and a rapid increase in activity to a level
exceeding the baseline for up to several tens of milliseconds
(pause-facilitation). Figure 5C quantifies the climbing
fiber pause in L7-PKCI/ and L7-PKCI+/ cells. Note that the
average pause duration was approximately 11 msec for both genotypes
(Student's t test; p = 0.7). Because the
climbing fiber pause reflects only a single event (i.e., the shortest
interval occurring between a complex spike and a subsequent simple
spike within a recording epoch), we also made histograms of the complex
spike-simple spike intervals for each cell (Fig. 5
A2, B2) and quantified the
mean and variance of these intervals. As shown in Figure 5D,
the mean duration of the complex spike-simple spike interval strongly
depends on the mean simple spike ISI (Pearson's correlation
coefficient, >0.9) in both the PKCI+/ (Fig. 5D, ) and
PKCI/ (Fig. 5D, ) cell population (2D KS test;
p = 0.2). The mean complex spike-simple spike interval
typically exceeds the mean simple spike ISI. This presumably reflects a
combination of prolonged refractoriness of the Purkinje cell membrane,
climbing fiber-evoked inhibition by basket cells, and reduced
excitation by parallel fibers because of the impact of climbing fiber
collaterals on Golgi cells, which inhibit the mossy fiber-granule cell
pathway (Armstrong, 1974; Simpson et al.,
1996). As a result, only part of the variance is explained by
the variance in simple spike ISI in most cells (Fig. 5E)
(Pearson's correlation coefficient, >0.8) of both genotypes (2D KS
test; p = 0.9). Note, however, that the inhibition of
various PKC isoforms and the consequent blockage of LTD induction
appear to have no effect on these short-term interactions.
View larger version (34K):
[in this window]
[in a new window]
|
Figure 5.
Short-term effect of climbing
fiber-mediated complex spikes on simple spike firing is unaltered in
L7-PKCI mutants. A1, Histogram of simple spikes
(SS) triggered by the occurrence of a spontaneous complex
spike (CS) for a wild-type Purkinje cell. Bin width, 1 msec
CFP in this example, 10 msec. A2, Histogram of
the interval between a complex spike and the first following simple
spike. Bin width, 1 msec. Mean ± SD of the distribution,
CSI = 21 ± 10 msec. B, Similar histograms for
a mutant Purkinje cell (CFP = 9 msec and CSI = 27 ± 15 msec). C, Average duration of the climbing fiber pause in
wild-type and mutant Purkinje cells. Error bars indicate 1 SD.
Mean ± SD, CFP = 11 ± 7 msec and CFP = 11 ± 6 msec for L7-PKCI/ and L7-PKCI+/ cells, respectively.
D, Mean duration of the complex spike-simple spike
intervals correlates well with the mean simple spike ISI
(p < 0.0001). Pearson's correlation
coefficients are 0.93 and 0.92 for the L7-PKCI/ and L7-PKCI+/
cells, respectively. Note the overlap in data for wild type () and
mutants (), and that most data points lie above the identity line
(dotted line). E, Variance in the duration of the
complex spike- simple spike interval correlates with the variance in
simple spike ISIs (p < 0.0001). Pearson's
correlation coefficients are 0.84 and 0.87 for the L7-PKCI/ and
L7-PKCI+/ cells, respectively, and the data for the two
genotypes overlap considerably. Note that the variance of complex
spike-simple spike intervals typically exceeds the variance of the
simple spike ISIs.
|
|
Mono climbing fiber innervation in old L7-PKCI mutants
Previous slice experiments have indicated that ~50% of the
Purkinje cells in 4- to 5-week-old L7-PKCI animals show a persistent multiple climbing fiber innervation (De Zeeuw et al.,
1998). We were surprised therefore that the mean complex spike
rate in the Purkinje cells of the alert L7-PKCI mice (3-12 months of
age) was normal ( 1.0
spikes/sec). More specifically, considering the measured complex spike
rates of the wild-type cells (receiving only one climbing fiber), the a
priori prediction was to find a significant increase in complex spike
rate to 1.4 ± 0.7 spikes/sec (mean ± SD;
p < 0.001) in the mutants (using Monte Carlo
simulation, zaq;/assuming that 50% of the PKCI+/ cells are excited
by two climbing fibers). Clearly, the increase in both mean and SD
should have emerged as a change in the distribution of complex spike
rates, but this was not the case either (Fig. 2E).
Although two distinctly different complex spike waveforms were
occasionally identified during spike discrimination, these different
complex spikes were never picked up from one and the same Purkinje cell
(as inferred from analysis of the climbing fiber pause) and were
therefore not included in the analysis. These findings raised the
question of whether the multiple climbing fiber innervation found in
cerebellar slices of young L7-PKCI animals [postnatal day 28 (P28) to
P35] (De Zeeuw et al., 1998) remains evident in
mice that are >3 months of age, which is the age used for our
electrophysiological in vivo recordings.
When slices were prepared from the cerebella of 3- to 6-month-old
L7-PKCI mice (n = 10) and their wild-type littermates
(n = 5), no gross differences in morphology were
observed at the light microscopic level (data not shown). Examination
of a number of basal electrophysiological parameters also revealed no
significant differences between L7-PKCI+/ and L7-PKCI/ Purkinje
cells (Table 1). As shown in Figure
6, multiple climbing fiber innervation was not found in either L7-PKCI+/ or L7-PKCI/ Purkinje cells. Thus, stimulation of the granular layer or the molecular layer near the
recorded Purkinje cell (50-70 µm away) evoked typical all-or-none
climbing fiber-mediated EPSCs in the Purkinje cells obtained
from slices of L7-PKCI+/ mice (Fig. 6 A1,A2)
(n = 34; 10 cells studied blind) or their wild-type
littermates (Fig. 6B) (n = 18; 16 cells studied blind). In the current-clamp mode, these responses
recorded at 70 mV consisted of an initial full spike followed by a
plateau of depolarization on which partial spikes were superimposed in
both L7-PKCI+/ (n = 5) and L7-PKCI/
(n = 5) cells (Fig. 6C).
View larger version (20K):
[in this window]
[in a new window]
|
Figure 6.
Mono climbing fiber innervation of Purkinje cells
in 3- to 6-month-old L7-PKCI mice. A1, Two subthreshold
responses and two superimposed sweeps of all-or-none climbing
fiber-mediated EPSCs evoked in a mutant Purkinje cell held at a holding
potential of 80 mV, by stimulating the granular layer with
progressively increasing intensity. A2, Plot of amplitudes
against time of climbing fiber-mediated EPSCs recorded at 80 mV in
the same cell as in A1. Stimulus intensity varied
progressively in strength during the recording period. B,
Two superimposed sweeps of all-or-none climbing fiber-mediated EPSCs
and two subthreshold responses elicited in a wild-type Purkinje cell.
C, Classical climbing fiber responses recorded in another
mutant Purkinje cell. The cell was held in current clamp and the
granular layer was stimulated with progressively increasing stimulus
intensities.
|
|
Mean amplitudes, mean rise times, and mean decay time constants of
climbing fiber-mediated EPSCs in L7-PKCI mice were not significantly
different from those observed in wild-type littermates (Table 1). These
parameters were measured in cells with no contamination of climbing
fiber-mediated EPSCs by underlying parallel fiber-mediated EPSCs, and
the decay time constants of these responses were obtained by fitting
the decay phases of EPSCs with single exponentials (Llano et
al., 1991). In addition, climbing fiber-mediated EPSCs were
unaffected by bath application of the NMDA receptor antagonist AP-5
(100 µM) in either L7-PKCI or wild-type mice, but they
were blocked by the non-NMDA receptor antagonist CNQX (10 µM), indicating that these EPSCs are mediated exclusively
by the non-NMDA receptors, as reported previously (Konnerth et
al., 1990).
LTD induction is blocked in slices of L7-PKCI mutants
In vitro experiments have shown that cultured Purkinje
cells from L7-PKCI mice show a complete blockage of LTD induction
(De Zeeuw et al., 1998). However, the signaling chain
underlying cerebellar LTD in cultures may partially differ from that
seen in slice preparations (Linden and Connor, 1995;
Lev-Ram et al., 1997). For example, presumably because
of the absence of sufficient synaptic inputs with the activity of
nitric oxide (NO) synthetase in the culture preparation, the
role of the NO-PKG pathway in LTD induction can be demonstrated in the
slice preparation but not in cultures of Purkinje cells. Therefore, we
initiated experiments to investigate whether the general
electrophysiological responses of Purkinje cells in cerebellar slices
of the L7-PKCI mutants are normal and whether the induction of
cerebellar LTD is blocked in these slices.
Stimulation of the parallel fiber parts of the granule cell axons
in the molecular layer of L7-PKCI+/ mutants evoked EPSCs that were
indistinguishable from those in the wild types; the amplitudes and
shapes of their EPSCs equally depended on stimulus intensity. Both
responses were totally abolished by bath application of 10 µM CNQX, and both were unaffected by 100 µM
AP-5 (data not shown). In the nine Purkinje cells prepared from six
wild-type mice, the pairing protocols of parallel fiber-mediated EPSCs
with Ca2+ spikes induced a clear-cut LTD of parallel
fiber-mediated EPSCs, which was apparent immediately after the period
of pairing and lasted for at least 30 min. The mean amplitude of the
parallel fiber-mediated EPSCs was 71.70 ± 3.41% of controls
(mean ± SEM) 20 min after the pairing period (Fig.
7A). In contrast, the same pairing protocols applied to eight Purkinje cells obtained from five
L7-PKCI mice did not induce any persistent depression of parallel
fiber-mediated EPSCs; only transient depressions were observed.
The mean amplitude of these responses was 95.96 ± 4.21% of the
control value 20 min after the pairing period (Fig. 7B). Thus, the relative changes in amplitude of parallel fiber-mediated EPSCs in wild-type mice 20 min after the pairing period were
significantly larger than in the L7-PKCI mice (Student's t
test; p < 0.01). Therefore, the present results show
that activation of PKC is necessary for the induction of LTD not only
in cultures but also in slices. Thus, although the NO-PKG pathway
could be operational for the induction of LTD in slices of the
L7-PKCI mutants (Daniel et al., 1998), apparently
the blockage of the activation of PKC is sufficient for the blockage of
LTD induction in slices.
View larger version (19K):
[in this window]
[in a new window]
|
Figure 7.
Induction of cerebellar LTD is suppressed in
slices prepared from L7-PKCI cerebella. A, B, The plots
represent the normalized amplitudes of parallel fiber-mediated ESPCs
against time before and after two successive pairing protocols
(P) (see Materials and Methods) in wild-type
(A) and in L7-PKCI+/ (B) Purkinje
cells. Each point is the mean ± SEM of separate blind experiments
in nine wild-type cells (A) and in eight mutant cells
(B). The insets show superimposed averaged
EPSCs in one of the Purkinje cells recorded under control conditions
(1), after the first pairing protocol (2), and
during expression of LTD (3) in wild-type
(A) and in L7-PKCI+/ (B) cells.
Experiments on either wild-type or L7-PKCI mice at 3-8 weeks of age
were intermingled and were done at the same time of the day.
C, Sodium and calcium action potentials evoked by a 200 msec
depolarizing somatic current pulse in an L7-PKCI+/ Purkinje cell. Note the prolonged
afterhyperpolarization, which follows the pulse break
(arrow). D, mGluR EPSCs in L7-PKCI+/ Purkinje
cells evoked by repetitive parallel fiber stimulation with eight pulses
at 100 Hz (arrow) in control saline (1), and
inhibition by 300 µM AIDA (2). Each trace is
the average of four consecutively evoked mGluR EPSCs.
|
|
It is possible that the LTD deficiency observed in the mutant Purkinje
cells in acute slices may be caused by some effects of the L7-PKCI
transgene that are independent of the direct inhibition of PKC. Because
the induction of LTD is triggered by the influx of
Ca2+ into Purkinje cells and by activation of
mGluR1, leading to intracellular cascades involving the release of
Ca2+ from inositol 1,4,5-trisphosphate
(IP3)-sensitive intracellular stores through
the production of IP3 (Daniel et al.,
1998), we have tested whether these two crucial signaling
pathways were altered in the mutant cells.
Depolarizing current pulses passed through the recording patch pipette
routinely evoked depolarizing plateau potentials with combined
Na2+ and Ca2+ spikes in the
mutant Purkinje cells (Fig. 7C). The presence of typical
Ca2+ spikes indicated that the voltage-dependent
Ca2+ channels of normal Purkinje cells were at least
qualitatively preserved in the L7-PKCI mice. After the plateau
potential, a prolonged after-hyperpolarization (Fig. 7C,
arrow) was always observed that was largely dependent on
Ca2+, because it increased by 29.6 ± 2.2%
(mean ± SEM; n = 5) when calcium spikes were
evoked during the plateau. Thus, the generation of a calcium-dependent
after-hyperpolarization in mutant Purkinje cells after
depolarization-induced spikes indicates that the
Ca2+-dependent potassium conductances, which are
involved in this membrane potential variation (Llinás and
Sugimori, 1980), are also at least qualitatively preserved in
the L7-PKCI mice.
Slow postsynaptic current recordings evoked by repetitive parallel
fiber stimulation and dendritic fluorometric measurements of the
intracellular Ca2+ concentration with the
high-affinity Ca2+ indicator fluo3 (see Materials
and Methods) were used to test the function of the mGluR1 receptor
pathway in mutant Purkinje cells. As established previously
(Tempia et al., 1998), brief trains of eight stimuli
delivered at 100 Hz to parallel fibers (see Materials and Methods)
routinely evoked mGluR1-mediated EPSCs with a mean amplitude of 59 ± 4 pA (mean ± SEM; n = 3) (Fig. 7D). These currents were blocked by bath application of 300 µM
(RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), a selective
mGluR1 antagonist. Because repetitive parallel fiber stimulation
clearly evokes mGluR1-mediated EPSCs, these experiments indicate that
mutant Purkinje cells express functional mGluR1 and that these currents
are independent of PKC activation (see also Tempia et al.,
1998). In addition, because activation of these receptors
increases the Ca2+ concentration through production
of IP3, we then tested the function of these
receptors with fluorometric measurements of Ca2+
variations in a second series of experiments. The cells were recorded
in voltage-clamp mode at 70 mV and stimulated with bath-applied group
I agonist
1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (100 µM), while
transient fluorescence changes were used to detect transient increases
in the intracellular free calcium concentration. Application of
1S,3R-ACPD induced transient changes in the
proximal dendrites of mutant cells, with a mean amplitude of relative
fluorescence variation (F/F) of 6.1 ± 0.4%
(mean ± SEM; n = 3). In addition, removal of
Ca2+ ions from the external medium failed to abolish
these 1S,3R-ACPD-induced transient increases,
supporting the presence of a functional mGluR1 pathway coupled to
internal Ca2+ mobilization. These results were not
significantly different from those recorded in wild-type cells; the
mean amplitude of the 1S,3R-ACPD-induced
transient was 6.3 ± 0.3% (mean ± SEM;
n = 3; F/F).
All these data in acute slices demonstrate that the blockade of LTD
induction in mutant Purkinje cells is not attributable to an indirect
effect of the L7-PKCI transgene on these two signaling systems, a
finding that is consistent with and that extends earlier results
obtained in cultured Purkinje cells (De Zeeuw et al., 1998).
| |
DISCUSSION |
The major findings of the present study are (1) that Purkinje
cell-specific inhibition of PKC leads to a blockage of LTD induction at
the parallel fiber-Purkinje cell synapse in slices, (2) that a
blockage of LTD induction does not lead to a disturbed baseline discharge of Purkinje cells in alert mice, and (3) that inhibition of
PKC does not prevent but only delays the developmental conversion from
multiple to mono climbing fiber innervation of Purkinje cells. As
explained below, all of these findings are in agreement with and
re-emphasize the importance of our tenet that LTD is necessary to reach
a high level of motor learning in a relatively short time (De
Zeeuw et al., 1998).
Consistent with earlier results obtained in the cultured Purkinje cells
(De Zeeuw et al., 1998), our present data show that the
specific expression of a PKC inhibitor in Purkinje cells results in an
almost complete loss of cerebellar LTD in the acute slice preparation
and that potential upregulation of PKC isoforms does not succeed in
blunting the biochemical effects of the L7-PKCI transgene in
situ. These findings also corroborate existing evidence that
cerebellar LTD induction has an absolute requirement for the activation
of PKC (Linden, 1996) and that the absence of this PKC
pathway cannot be sufficiently compensated for by the NO-PKG pathway
(Daniel et al., 1998).
Theoretically, it is possible that LTD was blocked by inhibition of PKC
in culture but not in slices, because NO-synthetase, which is
necessary for the NO-PKG pathway, is hardly present in the culture
system, whereas it is readily available in the parallel fibers in
slices. Such an outcome would obviously have precluded any further
interpretation of the relationship between LTD induction and motor learning.
The present finding that PKC inhibition inside Purkinje cells and a
consequent blockage of LTD induction do not directly corrupt their
simple spike and complex spike discharge properties is equally essential. If the short- and long-term dynamics of simple spike and
complex activities would have been abnormal in the L7-PKCI mutants,
their impaired motor learning might have reflected a secondary
abnormality of their Purkinje cells attributable to, for example, an
aberration of one of the kinase-regulated ion channels rather than to a
direct lack of LTD itself.
Although cerebellar LTD may be necessary for certain forms of
associative motor learning, it is unclear so far whether LTD is
essential for the maturation of motor coordination during development. A general assumption is that Purkinje cells may initially receive an
abundance of inappropriate or even erroneous parallel fiber inputs that
need to be attenuated to develop a normal level of motor performance.
However, the fact that simple spike firing rates in LTD-deficient
L7-PKCI mice are indistinguishable from those in their wild-type
littermates suggests that LTD probably does not contribute much to this
process. If this were the case, one would expect a reduction of
baseline simple spike activity in wild-type mice because the
excitability of their Purkinje cells to spontaneous parallel fiber
inputs would be attenuated because of the long-term synaptic
depression. Our findings suggest therefore that cerebellar LTD may be
used only for short-term adaptation, whereas other mechanisms, perhaps
in multiple brain areas, may serve to store and retrieve information
required for long-term adaptation and maturation of motor control
during development. This view is supported by the observation that
L7-PKCI mice do not exhibit general motor coordination deficits,
whereas rapid adaptation of their VOR after a few hours of
visuovestibular training is impaired (De Zeeuw et al.,
1998). Thus, apparently these LTD-deficient mice show deficient
motor learning in the short term, but in the long term they have other
sufficient learning mechanisms that allow them to obtain a normal motor
performance with normal dynamics of their simple spike and complex
spike responses.
An interesting hypothesis proposed by De Schutter
(1995) is that cerebellar LTD might act to keep the simple
spike rate of Purkinje cells within its physiological range. Because
the inhibitory circuitry onto Purkinje cells is to a large extent
feedforward, LTD could be the missing negative feedback mechanism
preventing overexcitation of Purkinje cells by their large number of
parallel fiber inputs. In this hypothesis, coactivation of adjacent
parallel fiber synapses alone would cause local increases in the
Ca2+ concentration that would be sufficient to
induce LTD at the activated synapses, without the need for conjunctive
complex spikes. However, our present data show that blocking the
activation of Ca2+-dependent PKC does not lead to
hyperactivity of Purkinje cells. Instead, both the short- and long-term
dynamics of simple spike firing in Purkinje cells of alert wild-type
and L7-PKCI mutant mice are indistinguishable. We conclude therefore
that cerebellar LTD is not critically involved in normalizing simple
spike rates in the long run, and thus probably is not operating as a
protective mechanism as proposed by De Schutter
(1995).
Although the L7-PKCI mutant shows normal simple spike and complex spike
discharge properties as well as normal motor performance, other mutants
can be found with both abnormal Purkinje cell responses and
coordination deficits. For example, Schiffman et al.
(1999) reported that mice lacking the Ca2+
binding protein calretinin exhibit impaired motor coordination, and
that the simple spike frequency, complex spike duration, and climbing
fiber pause of their Purkinje cells are severely affected in
vivo. Interestingly, no profound alterations were observed in the
slice preparation of these knock-out mice, suggesting that calretinin
primarily plays a major role at the network level. For example, the
climbing fiber pause probably results from a direct postexcitational
inactivation of the Purkinje cell membrane combined with the potential
impact of the climbing fiber collaterals on cortical interneurons such
as the Golgi cells and basket cells, which can directly inhibit the
transmission in the mossy fiber-granule cell pathway and the output of
Purkinje cells, respectively (Armstrong, 1974;
Simpson et al., 1996). In contrast to the calretinin
knock-outs, the L7-PKCI mutants showed no abnormalities in their
complex spike-simple spike intervals, including the climbing fiber
pauses. Thus, a blockage of LTD induction does not, unlike impairment
in Ca2+ homeostasis, disturb the basal firing
properties of the cerebellar circuitry.
Climbing fiber inputs play a central role in the
Marr-Albus-Ito theories of cerebellar motor learning (Simpson
et al., 1996). They are thought to guide the learning process by
signaling errors in performance (Ito, 1989). In the
L7-PKCI mutant, multiple climbing fiber innervation has been the only
developmental abnormality that could be identified (De Zeeuw et
al., 1998). Interestingly, however, our present results
demonstrate that the multiple climbing fiber innervation found in
younger L7-PKCI mutants (<3 months of age) does not persist in older
animals (>3 months of age). Because VOR adaptation is impaired in both
younger and older adult animals (De Zeeuw et al., 1998),
we conclude that the impaired motor learning observed in L7-PKCI mice
is not caused by an abnormal climbing fiber innervation of the Purkinje cells.
Persistent multiple climbing fiber innervation has been suggested to
underlie impaired motor coordination in PKC knock-outs (Chen
et al., 1995). This association is supported by the impaired motor coordination found in mGluR1- (Aiba et al., 1994;
Conquet et al., 1994), GluR |
TOP
ABSTRACT
INTRODUCTION
