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The Journal of Neuroscience, March 15, 2003, 23(6):2019
Coexistence of Excitatory and Inhibitory GABA Synapses in the
Cerebellar Interneuron Network
Joël
Chavas and
Alain
Marty
Laboratoire de Physiologie Cérébrale, Université
Paris 5, 75270 Paris, France
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ABSTRACT |
Functional GABA synapses are usually assumed to be inhibitory.
However, we show here that inhibitory and excitatory GABA connections coexist in the cerebellar interneuron network. The reversal potential of GABAergic currents (EGABA)
measured in interneurons is relatively depolarized and contrasts with
the hyperpolarized value found in Purkinje cells (
58 and 85 mV
respectively). This finding is not correlated to a specific
developmental stage and is maintained in the adult animal.
EGABA in interneurons is close to the mean membrane potential (56.5 mV, as measured with a novel "equal firing
potential" method), and both parameters vary enough among cells so
that the driving force for GABA currents can be either inward or
outward. Indeed, using noninvasive cell-attached recordings, we
demonstrate inhibitory, excitatory, and sequential inhibitory and
excitatory responses to interneuron stimulation [results obtained both
in juvenile (postnatal days 12-14) and subadult (postnatal days
20-25) animals]. In hyperpolarized cells, single synaptic GABA
currents can trigger spikes or trains of spikes, and subthreshold stimulations enhance the responsiveness to subsequent excitatory stimulation over at least 30 msec. We suggest that the coexistence of
excitatory and inhibitory GABA synapses could either buffer the mean
firing rate of the interneuron network or introduce different types of
correlation between neighboring interneurons, or both.
Key words:
cerebellum; Purkinje cells; basket cells; GABA; synapses; gramicidin-perforated patch
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Introduction |
In mammals, postsynaptic effects of
GABAergic synapses primarily result from the transient opening of
GABAA channels, which are permeant for
Cl
and bicarbonate ions. During
embryonic life, neurons accumulate Cl,
probably through the Na+-K+-Cl
cotransporter (NKCC1; Vardi et al., 2000
), resulting in a depolarized reversal potential (EGABA). Later in
development, however, Cl is extruded,
probably through the K+-Cl cotransporter
(KCC2; Rivera et al., 1999), so that the driving force for
Cl ions is inverted [hippocampus
(Ben-Ari et al., 1989), cortex (Owens et al., 1996), and cerebellum
(Brickley et al., 1996; Eilers et al., 2001)]. For this reason, mature
GABAergic and glycinergic synapses are generally viewed as inhibitory
(Rivera et al., 1999).
However, recent results indicate more depolarized
EGABA values for interneurons (INs,
mature or immature alike) than for principal cells in the dorsal
cochlear nucleus (Golding and Oertel, 1996) and in the sensory cortex
and amygdala (Martina et al., 2001). These results suggest that the
IN
IN synapses in these or in other central areas may be excitatory.
Determining EGABA is not sufficient to
know how the GABAergic postsynaptic potential (GPSP) affects the target
neuron. A GPSP primarily drives the membrane potential of the cell
(Vm) toward EGABA. If the driving force
EGABA - Vm is negative, this hyperpolarizes the cell and inhibits its firing, except when hyperpolarization-induced excitatory conductances are activated, giving rise to rebound excitation [as in deep cerebellar nuclei (Llinas and
Mühlethaler, 1988; Aizenman and Linden, 1999) or thalamic relay
cells (Bal et al., 1995)]. Conversely, if the driving force is
positive, the GPSP depolarizes the cell and is expected to increase its firing; however, the neuron can still be inhibited through shunting, as
long as the GABAA conductances are active (Staley
and Mody, 1992; Gao et al., 1998), or through longer-lasting,
depolarization-induced Na+ channel
inactivation (Zhang and Jackson, 1995) or
K+ channel activation (Monsivais et al.,
2000).
Sorting out these effects without perturbing the system is a challenge
in a slice preparation. Recording methods (e.g., using sharp
electrodes) may lead to a significant redistribution of Ca2+ and
Cl. In addition, application of a
GABAergic agonist or extracellular electrical stimulation can lead to
intracellular (Kaila and Voipio, 1987; Staley et al., 1995) or
extracellular (Kaila et al., 1997) ion redistribution. Accordingly, the
exact consequences of GPSPs on postsynaptic firing are often unclear or
controversial [e.g., in immature CA3 pyramidal cells (compare
Leinekugel et al., 1997; Lamsa et al., 2000)].
We addressed this question at the ININ synapses of the molecular
layer in cerebellar slices. This is an attractive preparation because
these INs include only two closely related cell types (Sultan and
Bower, 1998); moreover, quantal sizes are very large (Llano and
Gerschenfeld, 1993; Kondo and Marty, 1998a), so that firing of single
presynaptic neurons induces marked changes in the firing of the
postsynaptic cell (Häusser and Clark, 1997). Using a combination
of gramicidin-perforated patch and cell-attached recordings, we found
that ININ GPSPs display a variety of effects ranging from full
inhibition to full excitation.
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Materials and Methods |
General methods
Slice preparation. Cerebella from young rats (12-14
d old) were prepared as described earlier (Llano and Gerschenfeld,
1993). For older rats [postnatal day 20 (P20)-P25 or P35-P40], the
same procedure was used, except that animals were anesthetized with halothane (Sigma, St. Louis, MO), and that the cold
solution used for cutting contained (in mM): 248 D(+)-saccharose, 10 glucose, 4 KCl, 1 CaCl2, 5 MgCl2, 1.3 NaH2PO4, and 26 NaHCO3, pH 7.4 when equilibrated with a 5%
CO2 and 95% O2 mixture.
These special procedures, including the elimination of sodium from the
extracellular space during cutting, were designed to improve the
condition of INs in slices of mature animals.
The experimental chamber was continuously perfused at a rate of 1-1.5
ml/min with physiological saline containing, except where otherwise
stated (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, pH 7.4 when equilibrated with a 5% CO2 and 95% O2
mixture. All experiments were performed at room temperature (21°C;
SD, 2°C). Liquid junction potentials were corrected.
Cell selection. INs of the molecular layer were identified
as explained previously (Llano and Gerschenfeld, 1993). INs of the
lower third and upper two-thirds of the molecular layer were considered
basket cells and stellate cells, respectively.
Chemicals. The ionotropic glutamate receptor antagonists
6-nitro-7-sulfamoylbenzo[f] quinoxaline-2,3-dione (NBQX, 10 µM; Tocris) and D-APV (20 µM; Tocris) were applied to the recording
chamber throughout the experiments except when stated otherwise.
()-Bicuculline methochloride, (bicuculline, 10 µM; Tocris) or gabazine (10 µM; Research Biomedicals International, Natick,
MA) were used to block GABAA receptors.
Statistics. Values are mean ± SEM unless specified otherwise.
Whole-cell recordings
Pipette resistances used for whole-cell experiments were 3.5-5
M
. Series resistance values were kept in the range of 10-30 M
(Rs = 22 ± 4 M), and were not
compensated. The capacitance of the IN was in the range of 3-8 pF;
this gives a maximum time constant of the clamp of 0.24 msec. The total
input resistance observed at -60 mV was 1-10 G (2.5 M ± 1 G) and was therefore of the same order of magnitude as typical
pipette seal resistances. Indeed, the I-V curve reversed at
a depolarized value (35 ± 8 mV), confirming that the leak from
the pipette to the extracellular solution is not negligible.
Whole-cell experiments were performed with a "physiological"
intracellular solution containing (in mM): 144 K-gluconate,
6 KCl, 4.6 MgCl2, 10 K-HEPES, 1 K-EGTA, 0.1 CaCl2, 0.4 NaGTP, and 4 NaATP, pH = 7.2. With this solution, the Nernst equation gives -56 mV as the value for
ECl, not significantly different from the value measured for EGABA, the
reversal potential of GPSPs, as determined in gramicidin-perforated
patch recordings (58 mV).
GABA-sensitive channels are permeable not only to
Cl ions but also to
HCO
. Solving for the Goldman-Hodgkin-Katz equation, assuming ECl = 56 mV,
EHCO3 = 6 mV, and a permeability ratio of 0.2 between HCO and Cl
(Bormann et al., 1987; Kaila and Voipio, 1987), gives a value of 53
mV for EGABA. However, using the
physiological intracellular solution in the whole-cell configuration,
control muscimol (20 µM) puff experiments gave
a reversal value of 56 ± 0.6 mV, the value of
ECl. Two factors explain that the
contribution of bicarbonate ions is negligible: the relatively high
value of the Cl concentration, which
minimizes the impact of any additional bicarbonate, and the fact that
the pipette solution, which equilibrates with the cell interior, did
not contain any bicarbonate.
Gramicidin-perforated patch
Recording patch pipettes had a resistance of 6-11 M.
Experiments were started when series resistance was under 100 M
(80 ± 10 M), giving a maximum time constant of the clamp of
0.8 msec. The total input resistance measured at -60 mV was 1-5 G.
The I-V curve reversed at -25 ± 5 mV. On the basis
of these numbers, the maximum change of the membrane potential of the
clamped cell attributable to the flow of current through
Rs was calculated as 0.5 mV.
The intracellular solution contained (in mM): 155 K-gluconate, 1 MgCl2, 10 K-HEPES, 1 K-EGTA, 0.1 CaCl2, 600 µM fura-2, and 50 µg/ml gramicidin D (Dubos; (Sigma), pH 7.2. This
low-Cl solution was selected to ensure
that the excitatory responses that were recorded were not caused by
unwanted Cl exchange between the pipette
and cell compartments (see below). A 50 mg/ml stock of gramicidin in
DMSO was prepared freshly (<2 hr before recording) and sonicated. This
was diluted with gramicidin-free solution, sonicated again for 10-30
sec, and centrifuged. The tip of the patch pipette was filled with the
gramicidin-free solution. A gradual increase in pipette-cell
conductance was observed 10-40 min after seal formation. Spontaneous
passage to whole-cell recording was a very common occurrence in these
experiments, particularly with mature animals. This was detected as a
change in the polarity of GPSCs and by a somatic fluorescence increase
(recorded with a high-resolution CCD camera; Fig. 1B)
above the background level, reflecting the diffusion of fura-2
molecules from the pipette into the cell. Experiments were discontinued
as soon as such changes were noticeable.
Cell-attached experiments
Cell-attached pipettes were small (10 M) and filled either
with a K+-rich solution (containing in
mM: 150 K-gluconate, 4.6 MgCl2, 10 K-HEPES, 1 K-EGTA, and 0.1 CaCl2) or with a
Na+-rich solution (containing in
mM: 125 NaCl, 2.5 KCl, 0 or 2 CaCl2, 1 MgCl2, 1.25 NaH2PO4, 26 NaHCO3, and 10 glucose, preequilibrated with
O2 and CO2). The
K+-rich solution was initially selected to
allow the recording of GPSCs after breaking the seal at the end of the
experiments, as explained below. However, with the
Na+-rich solution, the cell was less
perturbed while recorded in cell-attached mode, and GPSCs could still
be recorded after breaking the seal.
With the K+-rich solution, it was observed
that the firing frequency of the IN depends on the pipette potential,
indicating the presence of a conductance link between the pipette and
cell compartments. This was not observed when the pipette was filled with the extracellular saline solution. For this reason, the results of
the experiments in the two conditions were kept apart in the analysis.
Extracellular stimulation (0.2 Hz; protocol described below) was
performed at least 50 times (and up to 300 times) both in control
conditions and in the presence of GABAA blockers.
For these experiments, the pipette potential was maintained at 0 mV.
At the end of most cell-attached experiments,
GABAA blockers were washed away, and whole-cell
recordings were performed to verify the effectiveness of the
presynaptic stimulation.
Extracellular stimulation
Extracellular stimulation was performed with glass pipettes
similar to those used for whole-cell recording.
Stimulating presynaptic stellate and basket cells. It was
essential for the interpretation of our cell-attached experiments that
extracellular stimulation would not produce any subthreshold excitation
of the recorded cell. Eventually the following protocol was adopted. In
a first step, we searched for the location of the axon of the recorded
cell by positioning the stimulation pipette at a distance of ~100
µm of the recorded soma, in the proximal molecular layer (close to
the Purkinje cell layer). The side where antidromic action potentials
were evoked was the side of the axon (Pouzat and Marty, 1999). Having
noted the value of the threshold for stimulation of antidromic action
potentials, the pipette was moved to the other side, again at a
distance of 100 µm of the recorded soma, and the stimulation
potential was set at 30 V above this threshold value. When using
whole-cell recording, this procedure does not result in any direct
potential change in the interneuron soma (Pouzat and Marty, 1999).
Under these conditions, any change in the firing pattern that was
observed can be ascribed to synaptic effects and not to direct
stimulation of the recorded cell. To verify this point, the same
protocol was then repeated in the presence of
GABAA blockers. As a further control, in most
experiments (including the examples shown in Fig. 5), a whole-cell
recording was finally established with the original recording pipette,
and it was verified (1) that the extracellular stimulation did not produce any cellular current change, and (2) that GABAergic currents were elicited after washing off bicuculline.
The number of presynaptic INs was estimated by counting failures in
this last part of the experiments. We found a failure rate ranging from
0 to 0.92, with an average of 0.31 (n = 17). On the
basis of the mean failure rate of individual connections as measured in
paired recordings (0.615; calculated from Kondo and Marty, 1998a, their
Table 1), and assuming independence, we obtain an average number of
presynaptic neurons of 2.38.
Stimulating presynaptic granule cells. To stimulate
presynaptic granule cells (or climbing fibers or both), a stimulating pipette was positioned in the granule cell layer ~100 µm away from
the recorded cell. Another pipette was positioned as explained above in
the molecular layer to stimulate stellate and basket cells. With this
arrangement, pure glutamatergic and GABAergic synaptic potentials could
be elicited in the same experiment without resorting to pharmacological blockers.
Estimate of the bias in the measure of the mean resting potential
of INs
Using the K+ channel reversal
method. We compared several methods to determine the mean resting
potential in unperturbed interneurons. We call this parameter
VCA because it applies to
cell-attached recordings. We initially used the method of
K+ channel reversal to measure
VCA (Zhang and Jackson, 1993;
Verheugen et al., 1999; Lu and Trussell, 2001). In this method, the
pipette is filled with a K+-rich solution
(composition as above) so that the reversal potential of
K+-sensitive channels is very close to 0 mV. The pipette potential for which
K+-selective single-channel
currents revert their polarity is the mean resting
potential of the cell (Vd = VCA - Vp = 0). In INs, this method seemed
particularly convenient at first, because all somatic patches contained
BK channels, which are highly selective for
K+ ions and display very high unitary
conductance. The seal conductance is irrelevant in these measurements,
because it affects the holding current value registered at each
potential but not the voltage for which reversal occurs. The pipette
holding potential was 0 mV; voltage pulses were given with a duration
of 500 msec, a frequency of 0.2 Hz, and amplitude increments of 10 mV.
At each test potential, single-channel currents were individually
measured, and an estimate of their amplitude was obtained by averaging.
The reversal potential was calculated by fitting a straight line to the
average single-current amplitude as a function of potential, yielding a
value of 71 ± 7 mV (n = 11). This is 14.5 mV
more hyperpolarized than the estimate of
VCA determined by the equal firing
method (Veq; see below). However,
while applying hyperpolarized steps in the pipette (Vp = 70-110 mV), although
the quality of the seals was good (several gigaohms), action potentials
were invariably stopped. This may have resulted in a change in the
dynamics of the voltage-activated channels and consequently in a change
in the membrane potential. A likely mechanism is provided by the
evidence presented in Results, indicating that the high pipette
K+ concentration used in these experiments
induced a conductance link between cell and pipette. This conductance
may have led to cell depolarization (because the pipette holding
potential was 0 mV) and therefore to an elevation of the resting
Ca2+ concentration. During the negative
voltage pulses that were used to determine VCA,
the depolarizing current through the patch membrane was removed, but
the counteracting K+ conductance
attributable to Ca2+-dependent
K+ channels presumably remained active,
leading to a membrane potential measurement below the value that would
apply for an undisturbed cell. Thus, the
K+ channel reversal method gave a value
that may have been too hyperpolarized.
Using the current-clamp gramicidin-perforated patch
configuration. The mean membrane potential,
Vm, for a given clamp current, was
defined as the mean potential of the cell, spikes excluded. Spikes were
identified with an automatic selection routine, and short sections were
eliminated around each spike by using a threshold slope value of 1 mV/msec (both at the spike onset and at the end of the
hyperpolarization potential). Similarly, the spike threshold was
determined at the time position when the slope crossed the value 1 mV/msec before an action potential.
To measure VCA, it is standard
practice to determine the zero current potential in the current-clamp
perforated patch configuration. But for compact cells, such as the INs
studied here, which have a very high input resistance (1-10 G),
this measurement of VCA is biased
because of the leakage resistance through the seal. The associated bias
may reach tens of millivolts (see Appendix, Eq. 4).
Therefore, another method was derived, in which the intensity
i of the clamp is set such that the firing rate equals the
mean spontaneous firing rate measured in the cell-attached
configuration in the same preparation (as detailed in Results). Our
estimation of VCA (which we call the
"equal firing potential," Veq) is
the mean membrane potential measured in these conditions, excluding spikes. The bias Veq VCA of such an estimate is a fraction
of VCA T, where
T is the spike threshold and is on the order of a few
millivolts (Appendix, Eq. 5). Therefore, the equal firing potential
Veq appeared as the best estimate of
VCA.
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Results |
Interneurons have more depolarized GABAA reversal
potentials than Purkinje cells
The EGABA of GPSCs was measured
using the gramicidin-perforated patch method, which allows
voltage-clamp experiments but preserves the anionic conditions of the
cell (Kyrozis and Reichling, 1995) (Fig.
1A,B). Presynaptic
stellate and basket cells were stimulated extracellularly (Vincent et
al., 1992). Ionotropic glutamate receptors were blocked with a
combination of NBQX and D-APV. In Purkinje cells
(PCs) at P12-P14, a classical pattern of reversing GPSCs was observed,
with a mean reversal potential of -85 ± 5 mV (n = 7; Fig. 1C), similar to that found for short applications of exogenous GABA (Eilers et al., 2001). However, in most of the IN
recordings, only inward current responses were obtained in the
potential range in which the postsynaptic cell could be held without
firing action potentials, and EGABA
could not be measured directly. Extrapolation from the points in the
nonspiking voltage range (-100 to -60 mV), using the
Goldman-Hodgkin-Katz equation, gave a mean reversal potential of 58
mV ± 5 mV (n = 5; Fig. 1D). Because the extracellular K+ concentration
(Ko) can influence the intracellular
Cl concentration (e.g., by interfering
with Cl transporters; Rivera et al.,
1999), we performed similar experiments with a Ko
of 5 instead of 2.5 mM. This, however, did not
significantly affect EGABA in INs
(61 ± 6 mV; n = 4; Fig. 1E),
showing that variations in Ko cannot explain the
depolarized value of EGABA observed in
INs.
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Figure 1.
Determination of EGABA
in INs and Purkinje cells. A, Schematics of the
experimental conditions. An interneuron or a Purkinje cell was studied
using a gramicidin-perforated patch, with a recording pipette that
contained a very low Cl concentration and 600 µM fura-2. An extracellular stimulation
(stim.) pipette was positioned in the molecular layer to
stimulate presynaptic INs. B, After seal formation,
capacitive currents evolved with time over tens of minutes (top
panel; from an interneuron recording), reflecting the
incorporation of gramicidin channels in the patch. Inspection of the
pipette-cell assembly allowed visualizationof spontaneous transitions
to the whole-cell recording mode (middle panel).
This was accompanied with a shift in the polarity of spontaneous and
evoked GPSPs (bottom panel; arrows mark
extracellular stimulations of presynaptic INs) as well as an increase
of the apparent size of action potentials attributable to the sudden
increase of the access conductance to the cell soma. C,
Averaged GPSCs obtained at various holding potentials in a Purkinje
cell experiment (P12-P14). Bottom panel,
I-V curve for this experiment. There is a clear
reversal at 87 mV. Pooled results gave
EGABA = 85 ± 7 mV;
n = 7; the thick line on the
Vp axis indicates the corresponding voltage
range). D, Similar experiment as performed on INs
(P12-P14). The reversal potential is obtained by extrapolation with
the Goldman-Hodgkin-Katz equation (54 ± 4.5 mV in the example
shown; 58 mV ± 4.5 mV for a series of 5 cells).
E, Similar experiment as performed on P12-P14 INs, with
a higher extracellular K+ concentration (5 mM instead of the standard 2.5 mM). Reversal
potential, 58 ± 6 mV in the example shown; 61 ± 6 mV
for a series of four cells. F, Similar experiment as
performed on P35-P40 INs. Here, responses to short puffs of muscimol
(10-30 msec; pipette concentration, 20 µM) were used
instead of synaptic currents. Reversal potential, 59 ± 8 mV in
the example shown; 60 ± 8 mV for a series of seven cells.
Thick lines in C-F indicate the range
comprising the mean ± 2 SD in each condition.
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The above experiments, combined with published ones (Eilers et al.,
2001), confirm that the negative shift of
EGABA with age is complete for PCs by
P12. However, it might not be the case for INs. To test this point,
puff applications of muscimol were performed on fully adult INs
(P35-P40; Fig. 1F), giving an
EGABA value of -60 ± 8 mV
(n = 7). Therefore, the depolarized value of
EGABA is not attributable to a late
change in intracellular Cl concentration
for this class of cells.
We conclude that, from P12, EGABA is
much more depolarized for INs than for PCs. The difference likely
reflects a higher intracellular Cl
concentration in INs than in PCs. If the contribution of bicarbonate ions to the GABAergic conductance is neglected (see Materials and
Methods), values of the intracellular Cl
concentration of 5.4 and 15 mM can be calculated
for PCs and INs, respectively. In the remainder of this paper, we
investigate the consequences of the latter value for GABAergic
signaling at the ININ synapses. We shall focus our attention on the
P12-P14 stage except when otherwise stated. Moreover, to restrict the possibility of variations linked to the nature of the postsynaptic cell, from this point on, we performed experiments exclusively on
postsynaptic basket cells.
Estimate of the mean membrane potential in unperturbed cells
Given the depolarized value of
EGABA for the INs, are GABAergic
currents inhibitory or excitatory? The effect depends primarily, but
not exclusively, on the driving force
EGABA - Vm for GABAergic currents (see
Introduction). Knowing the value of
EGABA, we aimed next at estimating the
value Vm in an unperturbed cell as
accurately as possible.
At this stage, a distinction is needed between the mean membrane
potential, designating the value that is experimentally measured (e.g.,
in current-clamp configuration) and that is noted
Vm, and the actual mean membrane
potential of unperturbed cells. The latter is noted
VCA because it applies to cells
recorded in the cell-attached configuration.
To estimate VCA, two main methods are
available in the literature: the zero current potential measurement and
the method of K+ channel reversal (Zhang
and Jackson, 1993; Verheugen et al., 1999; Lu and Trussell, 2001). As
explained in Materials and Methods, the first technique is clearly
inappropriate for cerebellar interneurons. The second technique was
performed on INs, but it turned out to yield seriously biased results
as well (see Materials and Methods).
Therefore, another method was derived on the basis of the fact that INs
are spontaneously active in our preparation (Llano and Gerschenfeld,
1993). In cell-attached recordings, INs fire irregularly at a mean
firing rate, f, that varies among cells in the
range of 0.1-20 Hz (Kondoand Marty, 1998a; results below). In
gramicidin-perforated patch recordings, the intensity of the clamp, i, was set such that f equaled the mean
spontaneous firing rate measured in the cell-attached configuration in
the same preparation. Our estimate of
VCA (which we called the equal firing
potential) is the mean membrane potential,
Veq, measured in such a configuration, excluding spikes. A quantitative argument is presented in Appendix to
show that the equal firing potential is a better estimate of VCA than the zero current potential.
The determination of Veq is
illustrated in Figure 2. In each
experiment, f and Vm values
were obtained at various holding currents. Resulting
f-Vm curves were very
steep, particularly at f >1 Hz, so that the frequency range
0.1-20 Hz was obtained within a small potential range. However, the
position of individual f-Vm curves varied
markedly from cell to cell along the potential axis. On the basis of
these results, Veq was estimated by
taking the intersects of
f-Vm curves with a
horizontal line (Fig. 2, dashed line), which represented the
mean firing frequency value obtained in cell-attached experiments
(i.e., 3.2 Hz; calculated from Kondo and Marty, 1998a, their Table 1;
results from cell-attached experiments described below are consistent
with this value). This estimate is a first approximation, because it
assumes that individual resting potentials of individual cells are not
correlated with their resting firing frequency. In reality, some
correlation exists, as shown in Figure 2, dotted slanted
line, which was determined as explained in Appendix. Because of
the steepness of individual f-Vm curves, it did not
really matter whether Veq was
estimated by taking intersects with the dashed or dotted line:
respective values were 56.5 ± 7.5 mV (n = 12)
and 56.1 ± 7.8 mV (n = 12). These values are
similar to EGABA (-58 ± 5 mV).
They are also similar to the values of the spike threshold, which were
determined in whole-cell and perforated patch recordings alike at
-53.7 ± 7.5 mV (n = 9).
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Figure 2.
Determination of the equal firing potential in
interneurons. A,
f-Vm curves. Interneurons
were recorded in the current-clamp mode with the gramicidin-perforated
patch configuration. The holding current was set in each cell so that
its mean firing frequency lay in the range of 0.1-20 Hz. For each
holding current, the mean frequency is plotted against the mean
potential of the cell. Points joined by a
line belong to the same cell. The equal firing potential
Veq is the intersection of the
f-Vm curve of each cell
either with the line f = 3.2 Hz (dashed
horizontal line), corresponding to the mean firing frequency as
measured in cell-attached experiments, or with the
fCA-VCA curve
(dotted line; calculated according to Eq. 8; see
Appendix). Veq = -56.5 ± 7.5 mV
(n = 12); this is our best measurement of the mean
membrane potential of the interneurons in this preparation.
B, Comparison of two Gaussian curves, with means and
variances corresponding to the measurements of
EGABA (thin line) and
Veq (thick line). Note that there is a high
degree of overlap between the two curves. The curves have been scaled
so that their integrals are identical.
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The driving force EGABA Vm determines whether the synapse is excitatory
or inhibitory
The fact that the membrane potential
VCA and the reversal potential
EGABA have a similar distribution
would imply that ININ synapses can be both excitatory and
inhibitory, assuming that the driving force is the main determinant of
the sign of the synapse.
To examine this point, we performed whole-cell recording experiments
with an intracellular Cl concentration
of 15 mM to obtain a value of
ECl matching the mean value of
EGABA, 58 mV.
GPSPs were induced through extracellular stimulation of a presynaptic
IN (see Materials and Methods). Figure
3A, left, illustrates a cell
with a low VCA value (see Fig. 2), in
which the holding current is adjusted so that the firing frequency is
low (f = 0.1 Hz). In three of three experiments,
the induced GPSPs resulted in the emission of action potentials. For
these three cells, the probability values of inducing an action
potential were, respectively, p = 0.2, 0.4, and 0.9. Thus, GPSPs are excitatory in these conditions. On the other hand,
Figure 3A, right, illustrates a cell with a more depolarized
VCA in which the holding current was
adjusted to produce repetitive firing. In this case, the induced GPSPs resulted in a clear delay of the following spike compared with the
position that would have been obtained without stimulation and were
therefore inhibitory.
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Figure 3.
Positive and negative modulation of cell firing as
measured in current-clamp experiments. A, Whole-cell
experiments, Cli = 15 mM. Left
panel, The holding current is set such that
Vm ~ 70 mV; in these conditions,
the resting firing frequency, f, is very small. An
action potential can be induced by the extracellular stimulation of a
presynaptic interneuron (stim. arrow; the probability to
obtain a spike was p = 0.2 in this recording).
Right panel, with a more depolarized cell
(Vm ~ 58 mV), the stimulation inhibits spontaneous
firing (from another experiment). B, Same type of
experiments in gramicidin-perforated patch recordings.
C, Summary results. Closed triangles
(n = 9) and open circles
(n = 5) represent, respectively, gramicidin and
whole-cell experiments. The y-axis represents
 n1, the net gain or loss of spikes
during the period of  /4 after the stimulation, where = 1/f. Results originating from the same cells are
connected with lines. The effect of a GPSP is shown to
shift from inhibition to excitation for
Vm ~ 60 mV. Whole-cell and
gramicidin results alike can be fitted with an exponential function,
the equation of which is given in Appendix (Eq. 6).
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The same types of experiments were then repeated in the
gramicidin-perforated patch configuration. And the same types of
results as in whole-cell recordings were obtained: at hyperpolarizing potentials, GPSPs were able to induce action potentials (either single
spikes or bursts of up to three spikes), whereas at more depolarizing
potentials, GPSPs were accompanied by an inhibition of the spontaneous
firing (Fig. 3B).
Both whole-cell and gramicidin results are summarized in Figure
3C. The response was quantified by measuring the difference between the observed number of spikes after the GPSP and that expected
from the background activity. For consistency with later results, this
difference, called n1, was
calculated over a period of /4, where is the mean control
interspike interval ( = 1/f). In a first
approach, we call a synapse excitatory if
n1 > 0, and we call it inhibitory
if n1 < 0. The relation of
n1 on membrane potential has
similar shapes in whole-cell recordings and in gramicidin experiments,
and a sign reversal occurs in both cases near -58 mV (Fig.
2C), which is at the value of
EGABA that was derived earlier (Fig.
1).
Note that most of the cells remained on one or the other side of the
dividing line for the entire frequency range 0.1 < f < 20 Hz and gave therefore rise only to inhibitory
or excitatory responses. Nevertheless, for a few recordings, sign
reversal could be obtained in the frequency range 0.1 < f < 20 Hz simply by altering the holding potential
[as previously shown using sharp electrode recordings in the INs of
the dorsal cochlear nucleus (Golding and Oertel, 1996)].
Unitary excitatory GPSPs
An extracellular stimulation may simultaneously activate more than
one presynaptic IN. We next asked whether unitary GPSPs can trigger
spikes in the postsynaptic IN.
To this end, we reexamined gramicidin-perforated patch recordings
between extracellular stimulations. If the holding potential was near
-70 mV, spontaneous GPSPs were invariably depolarizing and often led
to spikes or even bursts of spikes. An example is given in the
recording of Figure 4A
(holding current, 20 pA). Here the amplitude of spontaneous synaptic
potentials was 6.7 ± 0.6 mV (events with spikes excluded), and
the proportion of events leading to spikes and to bursts of spikes was
11.5% each. These results indicate that single-cell GPSPs can trigger
action potentials.
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Figure 4.
Excitatory spontaneous synaptic potentials.
A, Current-clamp recording with a gramicidin-perforated
patch. The cell was maintained near -68 mV by injecting a
hyperpolarizing current of 20 pA. Glutamatergic synaptic currents were
blocked with CNQX and APV. Left, Examples of spontaneous
synaptic potentials leading to a train of spikes (top),
a single spike (middle), or subthreshold
(bottom), with the corresponding occurrence rate.
Right, Superimposed plot of the two events with spikes
showing the full extent of the action potentials. B,
Spontaneous currents recorded in another cell near -66 mV (holding
current, 6.6 pA) led mostly to delayed single-spike discharges.
Stimulation of INs (stim.) leads to similar excitatory
responses. Action potentials are clipped for clarity.
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The shape of spontaneous events and their ability to trigger action
potentials were steeply voltage-dependent. In the cell illustrated in
Figure 3B, GPSPs near -70 mV holding potential (holding
current, 8.8 pA) were always subthreshold (results not shown), with a
shape very similar to that illustrated in Figure 4A,
bottom left record. However, when a slightly more depolarized holding potential was selected (approximately -66 mV; holding current,
-6.8 pA), most spontaneous GPSPs led to delayed spike firing (Fig.
4B). Similar events were triggered by electrical stimulation of presynaptic INs (Fig. 4B). They were
blocked by bicuculline (results not shown). Even though an analysis of
the cellular mechanisms underlying these events falls outside the scope
of this study, the results at hand clearly suggest that the
depolarization induced by the activation of GABA channels is enhanced
by excitatory voltage-dependent conductances. Therefore, the
depolarization is not limited to EGABA; the
record of Figure 4B suggests that the membrane
potential exceeds EGABA for tens of milliseconds
before each spike.
Mixed inhibitory and excitatory effects of ININ synapses as
measured in cell-attached experiments
Our main conclusion so far, that excitatory and inhibitory
synapses between INs coexist in this preparation, rests mainly on
current-clamp experiments. Because cerebellar INs have very large input
resistance (several gigaohms), however, small current changes may have
induced dramatic effects on the excitability of the cell; to confirm
the above results, less invasive methods were required.
Therefore, we investigated the effects of GPSPs on basket cell firing
in cell-attached experiments in which interference with cell
excitability is minimized. Care was taken to stimulate one or a few
presynaptic INs without injecting any significant current in the
recorded cell (see Materials and Methods). The pipette was filled with
high Na+ solution except in a series of
experiments to be described below.
Four typical examples are illustrated in Figure
5A-D. Left panels
show superimposed consecutive traces centered on stimulation time
(arrows), and right panels show
analysis of the results in terms of action potential frequency. In
Figure 5A, stimulation of GABAergic inputs resulted in a
pure inhibition of the cell firing. The right panel reveals
an inhibitory period (light gray, lasting 70 msec after the
stimulation) during which the frequency was much lower than the control
frequency f (horizontal line).
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Figure 5.
Variable effects of presynaptic stimulation on
cell firing as measured in cell-attached recordings. All recordings
were performed with a Na+-rich pipette solution.
A, Pure inhibitory response. Left,
Superimposed consecutive traces (n = 25) showing the effect of extracellular stimulation
(stim.) on spontaneous action potentials as recorded in
the cell-attached configuration. Right, Summary plot of
instantaneous spiking frequency across the stimulation period. The mean
control frequency, f, is represented by a thick
horizontal line. Stimulation results in a transient frequency
decrease (light gray area). B, Mixed
inhibitory-excitatory response. Stimulation results in a transient
frequency decrease (light gray area) followed by a
frequency increase (dark gray area). C,
Results from another experiment in which extracellular stimulation
mainly resulted in a frequency increase after a delay of ~20 msec.
D, Excitatory response measured in a cell with very low
resting frequency (0.3 Hz). Spikes were observed in 7 of 100 sweeps
with a latency of ~5 msec.
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In Figure 5B, the stimulation resulted in biphasic
inhibition-excitation sequences of cell firing. The right
panel reveals an inhibitory period, lasting 90 msec after the
stimulation, and a rebound excitatory period (dark gray,
with a sharp peak at 90-110 msec after the stimulation, followed by a
smaller tail) during which the frequency was clearly enhanced.
Figure 5C illustrates an experiment in which the main effect
was an enhancement of firing observed 20-60 msec after extracellular stimulation. There was an indication of a short inhibitory period (lasting ~20 msec), possibly reflecting shunt inhibition, but its
statistical significance could not be guaranteed in view of the scatter
of the control data. Thus the response of Figure 5C is rapid
and almost purely excitatory, whereas that of Figure 5B is
longer-lasting and of a mixed inhibitory and excitatory nature.
Whereas most recordings displayed a resting firing frequency between
0.5 and 20 Hz, allowing a simultaneous analysis of excitation and
inhibition, one-third of the cells (8 of 24) had a very low resting
firing frequency (f < 0.5 Hz), which precluded
in practice any detection of inhibition. Three of these eight cells
failed to manifest any response and will not be considered further.
However, the remaining five displayed a clear excitatory response, as
illustrated in Figure 5D. Here, single spikes were elicited
shortly after the stimulation (mean latency, 5 msec in the case shown;
range across cells, 5-80 msec). In two of the five responsive cells, trains of spikes (up to three spikes per train) were occasionally obtained (results not shown).
Spike balance after synaptic stimulation
The various effects of GABAA stimulation on
postsynaptic firing can be visualized by plotting
n(t), the difference between the mean number
of action potentials observed up to a certain time t after
the stimulation and that expected from the control frequency observed
before the stimulation. n(t) measures the spike balance, i.e., the integrated spike deficit or surplus after the stimulation.
Figure 6A shows plots
of the spike balance for four experiments. To facilitate comparisons
between cells, times have been normalized with respect to the mean
control interspike interval, . Cell a shows an excitatory
response (same recording as in Fig. 5C). Here there is a
very short inhibitory period, presumably corresponding to the time of
activation of GABA channels. This is followed by a sharp rise of
n, which then stabilized at the level of 0.4, indicating
a net increase in the number of action potentials. Cells b
and c display mixed inhibitory and excitatory action, in
which an initial inhibition is more or less exactly compensated by a
later excitation, so that at long times, the difference n
is close to 0. For these examples, the effect of stimulation can be
described as spike synchronization: after the stimulation, spikes are
clustered around a rather precise time (the point of maximum slope in
the plots of Fig. 6A) with a position that varied
among cells between 0.4 * and 0.8 * . Finally, cell
d illustrates a pure inhibitory action. In this case, there is no late excitation, and the initial inhibition remains as a net loss
of ~0.6 spike.
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Figure 6.
Spike surplus or deficit after synaptic
stimulation at P12-P14. A, Plot of the spike balance as
a function of time. The time is counted from the stimulation
(stim.) and is normalized with respect to . The
y-axis shows at each time value the difference between
the mean total number of observed spikes and the corresponding number
expected from the firing frequency measured before the stimulation. The
four traces show an almost purely excitatory response
(a), mixed inhibitory-excitatory responses
(b, c), and a pure inhibitory response
(d). The hatched area represents
the 95% confidence limit of such traces, measured
starting from random points in standard saline solution.
B-D, Summary results from 31 experiments.
Open and closed symbols represent,
respectively, experiments performed with pipettes filled with a
Na+- and K+-rich solution.
Circles and squares represent cells with
f  0.5 and f < 0.5 Hz,
respectively. Data represent the excess or deficit in the number of
spikes, which was calculated during an early period of /4 after
stimulation (B; n1)
and during a longer period of after stimulation (D;
n2). The first
columns show results obtained after extracellular stimulation
of GABAergic afferents. Second, third, and fourth
columns show control results respectively obtained from data
gathered after an arbitrarily chosen time point (located 300 msec
before the actual stimulation), after the stimulus in the presence of a
GABAA blocker (bicuculline or gabazine), and after an
arbitrarily chosen time point in the presence of a GABAA
blocker. In B and D, dotted
lines delimit the calculated 95% confidence interval on the
value of n1 and
n2, respectively.
C, n1 is significantly
correlated (p < 0.01) to the resting firing
frequency. Low-frequency cells tend to display excitatory responses,
and high-frequency cells display inhibitory responses. Large
squares in B and D indicate
average values across all experiments. In C and
D, dark gray areas contain cells
significantly inhibited by a GPSP; light gray areas
contain cells significantly excited by a GPSP (see Results).
excit., Excitation; inhib.,
inhibition.
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To characterize the early phase of the response, n was
computed at a time of 0.25 * , when the positive-negative effect of the stimulation on the spike balance was the strongest. We will note
it n1, so that
n1 = n(0.25 * ).
If the stimulation has no average effect,
n1 should lie inside the 95%
confidence interval (0.09, 0.11) calculated on the assumption of a
binomial distribution.
This procedure permits a provisional classification of the early
responses in three groups (Fig. 6B). One group (four
cells) had n1 > 0.11, corresponding to excitation; a second group (five cells) with
-0.09 < n1 < 0.11, was
considered neutral; and a third group (12 cells) had
n1 < 0.09, indicating inhibition.
A more refined analysis had to be performed with low-frequency cells
(f < 0.5 Hz), represented in Figure 6,
squares. Two of them had
n1 > 0.11 and were therefore
already classified as "excited" cells. Three had -0.09 < n1 < 0.11, and have been
classified as "neutral" cells. However, low-frequency cells
displayed effects in a time window (up to 100 msec) that was much
shorter than 0.25 * , so that the 0.09, 0.11 confidence interval
did not apply. Within the 100 msec window, a significant excitatory
effect could be demonstrated in all three neutral cells (including that
of Fig. 5C) in addition to the two cells in which
n1 was >0.11.
Finally, to analyze late phases of the response to the stimulation, the
spike balance was determined after the time . We note this overall
balance n2, with
n2 = n(). The time
was chosen as the time after which the spike balance did not
significantly change. Summary results are shown in Figure
6D. The confidence interval for
n2 was determined as 2 * SD, 2 * SD, SD = 0.05 being the SD of the value of
n2 when there was no stimulation (sham stimulation; see below). At first sight, the results are similar
to those of Figure 6B, but close inspection reveals
interesting changes. Although the excitatory responses are not
significantly modified from one analysis window to the other, the
initially inhibitory responses display a variety of late changes,
ranging from full compensation to increased inhibition. As
a result of this, four early inhibitory responses were reclassified as
neutral (inhibition followed by excitation), with no net effect on
n2.
Altogether, of the 21 cells that responded to stimulation, 7 (33%)
gave an excitatory response, 8 (38%) gave an inhibitory response, and
6 (29%) gave mixed responses (including four inhibitory-excitatory sequential responses, as in Fig. 5B, and two cases in which
the response was dependent on the spike frequency before stimulation) without any significant net gain or loss of action potentials.
Control cell-attached experiments
To test whether the frequency changes illustrated in Figure 6,
A and B, were genuine, we compared the results
with those obtained when starting the analysis from an arbitrary point
in time without stimulation. For
n1, no significant departure from
the control frequency f was observed, confirming that the
changes were linked to the stimulation (Fig. 6B, second
column of points). Likewise, the variations of
n2 were an order of magnitude
smaller after a random point than after the stimulation (Fig.
6D).
Next we asked whether the stimulation was producing its effect
exclusively through activation of GABAergic fibers. To answer this
question, we repeated the stimulations in each experiment after
perfusion of bicuculline or gabazine in the bath (10 µM of either blocker). This invariably abolished the effects, as can be
seen in the summary data of Figure 6B-D
(third, fourth columns; the slightly larger scatter seen for
these data in Fig. 6D is attributable to a reduced
number of trials compared with the control runs). Thus, both the
positive and negative frequency changes illustrated in Figure 6,
A and B, are attributable to activation of
GABAA receptors. These results exclude any
participation of direct subthreshold stimulation of the recorded cell
through the stimulation pipette. They also exclude the participation of electrical junctions, which have been shown to play a significant role
in IN networks of the hippocampus and cortex (Strata et al., 1997;
Galarreta and Hestrin, 1999; Gibson et al., 1999) (for review, see
McBain and Fisahn, 2001), as well as in the cerebellum of adult guinea
pigs (Mann-Metzer and Yarom, 1999). We argued earlier that, in juvenile
rats, electrical junctions are occasionally present but play a modest
functional role (Pouzat and Marty, 1998).
Cell-attached experiments performed using a
K+-rich pipette solution
Noteworthy additional results were obtained using a
K+-rich pipette solution, which was used
in an early set of experiments to obtain better whole-cell recordings
at the end of the experiment as a check of the effectiveness of the
extracellular stimulation (see Materials and Methods). However, in
these conditions, it was noted that the frequency of firing depended on
the pipette potential, indicating an electrical link between the
pipette and the cell interior. Accordingly, because the pipette voltage
was held at 0, the experiments performed with the
K+-rich solution (Fig.
6B-D, gray dots) tended to have a high resting frequency (> 5 Hz; Fig. 6C), and the stimulation uniformly
led to an early inhibitory response (n = 10), whereas
the experiments performed with the
Na+-rich solution led to a variety of
responses, including excitatory ones, as detailed above. Our
interpretation of the results, in accordance with Figure 3,
A and B, is that a
K+-rich solution in the cell-attached
pipette depolarizes the cell, thus shifting the GPSPs to their
inhibitory mode.
P20-P25 cells
All experiments illustrated in Figures 2-6 have been performed at
P12-P14, at a stage during which the molecular layer undergoes extensive synaptic development. Even though
EGABA does not appear to change with
age (Fig. 1), it seemed possible that the basket cell excitability
could change after P14, thus altering the proportion of cells that
would respond in one or the other way to GABAergic stimulation. We
therefore performed additional cell-attached experiments at a more
advanced age (P20-P25) and compared the results with those at
P12-P14.
The mean firing frequency of the interneurons was higher at P20-P25
(fCA = 7.1 Hz;
n = 15) than at P12-P14, but responses to a GABAergic
input were similar at both ages. Figure
7A illustrates a clear-cut
case, obtained from a P24 animal, in which GPSPs led to an increased
frequency of postsynaptic firing, peaking a few milliseconds after the
presynaptic stimulation. This effect was abolished by bicuculline
(results not shown). In general, P20-P25 experiments yielded all four
classes of responses illustrated in Figure 5. Summary results are given
in Figure 7, B and C. Of 15 P20-P25 basket
cells, 5 (33%) gave excitatory responses; 5 (33%) gave inhibitory
responses; and 5 (33%) gave mixed responses (including 3 inhibitory-excitatory sequential responses). Thus the proportions of
the principal response types appear identical at P20-P25 and at
P12-P14.
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Figure 7.
Effects of presynaptic stimulation measured in
cell-attached recordings at P20-P25. Symbols are
identical to those of Fig. 6. A, Example of a cell
excited by a GPSP. Top, Superimposed consecutive traces
(n = 25), showing the effect of a spontaneous GPSP
on the cell firing as measured in a basket cell of a P24 animal.
Bottom, Summary plot of instantaneous spiking frequency
across the stimulation (stim.) period. Stimulation
mainly results in a strong frequency increase (dark gray
area). This was abolished by bicuculline (see
B). B, C, Summary results from 15 experiments. Both inhibition (dark gray areas) and
excitation (light gray areas) are observed at /4 and
. Note that the proportion of low-frequency cells (open
squares) is lower at P20-P25 than at P12-P14 (compare with
Fig. 6C,D).
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Subthreshold excitatory GPSPs enhance the basket cell response to
an excitatory input
The results illustrated in Figures 3-7 indicate that for
hyperpolarized cells, slowly discharging cells, or both, activation of
ININ synapses can lead to the firing of action potentials in the
postsynaptic cell. In such recordings, however, effects of subthreshold
depolarizations may still have been inhibitory because of
depolarization-induced Na+ current
inactivation or K+ current activation
(Zhang and Jackson, 1995; Monsivais et al., 2000; Lu and Trussell,
2001). To address this issue, we tested how GPSPs would alter the
postsynaptic excitability after a delay of 30 msec. This delay was
chosen such that GABAergic currents were just over, as illustrated
below, to avoid shunting inhibition (Gao et al., 1998).
We examined the cell excitability by measuring the current threshold,
i.e., the minimal amplitude of depolarizing current, using 2-msec-long
pulses, which could induce an action potential. In the example shown
(Fig. 8A),
approximately half of the extracellular stimulations resulted in
failures of synaptic transmission. The results were separated depending
on the outcome of extracellular stimulation, and the current thresholds
were compared after successful stimulations and after failures. As may
be seen, in all cases of failures of the synaptic potential, the
current injections resulted in a depolarization that did not induce an
action potential, but after the induction of a synaptic potential, the
same current injection led to an action potential, suggesting that
GPSPs had a strong excitatory effect. Full analysis of the results
obtained in this experiment revealed that GPSPs induced a 55 pA shift
of the current threshold (Fig. 8B).
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Figure 8.
Enhanced excitability of the postsynaptic cell
after presynaptic stimulation. A, A 1 msec current pulse
is induced 30 msec after the stimulation (stim.) of a
presynaptic interneuron. The current pulse does not reach threshold
when synaptic transmission fails but always induces an action potential
after a successful stimulation (whole-cell recording;
Vm = 71 mV; Cli = 15 mM). B, For this cell, the current threshold
IT for stimulation, defined as the current
needed to induce an action potential with a probability of
p = 0.5, is 55 pA smaller after a successful
GABAergic stimulation than after a failure. C, The mean
difference IT between the current
threshold measured after the stimulation and its control value measured
before the stimulation is plotted for five cells in saline solution
(IT = 12.9 ± 3.2 pA) and in
bicuculline (bicu; IT = 6.5 ± 4.8 pA). In this plot, no distinction is made according
to whether the stimulation elicited a synaptic current or led to a
failure. On average, IT increases by
19.3 ± 3 pA between the two conditions
(p < 0.005).
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The results of five such experiments are summarized in Figure
8C. To pool all results together, we compared the current
thresholds with and without extracellular stimulation, instead of
separating the traces obtained after extracellular stimulation
according to the absence or presence of failures. In normal saline
(containing NBQX and APV), the current threshold was on average smaller
by 19 ± 3 pA (p < 0.005) with presynaptic
stimulation than without stimulation. After perfusion of bicuculline in
the bath, the difference in current threshold was abolished (Fig.
8C). We conclude that, in whole-cell recordings performed
under physiological conditions (with an intracellular Cl
concentration of 15 mM) at a holding potential
negative of the firing range, the excitability of INs to depolarizing
current injection is enhanced 30 msec after the GABAergic stimulation. These results are thus fully consistent with those of Figure 3, A and B, left panels, and Figure 5D,
which all displayed enhanced firing under similar conditions.
Interactions between parallel fiber and interneuron
synaptic inputs
We finally asked whether the effects shown in Figure 8 could
result in significant interactions between interneuron and parallel fiber inputs. NBQX was omitted from the bath solution, and current injection in the postsynaptic cell was replaced by an extracellular stimulation of a presynaptic granule cell axon. One pipette was in the
molecular layer, at ~100 µm from the recorded cell, to recruit
axons from presynaptic basket cells, stellate cells, or both. The other
pipette was located in the granule cell layer, at the same level as the
recorded cell, to recruit the ascending axon of presynaptic granule
cells (Fig. 9A). In the
example shown, stimulation of the granule cell axons alone (stim
GC) triggered an action potential in 43% of the trials. If the
same stimulus was preceded with a stimulation of presynaptic INs
(stim IN), the percentage of action potentials
increased to 72% (Fig. 9B). In two other cells, this
percentage increased from 52 to 62%, and from 79 to 97%
(n 60 stimulations).
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Figure 9.
Interactions between glutamatergic and GABAergic
synaptic inputs. A, Schematics of the experimental
conditions: one extracellular stimulation pipette (stim
IN) was positioned in the molecular layer near the
Purkinje cell layer (PCL) to stimulate presynaptic INs,
and another one (stim GC) was placed in the granular
cell layer to stimulate ascending axons from granule cells or climbing
fibers. The postsynaptic IN is recorded in whole-cell configuration
using a physiological Cl concentration
(Cli = 15 mM). B,
Current-clamp experiment (Vm ~ 70 mV).
Superimposed traces (10 in each
panel) show that the probability for a successful
glutamatergic stimulation to induce an action potential increases from
p = 0.43 to p = 0.72 (n = 60 trials in each condition;
p < 0.01) if it occurs 30 msec after a successful
GABAergic stimulation. Therefore, 30 msec after a GPSP, the
excitability of the cell is increased. C, Synaptic
currents recorded under voltage-clamp conditions for the same cell as
in B. The two panels show superimposed
traces of postsynaptic currents induced by stim
GC and stim IN, respectively. Exponential fits
to the decays of the synaptic currents gave time constants of 1 and 7 msec, respectively. The marked differences in the time courses of decay
for the two sets of traces demonstrate that stim
GC and stim IN respectively induce only EPSCs
and only GPSCs. Note that at the time chosen for the second stimulus,
GABAergic currents have fully subsided, so that shunting inhibition
does not play a role here.
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To ascertain that the stim GC and stim IN stimulations were indeed
respectively purely glutamatergic and GABAergic, voltage-clamp recordings were performed in second parts of the experiments, and the
synaptic currents elicited were examined (Fig. 9C). The results show that stim IN and stim GC were invariably associated with
slow- and fast-decaying synaptic currents, respectively. Very distinct
decay kinetics are associated with inhibitory and excitatory inputs,
respectively (Llano and Gerschenfeld, 1993), demonstrating that the
separation between the two kinds of input was fully effective.
In summary, the results show that under the conditions of these
experiments (physiological intracellular
Cl concentration, and holding potential
near 70 mV), stimulation of IN afferences enhances the responsiveness
to subsequent granule cell stimulation in a time range of tens of milliseconds.
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Discussion |
This study shows that, when taking into account the entire cell
population, ININ synapses are globally neutral in the sense that
they do not induce any net gain or loss of action potentials. However,
individual synapses are not. According to our best estimate, approximately one-third of the synapses are excitatory; one-third are
inhibitory; and one-third are mixed (mostly displaying an
TOP
ABSTRACT
Introduction
