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INTRODUCTION |
Efficient GABAergic1
synaptic transmission requires the presence of clustered postsynaptic
GABAA receptors localized in precise apposition to
presynaptic releasing sites. To form and maintain postsynaptic
clusters, neurons must possess the ability to appropriately sort,
target, cluster, recycle, and degrade GABAA receptors (1). All these processes are highly regulated and have been shown to rely on
the presence of the intact cytoskeleton and on the interplay of
functional proteins and intracellular factors (2, 3).
Among the proteins involved in delivering GABAA receptors
to the membrane, GABAA receptor-associated protein (4) is
thought to be crucial. In fact, not only does it show tubulin-binding activity (5) and interaction with the
2 subunit of
GABAA receptors (4), but it also binds
N-ethylmalemide sensitive factor, a protein that plays an
essential role in intracellular membrane trafficking (6). The possible
role of GABAA receptor-associated protein in
GABAA receptors trafficking is strengthened by its sequence
and structural similarities with mammals and yeast proteins involved in
membrane dynamics and vesicular transport (7-10), suggesting that this
protein could be specialized to recruit GABAA receptors
into budding vesicles targeted to the postsynaptic membrane (3).
After the insertion into the plasma membrane, GABAA
receptors may undergo clusterization. One of the major candidate
molecules for synaptic GABAA receptor clustering is
gephyrin (11-14), a tubulin-binding protein that has been shown to
co-localize with GABAA receptors at postsynaptic sites
(15). However, additional proteins must be involved in the clustering
process (3), because biochemical approaches failed to show a direct
interaction between gephyrin and GABAA receptors (16).
An increasing body of evidence indicates that the cytoskeleton is
essential for clustering GABAA receptors (4, 17-20). The cytoskeleton is formed by a complex meshwork of microtubules, actin
microfilaments, intermediate filaments, and many associated proteins.
The state of polymerization of microtubules appears to contribute in
modulating the activity of GABAA receptors (17-19). The
disruption of microtubules with colchicine, vinblastine, and nocodazole
has been shown to block muscimol-stimulated
36Cl
uptake into cortical microsacs and to
inhibit GABA-mediated currents in Xenopus laevis
oocytes expressing GABAA receptor subunits (17). Moreover,
in cultured hippocampal neurons nocodazole treatment induced a rundown
of muscimol-induced currents (18). Despite the efforts to clarify the
cellular and molecular mechanisms regulating cytoskeleton-receptor
interactions, this topic is still a matter of debate. In particular, it
is unknown whether receptor clusterization may interfere with the
gating properties of GABAA receptors.
In this study we induced the depolymerization of microtubules with
nocodazole to alter the organization of GABAA receptor clusters in cultured hippocampal neurons. We found that
declusterization of GABAA receptors was associated with an
acceleration of the rise time of mIPSCs. Moreover, in diffuse
GABAA receptors, current responses to ultrafast
applications of GABA showed a faster onset and an accelerated
desensitization. Model simulations suggested that the increased rate of
entry into the desensitized state might account for the accelerated
onset kinetics of GABAergic currents.
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EXPERIMENTAL PROCEDURES |
Cell Culture--
Primary cell cultures were prepared as
previously described (21). Briefly, 2- to 4-day-old (P2-P4) Wistar
rats were decapitated after being anesthetized with an intraperitoneal
injection of urethane (2 mg/kg). This procedure is in accordance with
the regulation of the Italian Animal Welfare Act and was approved by
the local veterinary service authority. Hippocampus were dissected
free, sliced, and digested with trypsin, mechanically triturated,
centrifuged twice at 40 × g, plated in Petri dishes,
and cultured for up to 14 days. Experiments were performed on cells
cultured for at least 7 days.
Nocodazole Treatment--
Nocodazole (purchased from Sigma,
Milano, Italy) was used to disrupt microtubules. It was applied at the
concentration of 10 µg/ml (22) from a 100% Me2SO stock
solution. The final concentration of Me2SO in the
working solutions was 0.1% (v/v). Nocodazole was applied in two
different ways: in the culture medium (bath treatment) and via the
patch pipette (intrapipette application). Bath treatment consisted of
adding the drug to the neuronal culture medium and incubating the cells
at 37 °C for 2 h. Because the effects of nocodazole are
reversible, in electrophysiological experiments the drug was added to
the extracellular and intracellular solutions (10 µg/ml). To verify
whether Me2SO itself could affect synaptic transmission,
some electrophysiological experiments (n = 12) were performed also on cells incubated with Me2SO alone in the
culture medium at 37 °C for 2 h. Me2SO 0.1% (v/v)
did not produce any change in the kinetic properties of mIPSCs.
Intrapipette application consisted of adding nocodazole to the
intracellular solution to apply the drug only to the recorded cell via
the patch pipette (18, 23).
Immunofluorescence Staining--
Immunocytochemistry for the
2 subunit of the GABAA receptor was
performed to analyze the organization of GABAA receptors on
the neuronal membrane both before and after bath treatment with
nocodazole as described above. After fixation with 4%
paraformaldehyde, hippocampal neurons seeded on coverslips were washed
with phosphate-buffered saline, blocked with 5% normal serum, and
incubated with an affinity-purified polyclonal antibody raised against
the amino-terminal region of the
2 subunit
(
2[1-33]R13/7, kindly provided by W. Sieghart, University of Wien, Austria). The resulting immune complex was visualized with fluorescein isothiocyanate-labeled goat anti-rabbit IgG
(Sigma, Milano, Italy). Note that the receptors labeled were only those
expressed on the cell surface, because the incubation with the
anti-GABAA receptor
2 subunit antibody was
performed on non-permeabilized cells. To evaluate the disruptive effect of nocodazole on the microtubular network, the same hippocampal neurons
were then used for a second immunocytochemical experiment using an
antibody against tubulin. Cells were then permeabilized with 0.1%
Nonidet P-40, washed with phosphate-buffered saline, blocked with 5%
normal serum, and incubated with a rat monoclonal anti-tubulin
antibody. The resulting immune complexes were visualized with
tetramethyl rhodamine isothiocyanate-labeled rabbit anti-rat IgG
(Sigma, Milano, Italy). Neurons were imaged with the Olympus (BX51WI)
confocal system by using sequential dual-channel recording of
double-labeled cells.
Electrophysiological Recordings--
Currents were recorded in
the whole-cell and outside-out configurations of the patch-clamp
technique using an EPC-7 amplifier (List Medical, Darmstadt, Germany).
In the case of whole-cell recordings, the stability of the patch was
checked by repetitively monitoring the input and series resistance
during the experiments. Cells exhibiting changed values were excluded
from the analysis. The series resistance (Rs)
was in the range of 4-8 M
. Both mIPSCs and GABA-evoked
currents were recorded at a holding potential of
70 mV. The
intrapipette solution contained (in millimolar): CsCl 137, CaCl2 1, MgCl2 2, 1,2-bis(2-aminophenoxy)ethane-N,N,N9-tetraacetic acid 11, ATP 2, and HEPES 10, pH 7.2, with CsOH. The composition of the
external solution was (in millimolar): NaCl 137, KCl 5, CaCl2 2, MgCl2 1, glucose 20, and HEPES 10, pH
7.4, with NaOH. mIPSCs were recorded in the presence of tetrodotoxin (1 µM) and kynurenic acid (1 mM) to block fast
sodium spikes and fast glutamatergic excitatory postsynaptic events,
respectively. To gain a better resolution of the kinetic properties of
GABAA receptors, all the electrophysiological experiments
have been performed at room temperature (22-24 °C) rather than at
physiological temperatures (35 °C). This choice is also justified by
the fact that, at higher temperatures, the recordings (particularly in
the excised patch mode) are very unstable and therefore not
particularly suitable for the kinetic analysis performed in the present
experiments. For the analysis requiring a high temporal resolution
(e.g. rise time kinetics of synaptic and evoked currents)
signals were low pass-filtered at 10 kHz with a Butterworth filter and
sampled at 50-100 kHz using the analog-to-digital converter CED 1401 (Cambridge Electronic Design, Cambridge, UK) and stored on a computer
hard disk. The data and acquisition software were kindly given by Dr.
J. Dempster (Strathclyde University, Glasgow, UK).
GABA was applied to excised patches using an ultrafast perfusion system
based on a piezoelectric-driven theta glass application pipette (24).
The piezoelectric translator was from Physik Instrumente (Waldbronn,
Germany), and theta glass tubing was from Hilgenberg (Malsfeld,
Germany). The time course of the solution exchange was estimated by
liquid junction potential measurements. The application of a
10%-diluted external solution to the open tip patch pipette evoked a
junctional current. The establishment of this current represents the
complete solution exchange around the patch pipette. The 10-90% of
this process occurred in 40-80 µs (10-90% solution exchange time).
The speed of the solution exchange was also estimated around the
excised patch by the 10-90% onset of the membrane depolarization induced by application of high (25 mM) potassium saline. In
this case the 10-90% rise time value (60-90 µs) was very close to
that found for the open tip recordings.
Data Analysis--
Miniature synaptic currents were analyzed
with the AxoGraph 3.5.5 program (Axon Instruments, Foster City, CA).
This program uses a detection algorithm based on a sliding template.
The template did not induce any bias in the sampling of events, because
it was moved along the data trace one point at a time and was optimally scaled to fit the data at each position. The detection criterion was
calculated from the template-scaling factor and from how closely the
scaled template fitted the data. The threshold for detection was set at
3.5 times the S.D. of the baseline noise. Using the same program, the
decay time constant of averaged mIPSCs was taken from the biexponential
fit of the decay time. The rise time was estimated as the time needed
for 10-90% increase of the peak current response.
The decaying phase of the mIPSCs and GABA-evoked currents was fitted
with exponential functions in the form,
|
(Eq. 1)
|
where
i and Ai are the
time constants and relative fractions of the respective components In
the case of analysis of normalized currents, the fractions of kinetic
components fulfilled the normalization condition, as in Equation 2.
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(Eq. 2)
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Deactivation time courses of mIPSCs and GABA-evoked currents
were fitted with a sum of two and three exponentials, respectively (n = 2 and n = 3).
The mean time constant calculated as,
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(Eq. 3)
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was used to estimate the speed of the decaying process. In the
case of current responses elicited by long (250 ms) GABA pulses, the
desensitization onset was described by,
|
(Eq. 4)
|
where Afast and
Aslow are the fractions of the fast and the slow
component, respectively, and
fast and
slow are the fast and the slow time constants.
As is the steady state current.
The goodness of the fit was assessed by minimizing the sum of
the squared differences. Brief (2 ms) paired pulses separated by a
variable time interval were used to test whether or not the entrance of
bound receptors into the desensitized state proceeded after the agonist
removal. The parameter R was calculated according to the
formula,
|
(Eq. 5)
|
where I1 is the first peak amplitude,
Iend is the current value immediately before the
application of the second pulse, and I2 is the
second peak amplitude. During the 2-ms pulse the onset of the
use-dependent desensitization is minimal. Thus, in the case
of continued entrance into the desensitized state after the first short
agonist pulse, the peak of the second response
(I2) was smaller than the first one resulting in
R < 1. Simulation experiments were performed using the
Bioq software (kindly provided by Dr. H. Parnas, Hebrew University,
Jerusalem). The Bioq software converted the kinetic model (see Fig.
8A below) into a set of differential equations and solved
them numerically. Because in the absence of agonist receptors can
spontaneously open at very low probability (25-27), for
simulation convenience it was assumed as the initial condition,
i.e. at t = 0 no bound or open receptors were present. Various experimental protocols were investigated by
"clamping" the agonist concentration time course in the form of
square-like pulses (ultrafast perfusion experiments). The solution of
such equations yielded the time courses of probabilities of all the
states assumed in the model. The fit of the experimental data was
performed by optimizing the values of rate constants. The procedure for
the rate constants optimization was based on the comparison of the time
course of recorded currents and that of simulated responses. As
described in detail under "Results," specific experimental
protocols were used to estimate different sets of rate constants.
Data are expressed as mean ± S.E., and all the values included in
the statistics represent recordings from separate cells. Unless
otherwise stated, statistical comparisons were made with the use of
unpaired t test, Wilcoxon signed rank test, and
Kolmogorov-Smirnov test (p < 0.05 was taken as significant).
 |
RESULTS |
Nocodazole Treatment Induces the Depolymerization of Microtubules
and the Declusterization of GABAA
Receptors--
GABAA receptor clusterization is known to
be dependent on the presence of the intact cytoskeleton. Nocodazole was
tested on cultured hippocampal neurons for its ability to depolymerize
microtubules and disrupt the cytoskeleton. Neurons were incubated with
nocodazole (10 µg/ml) for 20, 40, 60, and 120 min at 37 °C and
then immunostained for tubulin. Progressive disruption of microtubules
occurred with the increasing duration of the incubation time. By 40 min
most of the microtubules were depolymerized (data not shown). Bath application of nocodazole for 2 h at room temperature and 37 °C produced similar microtubule disruption (n = 4, data
not shown), indicating that incubation temperature does not influence
nocodazole effect.
Then the organization of GABAA receptors in the plasma
membrane was analyzed upon nocodazole-induced depolymerization of
microtubules. In control conditions, the typical branched microtubule
bundles were associated with brightly stained clusters of
GABAA receptors on the soma and dendrites (Fig.
1, A and B). After
nocodazole treatment for 1-2 h, the disassembly of the microtubular
network was accompanied by a redistribution of GABAA
receptors, because they were diffusely expressed throughout the surface
of the cells or still belonged to residual puncta (Fig. 1, C
and D). These experiments provide evidence that nocodazole
is able to depolymerize microtubules and to induce GABAA
receptors declusterization in cultured hippocampal neurons.

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Fig. 1.
Nocodazole treatment promotes
GABAA receptor clusters disassembly. A and
C, surface staining of GABAA receptor
2 subunit in unpermeabilized control neurons
(A) and nocodazole-treated (C) cells visualized
using an affinity-purified rabbit polyclonal antibody followed by
fluorescein isothiocyanate-labeled goat anti-rabbit IgG. B
and D, after permeabilization the same cells shown in
A and C were labeled with a rat monoclonal
anti-tubulin antibody followed by rhodamine-labeled rabbit anti-rat
IgG. Samples were analyzed by confocal microscopy. Scale
bars = 10 µm. Insets in A-D show a
low magnification of the corresponding fields. Scale
bar = 20 µm.
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Nocodazole Treatment Affects the Onset Kinetics of
mIPSCs--
mIPSCs were recorded from cultured hippocampal
neurons in the whole-cell configuration of the patch-clamp technique at
a holding potential of
70 mV in the presence of tetrodotoxin (1 µM) and kyneurenic acid (1 mM). Miniature
events were reversibly blocked by bicuculline (10 µM)
indicating that they were GABAA-mediated (data not shown).
mIPSCs were recorded from untreated neurons (control), and cells were
incubated for 2 h with nocodazole (10 µg/ml). Nocodazole
treatment induced a significant (p < 0.0001) acceleration of the current onset: in control conditions, the mean
10-90% rise time of mIPSCs was 0.96 ± 0.03 ms
(n = 35), whereas in cells treated with nocodazole it
was 0.72 ± 0.02 ms (n = 29, Fig.
2A). This acceleration
resulted in a significant (p < 0.001) shift to the
left of the cumulative rise time distribution (Fig. 2B).
Nocodazole treatment did not significantly (p > 0.05)
affect the mean frequency of mIPSCs (0.67 ± 0.11 and 0.46 ± 0.10 Hz, in control and nocodazole, respectively, data not shown).
Although nocodazole treatment induced a slight reduction of the mean
peak amplitude (from 63.2 ± 3.4 to 58.6 ± 4.8 pA), and this
difference was not significant (p > 0.05). Moreover,
the cumulative amplitude distribution of mIPSCs was not significantly
different from control (p > 0.05; Fig. 2D).
The decay of mIPSCs was unaffected by nocodazole: current decay was
fitted with a biexponential function with time constants
fast and
slow of 8.0 ± 0.7 and
39.7 ± 1.9 ms (Afast = 0.35) and 6.3 ± 0.8 and 36.4 ± 3.6 ms (Afast = 0.33) in
control and in nocodazole treated neurons, respectively (Fig.
2C). This resulted in a mean time constant
(
mean) of 28.1 ± 1.2 (control) and 26.1 ± 2.3 ms (nocodazole). Altogether, these results indicate that the
declusterization of GABAA receptors subsequent to
microtubule disruption accelerates the onset kinetics of mIPSCs.
Moreover, the lack of a clear effect of nocodazole on mIPSCs frequency
suggests that this drug does not have a main effect on transmitter
release.

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Fig. 2.
Bath treatment with nocodazole accelerates
the kinetics of mIPSCs. A, normalized onset of mIPSCs
recorded from a control (thick line) and nocodazole-treated
(thin line) neurons. Each trace is the average of
55 individual events. B, cumulative 10-90% rise time
distribution of mIPSCs in control conditions (thick line)
and after nocodazole treatment (thin line). C,
normalized and superimposed traces of mIPSCs recorded in control
(thick line) and in nocodazole-treated (thin
line) neurons. Each trace is the average of 55 mIPSCs.
D, cumulative amplitude distribution of mIPSCs in control
and nocodazole (thick and thin lines,
respectively).
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To confirm this hypothesis, further experiments were performed by
adding nocodazole into the patch pipette, so as to disrupt microtubules
only in the recorded cell. Currents were recorded for at least 50 min
to allow nocodazole to produce its effect (as detected from
immunocytochemical experiments). mIPSCs recorded in 10-min
epochs were pooled together and then analyzed. In control conditions
(n = 10) the onset of mIPSCs did not significantly change with time, and no signs of rundown were observed. During intrapipette application of nocodazole (n = 11) a
progressive acceleration of mIPSCs onset was observed (Fig.
3A). Compared with controls,
in the presence of nocodazole a significant (p < 0.05)
reduction of mIPSCs rise time was detected from 30 min on. The linear
regression curves through the data points obtained in control and in
the presence of nocodazole had almost the same intercept, indicating
that, starting from similar conditions, it took some time for
nocodazole to produce its effect. If we considered the first 10 min of
intrapipette application of nocodazole as internal control, the
progressive reduction of the rise time became significant
(p < 0.05) after 40 min. The cumulative rise time
distribution of the events recorded during the last 10 min was
significantly (p < 0.001) shifted to the left compared
with that relative to the first 10 min (Fig. 3B). This
result was consistent with the immunocytochemical data showing that it
takes less than 40 min for nocodazole to considerably disrupt
microtubules and to induce the declusterization of GABAA
receptors.

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Fig. 3.
Intrapipette application of nocodazole
affects the onset kinetics of mIPSCs. A, each data
point represents the average 10-90% mean rise time of mIPSCs recorded
during 10-min epochs in control conditions (filled circles,
n = 10) and in the presence of nocodazole in the
pipette (open circles, n = 11). *,
p < 0.05. B, cumulative 10-90% rise time
distribution of the miniature events recorded during the first 10-min
epoch (thick line) and the last 10-min epoch (thin
line) during intrapipette application of nocodazole
(n = 11). C, mean amplitude and averaged
mean ( m) of mIPSCs recorded during the
last 10 min, normalized to the corresponding values recorded during the
first 10 min (n = 11). D, cumulative
interevent interval distribution of mIPSCs recorded during the first
(thick line) and the last (thin line) 10-min
epochs during intrapipette application of nocodazole in 11 neurons.
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No significant changes (p > 0.05) in the peak
amplitude and decay of mIPSCs occurred during intrapipette application
of nocodazole. During the first and the last 10-min epochs, the peak
amplitude was 58.4 ± 2.7 and 52.5 ± 4.9 pA, respectively,
whereas
mean was 28.2 ± 2.3 and 29.9 ± 4.0 ms, respectively (Fig. 3C).
Similarly to the bath treatment, the intrapipette application of
nocodazole did not alter the frequency of mIPSCs, because the
cumulative interevent interval distributions of mIPSCs recorded at 10 and 50 min were similar (p > 0.05, Fig.
3D). The lack of changes in mIPSCs frequency obtained after
bath treatment and during intrapipette application of nocodazole
suggests that the main site of action of this drug is postsynaptic.
Nocodazole Accelerates the Onset Kinetics of Currents Evoked by
Ultrafast Applications of GABA--
The use of the ultrafast agonist
application system allows determination of the microscopic gating of
GABAA receptors with a time resolution adequate to synaptic
events (28-30). Firstly we have investigated the possible direct
effect of nocodazole on the kinetic properties of GABAergic currents.
GABA was applied alone or with nocodazole to the same patch excised
from untreated neurons (n = 6). The co-application of
GABA (10 mM) and nocodazole (10 µg/ml), after a
pre-equilibration with nocodazole for 1 min, did not alter the peak
amplitude and the kinetic properties of the currents, compared with
controls (p > 0.05; Fig.
4A). In particular, in the
absence or presence of this drug, the mean 10-90% rise time and the
mean peak amplitude values of GABA-evoked responses were similar
(312 ± 25 µs and 375 ± 48 pA in control,
versus 302 ± 32 µs and 412 ± 74 pA in
nocodazole, respectively, n = 6, paired t
test p > 0.05, Fig. 4A). Single
applications of GABA (n = 10) or GABA plus nocodazole
(n = 9) were also tested in different patches. Again
nocodazole (10 µg/ml) did not induce any modification in the shape of
GABA-evoked currents. The 10-90% rise time was 309 ± 15 and
314 ± 19 µs and the peak amplitude was 371 ± 78 and 352 ± 89 pA, in control and co-application of nocodazole,
respectively (p > 0.05). Altogether these data
demonstrate that nocodazole does not have a direct effect on
GABAA receptors.

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Fig. 4.
Microtubule disruption accelerates the onset
of GABA-evoked currents, only at saturating concentration of
agonist. A (left), normalized and
superimposed onset of current responses to 2-ms application of 10 mM GABA (thick line) and co-application of 10 mM GABA plus 10 µg/ml nocodazole (thin line)
after pre-equilibration with nocodazole for 1 min obtained from the
same patch. Note that the two traces overlap. A
(right), mean 10-90% rise time of currents evoked by 10 mM GABA in control and by co-application of GABA plus
nocodazole on the same patches (n = 6). B
(left), normalized and superimposed onset of current
responses to 2-ms application of 10 mM GABA from a control
(thick line) and a nocodazole-treated neuron (thin
line). Each trace is the average of three responses.
B (right), mean 10-90% rise time of currents
evoked by 10 mM GABA in control (n = 26)
and in nocodazole (n = 29). C
(left), normalized and superimposed onset of responses to
100 µM GABA in control (thick line) and after
nocodazole treatment (thin line). Each trace is
the average of four responses. Note that the two traces overlap.
C (right), mean 10-90% rise time of currents
evoked by 100 µM GABA in control (n = 13)
and nocodazole (n = 15). D, mean 10-90%
rise time of currents evoked by different concentrations of GABA in the
presence of nocodazole (n = 7-29), normalized to the
corresponding control values (n = 8-26). *,
p < 0.05; **, p < 0.01.
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To investigate the mechanisms underlying the changes of mIPSCs kinetics
induced by GABAA receptor declusterization, currents evoked
by ultrafast applications of GABA were studied with different protocols
in patches excised from nocodazole-treated neurons. Similarly to
mIPSCs, nocodazole treatment (10 µg/ml, for 2 h) induced an
acceleration of the onset of currents evoked by brief (2 ms) pulses of
a saturating concentration of GABA (10 mM, Fig. 4B, left). In these conditions, the value of the
mean 10-90% rise time of GABA-evoked currents was significantly
(p < 0.01) smaller than control (297 ± 9 and
245 ± 8 µs, in control, n = 29, and nocodazole,
n = 26, respectively, Fig. 4B,
right). According to the kinetic model proposed by Jones and
Westbrook (28) (see Fig. 8A), the activation of
GABAA receptor is the result of two kinetically separated
steps: the binding of the agonist to the receptor and the
conformational change from the bound-closed to the bound-open state. It
is worth noting, however, that, once the binding step is completed, the
receptor may enter either the fully bound open or desensitized state
and therefore the rate of entrance into the desensitized state directly
affects the occupancy of the open state. Among the steps reported in
the model we are referring to, the only one whose kinetics is dependent
on GABA concentration is the binding (the effective rate of binding is proportional to kon × [GABA]). Thus,
at low GABA concentrations, the binding step becomes much slower than
the conformational change and is thus rate-limiting. In this way the
study of macroscopic current onset at low concentrations of GABA sheds
light on the binding step. For this reason GABA responses elicited by
GABA concentrations ranging between 3 and 300 µM were
recorded from control and nocodazole-treated neurons. In this range of
agonist concentrations any difference in the onset kinetics could be
ascribed to a modification of the binding rate constant. Nocodazole did not affect the onset of current responses evoked by low concentrations of GABA up to 100 µM (Fig. 4, C and
D) but significantly (p < 0.05) reduced the
mean 10-90% rise time of currents evoked by GABA 300 µM
(Fig. 4D). A modification in the binding rate after nocodazole treatment would be expected to affect the onset of currents
evoked by a broad spectrum of non-saturating GABA concentrations. Moreover, at 300 µM GABA, current onset is close to
saturation and it is possible that the nocodazole-induced acceleration
of current rising phase results from the modulation of conformational transition rather that from faster binding (at this GABA concentration the binding rate is comparable to that of the conformational change). These data suggest that nocodazole-induced microtubule disruption and
GABA receptor declusterization do not influence the binding process and
thus the binding rate constant kon.
Effect of Nocodazole on the Peak Amplitude and Decay of GABA-evoked
Currents--
Short (2 ms) pulses of a saturating concentration of
GABA (10 mM) were applied to patches excised from control
and nocodazole-treated neurons. A non-statistically significant
(p > 0.05) reduction of the mean peak amplitude of
GABA-evoked currents was observed after microtubule disruption (from
435 ± 45 to 398 ± 34 pA, in control and in nocodazole,
respectively; Fig. 5, A and
B). Similarly to mIPSCs, the decay of GABA-evoked currents
was not significantly (p > 0.05) affected by
nocodazole treatment, being the
mean 34.3 ± 4.3 ms
and 45.7 ± 4.4 ms (n = 12-18), in control and
nocodazole, respectively (Fig. 5, C and D).

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Fig. 5.
Nocodazole treatment does not affect the peak
amplitude and the decay of GABA-evoked currents. A,
current responses to short (2 ms) pulses of 10 mM GABA
(upper trace) in control (thick line) and
nocodazole (thin line). B, mean peak amplitude of
GABA-evoked currents in control conditions and after nocodazole
treatment. C, normalized and superimposed traces of currents
evoked by brief pulses (2 ms) of GABA (10 mM) in control
conditions (thick line) and after nocodazole treatment
(thin line). Note that the two traces overlap. D,
averaged mean ( m) of GABA-evoked currents
in control (n = 12) and in nocodazole
(n = 18).
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Nocodazole Accelerates the Desensitization Kinetics of GABA-evoked
Currents--
It is known that GABAA receptor rapidly
undergoes desensitization during activation. This process is thought to
be involved in shaping synaptic currents (28, 31). For this reason an ultrafast agonist application system is necessary to resolve the fast
desensitization of GABAA receptors. To address this issue, a set of experiments was performed applying long pulses (250 ms) of
saturating GABA concentration (10 mM). Current
desensitization was fitted with biexponential functions with time
constants
fast = 3.8 ± 0.4 ms and
slow = 67.1 ± 11.0 ms
(Afast = 0.64 ± 0.02; As.s. = 0.18 ± 0.02, n = 9; Fig. 6A) and
fast = 3.3 ± 0.1 ms and
slow = 63.0 ± 5.9 ms (Afast = 0.63 ± 0.04;
As.s. = 0.13 ± 0.02, n = 8; Fig. 6A) in control and nocodazole, respectively. Because
the slow component of desensitization is unlikely to influence the
shape of synaptic currents, we focused only on the first 15 ms when the
fast component is predominant. In this case current desensitization was
fitted by a monoexponential function (Fig. 6B). Nocodazole
treatment produced a significant acceleration of the desensitization
onset as the time constant of the fitted current was reduced from
3.12 ± 0.23 ms (A1 = 0.69 ± 0.02) in control to 2.56 ± 0.09 ms (A1 = 0.71 ± 0.02) in nocodazole (p < 0.05) (Fig.
6C).

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Fig. 6.
Microtubule disruption accelerates the onset
of desensitization of GABA-evoked currents. A, currents
evoked by long (250 ms) pulses of GABA in control (thick
line) and nocodazole-treated (thin line) neurons.
B, normalized and superimposed traces of the first 15 ms of
currents evoked by long pulses of GABA in control (thick
line) and after nocodazole (thin line) treatment.
C, mean time constant ( ) of the fitted first 15-ms
current responses in control neurons (n = 10) and
in nocodazole-treated neurons (n = 11, *,
p < 0.05). In this time range current desensitization
onset was fitted with a monoexponential function.
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Nocodazole Treatment Does Not Affect GABA Responses to Paired Pulse
Protocols--
To analyze the recovery from desensitization, paired
pulse protocols are commonly used. Current responses to the second
pulse reveal the fraction of receptors able to be activated when the second pulse is applied, and therefore they are influenced by the
number of receptors that have recovered from the desensitized state. It
should be stressed, however, that the recovery of the second peak
reflects not only the recovery from desensitization but a more complex
process, including multiple re-entries into the open and desensitized
states due to the functional coupling between
desensitization-resensitization, opening-closing, and unbinding, and
therefore it reveals important information about the proportions
between respective rate constants (28, 30, 31).
Paired pulses (2-ms duration each) of saturating GABA (10 mM) were applied at different time intervals ranging
between 5 ms and 3 s (Fig.
7A). The percentage of
recovery of the second peak at each time interval was calculated
according to the formula reported under "Experimental Procedures."
After microtubule disruption the time course of the recovery of the
second peak was not changed with respect to control (p > 0.05, Fig. 7B).

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Fig. 7.
Nocodazole treatment does not affect the
recovery of the second peak in the paired pulse protocols.
A, paired pulses of GABA (2 ms, 10 mM) elicited
at a 15-ms interval in control conditions (thick line) and
after microtubule disruption (thin line). B,
normalized recovery of the second peak evoked in control (filled
circles) and after nocodazole treatment (open circles).
Each point represents the mean of five to nine experiments. In
the inset a part of the plot has been enlarged to show the
recovery of the second peak at the shortest time intervals.
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Model Simulations--
The present findings demonstrate that
declusterization of GABAA receptors following microtubule
disruption produces similar effects on both mIPSCs and GABA-evoked
currents, namely it accelerates the current onset without significantly
affecting the current decay or the peak amplitude. Moreover, the
experiments using an ultrafast agonist application system provided
evidence that nocodazole accelerates the onset of desensitization of
GABA-evoked currents without affecting the recovery of the second peak
in paired pulses protocols. In the attempt to reconstruct the gating
properties of declustered GABAA receptors, model
simulations were used. We referred to the kinetic model proposed by
Jones and Westbrook (28) (Fig.
8A), which fulfills the
minimum requirement to adequately reproduce the gating of
GABAA receptors in different experimental protocols. As
shown in the model, all the kinetic states that the receptor may occupy
are functionally coupled with the others, and thus current responses
are inevitably influenced by all the rate constants that govern the
transition from one state to the other (27, 31, 32). However,
particular rate constants can be estimated by analyzing current
responses obtained in different experimental conditions. Two sets of
rate constants that could well reproduce experimental data both in
control and after microtubule disruption were chosen. The value of
kon was estimated from the current onset
kinetics at low GABA concentrations when the binding is the
rate-limiting step. The values of the rate constants governing the
entry and the exit from the desensitized state
(d2 and r2, respectively)
were evaluated from the time course of currents evoked by long agonist
application and paired pulse protocols. The value of the close-to-open
rate constant (
2) and d2 were adjusted according to the current onset at saturating concentrations of
agonist. The open to close rate constant (
2) and the
unbinding rate constant (koff) were optimized to
reproduce the decay of currents elicited by 2-ms pulses of saturating
concentration of GABA and paired pulse protocols. The rate constants
governing the transitions of singly bound states were adopted from
Jones and Westbrook (28). It should be noticed, however, that at
agonist concentrations of >30 µM the probability of
occupancy of singly bound receptors is negligible. Once a complete set
of rate constants is found to properly reproduce the experimental data
obtained from all the protocols in control conditions, the effect of
microtubule disruption on GABA-evoked currents was included in the
simulations.

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Fig. 8.
Model simulations of the effect of nocodazole
treatment on the kinetics of GABA-evoked currents. A,
kinetic model proposed by Jones and Westbrook (28). According to their
model, the receptor (R) binds in sequence two molecules of
agonist (A), reaching the doubly bound and closed state
(A2R). From this state it can open or
desensitize (A2R* or
A2D, respectively). The singly bound open
and desensitized states are also present (AR* and
AD, respectively). B, values of the rate
constants producing the simulated current responses in control and
nocodazole. The values of rate constants of the singly bound states
were adopted by Jones and Westbrook (28) and were assumed not to be
affected. C, simulations of current responses to brief (2 ms, upper line) pulses of GABA (10 mM) in
control conditions (thick line) and in nocodazole
(thin line). D and E, normalized onset
(D) and decay (E) of simulated responses to brief
pulses (2 ms, upper line) of GABA (10 mM) in
control (thick line) and in nocodazole (thin
line). F, normalized simulation of responses to long
pulses (250 ms, upper line) of GABA (10 mM) in
control (thick line) and in nocodazole (thin
line). Only the first 15 ms are represented.
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Surprisingly, increasing the value of d2 was
sufficient to reliably simulate the experimental current responses to
different protocols (Fig. 8B). The increase in
d2 may account for the accelerated onset of
desensitization (Fig. 8F). Moreover, according to the basic
properties of bifurcating reactions, the increase in
d2 may result in a faster current onset (Fig.
8D). In fact, the rate of entry into the doubly bound open
state at saturating [GABA] can be approximated as
2 + d2 (assuming
2
2 and d2
r2), and therefore an increase in
d2 would be expected to accelerate the current
onset rate. Indeed also an increase of
2 (leaving d2 unchanged) could reproduce the accelerated
current onset, but, contrary to our observations, this would have led
to an increased peak amplitude. It should be noticed, however, that
good resolution could also be obtained by properly increasing the
values of both
2 and d2, but this
set of parameters was excluded as it resulted in a faster recovery of
the second peak in the paired pulse protocols. In our simulations the
increase in d2 led to a reduced occupancy of the
open state that resulted in a 10% reduction in the peak amplitude of
the simulated current. This effect, although present as a trend in our
experimental data, was below the significance threshold due to the
large variability of the current peak amplitude (Fig. 8C).
Any change in the resensitization rate constant
(r2) did not produce any significant improvement
in the steady state of desensitizing currents (Fig. 8F) or
in the recovery of the second peak in the paired pulse simulations
(data not shown), so this parameter was kept unchanged. The lack of
effects of nocodazole on the rising phase of currents evoked by
non-saturating [GABA] indicates that the binding rate
(kon) is not affected, hence this parameter was not modified in our model. The value of
koff was kept unchanged as the current
deactivation was unaffected after nocodazole treatment (Fig.
8E). Although the deactivation process is known to be shaped
by GABAA receptor desensitization, in our simulations, it
was not relevantly affected by the proposed increase of
d2 after nocodazole treatment.
 |
DISCUSSION |
The present experiments clearly demonstrate that, in cultured
hippocampal neurons, nocodazole-induced declusterization of GABAA receptors is associated with an acceleration of the
onset kinetics of mIPSCs. The quantitative analysis of current
responses to ultrafast applications of GABA and model simulations
suggest that this effect results from changes in the microscopic gating of GABAA receptors and in particular from an accelerated
entry into the desensitized state.
It is known that the clusterization of native and recombinant
GABAA receptors depends on the presence of the intact
cytoskeleton, because microtubule disruption alters the punctuate
expression of GABAA receptors on the plasma membrane
(18-20). Among the drugs used to disrupt the cytoskeleton, nocodazole
and colchicine are the most frequently employed (12, 33, 34). Because
colchicine has been shown to competitively interact with
GABAA receptor binding site (22, 35), we have used
nocodazole. Acute application of nocodazole showed that it does not
directly interfere with GABAA receptor function. In
agreement with other studies (18, 19) nocodazole treatment considerably
altered the organization of GABAA receptors clusters at the
plasma membrane by breaking down the microtubules. GABAA
receptor clusterization has been mainly addressed under a molecular
point of view in the attempt to identify the proteins involved in the
anchoring and clustering (4, 13, 16, 36-40). However, an accurate
analysis of the effect of receptor clusterization on GABAergic
transmission has been often neglected, and therefore the present study
has focused mainly on the gating properties of diffuse receptors. In a
previous study it has been reported that, in declustered recombinant
receptors, GABA-evoked currents showed a slower deactivation and a
faster desensitization (19), compared with clustered ones. However, as
pointed out by the authors, the limited time resolution of the agonist
application system precluded the assessment of the kinetic changes
occurring at the submillisecond time scale.
A novel and unexpected finding of the experiments reported here was a
nocodazole-induced acceleration of the onset kinetics of mIPSC in the
absence of any significant effect on the peak amplitude and decay
kinetics. These results were related to receptor declusterization
following microtubule disruption. However, the widespread effect of
nocodazole raises the possibility that this drug affects other targets
that in turn may influence GABAergic currents. In fact it has been
recently demonstrated that GABAA receptors clusterization
can be regulated in an activity-dependent manner (41, 42).
Thus, there is the possibility that bath incubation with nocodazole may
increase receptor declusterization by reducing the incoming inputs from
presynaptic nerve terminals. However, this possibility is unlikely,
because nocodazole treatment did not significantly modify the frequency
of spontaneous miniature events. Moreover, the observation that the
same results could be achieved when nocodazole was applied into the
pipette or in the bath argues against a presynaptic site of action of
the drug.
As already mentioned, the analysis of the current evoked in excised
patches by ultrafast application of GABA allows to dissect out the
kinetic properties of GABAA receptors under non-equilibrium conditions such as those occurring at the synapse (29, 30). It should
be stressed that upon patch excision the detached portion of the
membrane is no longer interacting with the intracellular structural and
functional proteins and/or intracellular factors. However, the evidence
that despite patch excision nocodazole treatment was still able to
affect the kinetic properties of GABA-evoked currents suggests that
this procedure did not alter GABAA receptors clusterization. Moreover, the observation that GABA-evoked currents and
mIPSCs exhibited similar kinetic changes after nocodazole treatment
indicates also that extrasynaptic receptors are clustered in control
conditions (40).
Similarly to mIPSCs, currents evoked by saturating concentrations of
GABA exhibited a faster onset when elicited from declustered GABAA receptors. The analysis of the current onset at
different agonist concentrations is very useful to distinguish the
binding of the agonist (kon) from the
conformational change from the doubly bound closed state to the open
state (
2). As already mentioned, the only step in the
activation kinetics of GABAA receptors that is
concentration-dependent (28) is the binding of the agonist to the receptor (kon). The lack of changes in
the rise time at low agonist concentrations suggests that
kon is not altered in declustered receptors.
This hypothesis is not in contrast with the accelerated onset of
currents evoked by higher concentrations of GABA (starting form 300 µM) when microtubules are disrupted. In those cases in
fact the concentration of GABA makes the binding occur faster than the
transitions to the open and desensitized states, and so the current
onset would reflect the kinetics of the slower steps. According to the
Jones and Westbrook model (28), the open and the desensitized states
are arranged in a bifurcating reaction and thus, if the values of
2 and d2 were comparable, during
the activation of GABAA receptors, the two processes would occur at the same time. For this reason currents have to be analyzed under non-equilibrium conditions, because the recovery from the open
and the desensitized states may influence the gating of
GABAA receptors. In line with the basic properties of
bifurcating reactions, the onset rate of the two processes should be
2 +
2 + d2 + r2. However, being
2
2 and d2
r2, the values of
2 and
r2 can be neglected. For this reason the onset
rate of both the open and the desensitized states can be approximately
defined as
2 + d2. The
nocodazole-induced acceleration of the current onset at high GABA
concentrations suggests the increase of at least one of these rate
constants. However, model simulations argue against enhancement of
2, because it would lead to an increase in current
amplitude in contrast with the unchanged amplitude of currents recorded
after nocodazole treatment. Although
2 may play an
important role in shaping GABAergic currents, in the present experiments its contribution was not crucial, because an increase in
its value did not significantly improve the fit of experimental data.
An increased value of d2, as suggested by the
accelerated desensitization onset, may account for the accelerated
current onset. The increase of d2 also led to a
10% reduction of the peak amplitude of the simulated current. In fact,
during receptor activation, the entry into the open and
desensitized states proceeds together. Thus an increased rate of entry
into the desensitized state produces a lower occupancy of the open
state leading to a reduction of the open peak probability. However, our
experimental data did not show any significant difference in the mean
peak amplitude of GABA-mediated currents after nocodazole treatment,
possibly due to the large responses variability. Hence, we propose that after nocodazole treatment the promoted entry of declustered
GABAA receptors into the desensitized state reduces the
number of "ready to open" receptors and accelerates their onset
kinetics. The lack of effect on the decay and on the recovery of the
second peak in the paired pulse protocols suggests that nocodazole
treatment does not alter the unbinding process
(koff). This hypothesis is in line with the
"two arms binding site" theory (28, 43, 44), which states that
kon and koff are
inversely correlated. Thus, the unchanged value of
kon could be reasonably associated with an
unchanged value of koff, suggesting that the
declusterization does not affect the affinity
(koff/kon) of
GABAA receptors.
 |
CONCLUSIONS |
In the present report we demonstrate that the cytoskeleton is
essential for promoting and maintaining GABAA receptor
clusterization and this contributes to the modulation of the GABAergic
currents in cultured hippocampal neurons. Differently from previous
studies (17, 19) we investigated declustered native GABAA
receptor function under more physiological conditions by analyzing
mIPSCs and GABA-evoked currents in cultured hippocampal neurons.
From experimental data and model simulations we propose that the faster
current onset may result from an accelerated entrance into the fully
bound desensitized state of declustered GABAA receptors. Our results are in line with the progressively more accepted idea that
receptor desensitized state plays a crucial role in shaping synaptic
currents (28, 31) and tonic inhibition (45-48).