J Neurophysiol 91: 1036 –1049, 2004.
First published September 10, 2003; 10.1152/jn.00364.2003.
Developmental-Dependent Action of Microtubule Depolymerization on the
Function and Structure of Synaptic Glycine Receptor Clusters in Spinal
Neurons
Brigitte van Zundert,1,2 Francisco J. Alvarez,2 Juan Carlos Tapia,1 Hermes H. Yeh,3 Emilio Diaz,1 and
Luis G. Aguayo1
1
Laboratory of Neurophysiology, Department of Physiology, University of Concepción, Concepción, Chile; 2Department of Anatomy,
Wright State University, Dayton, Ohio 45435; and 3Center for Aging and Developmental Biology and Department of Pharmacology and
Physiology, University of Rochester Medical Center, Rochester, New York 14627
Submitted 11 April 2003; accepted in final form 2 September 2003
van Zundert, Brigitte, Francisco J. Alvarez, Juan Carlos Tapia,
Hermes H. Yeh, Emilio Diaz, and Luis G. Aguayo. Developmen-
tal-dependent action of microtubule depolymerization on the function
and structure of synaptic glycine receptor clusters in spinal neurons. J
Neurophysiol 91: 1036 –1049, 2004. First published September 10,
2003; 10.1152/jn.00364.2003. Microtubules have been proposed to
interact with gephyrin/glycine receptors (GlyRs) in synaptic aggre-
gates. However, the consequence of microtubule disruption on the
structure of postsynaptic GlyR/gephyrin clusters is controversial and
possible alterations in function are largely unknown. In this study, we
have examined the physiological and morphological properties of
GlyR/gephyrin clusters after colchicine treatment in cultured spinal
neurons during development. In immature neurons (5–7 DIV), dis-
ruption of microtubules resulted in a 33 ⫾ 4% decrease in the peak
amplitude and a 72 ⫾ 15% reduction in the frequency of spontaneous
glycinergic miniature postsynaptic currents (mIPSCs) recorded in
whole cell mode. However, similar colchicine treatments resulted in
smaller effects on 10 –12 DIV neurons and no effect on mature
neurons (15–17 DIV). The decrease in glycinergic mIPSC amplitude
and frequency reflects postsynaptic actions of colchicine, since
postsynaptic stabilization of microtubules with GTP prevented both
actions and similar reductions in mIPSC frequency were obtained by
modifying the Cl⫺ driving force to obtain parallel reductions in
mIPSC amplitude. Confocal microscopy revealed that colchicine re-
duced the average length and immunofluorescence intensity of syn-
aptic gephyrin/GlyR clusters in immature (approximately 30%) and
intermediate (approximately 15%) neurons, but not in mature clusters.
Thus the structural and functional changes of postsynaptic gephyrin/
GlyR clusters after colchicine treatment were tightly correlated. Fi-
nally, RT-PCR, kinetic analysis and picrotoxin blockade of glyciner-
gic mIPSCs indicated a reorganization of the postsynaptic region from
containing both ␣2␤ and ␣1␤ GlyRs in immature neurons to only ␣1␤
GlyRs in mature neurons. Microtubule disruption preferentially
affected postsynaptic sites containing ␣2␤-containing synaptic
receptors.
INTRODUCTION
Postsynaptic densities (PSD) harbor neurotransmitter recep-
tors that are anchored in the membrane at precise locations
opposite to presynaptic active zones releasing neurotransmit-
ters. Cytoskeletal components are believed to stabilize these
molecular complexes. As such, both tubulin and actin are
major components of the PSD (Cotman et al. 1974; Kelly and
Address for reprint requests and other correspondence: L. G. Aguayo, Dept.
of Physiology, University of Concepción, P.O. Box 160-C, Concepción, Chile
(E-mail: laguayo@udec.cl).
1036 0022-3077/04 $5.00 Copyright © 2004 The American Physiological Society
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
Cotman 1978; Matus and Taff-Jones 1978), and several bridg-
ing proteins have been proposed to link membrane macromol-
ecules to the underlying subsynaptic cytoskeleton within the
PSD (reviewed in Sheng and Pak 2000).
Gephyrin, the first protein identified as a synaptic bridging
component, is a key constituent of the PSD at inhibitory
synapses (Triller et al. 1985, 1987). Gephyrin antisense and
knock-out experiments indicated that gephyrin is associated
with glycine receptors (GlyRs) and GABAA receptors
(GABAARs) in postsynaptic clusters (Essrich et al. 1999; Feng
et al. 1998; Kirsch et al. 1993; Kneussel et al. 1999). While it
is known that gephyrin strongly binds GlyRs through a peptide
domain within the M3-M4 cytoplasmic loop of the GlyR ␤
subunit (Meyer et al. 1995), the mechanism of interaction with
GABAARs is not understood. In addition, in vitro binding
studies suggested possible interactions of gephyrin with the
cytoskeleton. Gephyrin binds tubulin and microtubules directly
with high affinity and significant cooperativity (Kirsch et al.
1991) and can interact with microfilaments through intermedi-
ate actin-binding proteins such as profilin (Mammoto et al.
1998) or regulators of actin filament dynamics like collybistin
(Kins et al. 2000). Hence, a commonly proposed model for the
structure of glycinergic synapses hypothesizes that gephyrin
anchors GlyRs to subsynaptic cytoskeleton (Kirsch 1999; Kneus-
sel and Betz 2000). Tubulin depolymerization would then be
expected to disrupt synaptic GlyR clusters.
Morphological studies performed in spinal cord cultures
showed that microtubule depolymerization affected the num-
ber, structure, and intensity of gephyrin/GlyR postsynaptic
clusters (Kirsch and Betz 1995). However, microtubule disrup-
tion in hippocampal cultures failed to alter gephyrin/GABAA
clusters (Allison et al. 2000) and led to the conclusion that
neither microtubules nor cytoplasmic tubulin-dimers play a
significant role in the organization of gephyrin-containing in-
hibitory synapses. The reasons for these conflicting results are
unknown.
Using the whole cell patch-clamp technique, we previously
reported that the function of synaptic GlyRs, but not extrasyn-
aptic receptors, was changed in cultured spinal cord neurons
dialyzed with the microtubule depolymerization agent colchi-
cine (van Zundert et al. 2002). In contrast, the function of
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked ‘‘advertisement’’
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
www.jn.org
 COLCHICINE AFFECTS SYNAPTIC GLYRS IN IMMATURE NEURONS
synaptic ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionic
acid receptors (AMPARs) and gamma amino butyric acid type
A receptors (GABAARs) was unchanged. In this study, we
have further analyzed the developmental profile of colchicine
action on the function and structure of synaptic GlyR clusters.
We found a strong inhibitory effect of colchicine on immature
glycinergic synapses, which contain the neonatal ␣2␤ receptor.
After synaptic maturation, however, mainly the adult ␣1␤
GlyR remained in the postsynaptic membrane and colchicine
was not effective. GABAARs postsynaptic clusters were unal-
tered at any developmental stage. Preliminary results were
presented in abstract form (van Zundert et al. 2001).
METHODS
Cell culture
Mouse (C57BL/J6) spinal cord neurons obtained from five to six
embryos (13–14 days) were plated at 300,000 cells/ml into 35-mm
tissue culture dishes coated with poly-L-lysine (MW ⬎ 350 kDa,
Sigma Chemical, St. Louis, MO). The neuronal feeding medium
consisted of 90% minimal essential medium (GIBCO, Grand Island,
NY), 5% heat-inactivated horse serum (GIBCO), 5% fetal bovine
serum (GIBCO), and a mixture of nutrient supplements (Aguayo and
Pancetti 1994). Fresh media was replaced every 3 days. Experiments
were performed on 5–17 DIV neurons. The microtubule interacting
molecules colchicine, ␥-lumicolchicine, nocodazole, and taxol (20
␮M) were added to the culture media for 3 h or dialyzed into the
neurons via the recording patch pipette as previously reported (Kirsch
and Betz 1995; Rosenmund and Westbrook 1993; van Zundert et al.
2002).
Electrophysiology
For voltage- and current-clamp recordings in the whole cell con-
figuration, patch electrodes were filled with (in mM) 140 KCl, 10
BAPTA, 10 HEPES (pH 7.4), 4 MgCl2, and 2 ATP-Na2. The external
solution contained (in mM) 150 NaCl, 10 KCl, 2.0 CaCl2, 1.0 MgCl2,
10 HEPES (pH 7.4), and 10 glucose. After the whole cell configura-
tion was established, the capacitance of the cell and the series resis-
tance were compensated using the amplifier (⬎80%). Spontaneous
glycinergic miniature postsynaptic currents (mIPSCs) were isolated
by the addition of CNQX (2 ␮M, RBI), bicuculline (2 ␮M, RBI), TTX
(0.1 ␮M, Sigma), and MgCl2 (2 mM) to the external solution (van
Zundert et al. 2002). They were confirmed as glycinergic mIPSCs by
a complete blockade with 750 nM strychnine. At 1 and 25 min after
establishing the whole cell configuration, the spontaneous events
occurring during a 3-min interval were analyzed. The properties of the
mean mIPSC amplitude and frequency at 1 min were used as an
internal control (van Zundert et al. 2002). By presenting the data as
normalized (25 min/1 min), the results obtained with different drug
treatments were pooled and analyzed in terms of their statistical
differences. The noise was not significantly changed when neurons
were dialyzed with any of the cytoskeletal disrupters. Synaptic cur-
rents were recorded using Axotape 7.0 software and analyzed off-line
(Axon Instruments, Union City, CA). Every identified synaptic event
collected during the test intervals displaying an amplitude above the
background noise (12–15 pA) was analyzed using MiniAnalysis 5.0
software (Synaptosoft). Data were excluded if the uncompensated
series resistance (⬍8 M⍀) increased by ⬎15% during the recordings.
Unless otherwise noted, all reagents were purchased from Sigma.
Analysis of mIPSC properties included peak amplitude, inter-event
interval, rise-time, and decay-time constant. The decay-phase of
mIPSCs was best fitted with a single exponential curve. To avoid
analysis of data points with excess noise at the peak and baseline, the
rise and decay-phase were fitted between 10 and 90% of its amplitude.
J Neurophysiol • VOL 91 • FEBRUARY 2004 •
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
1037
Immunocytochemistry
Neurons were fixed for 5 (GlyR) or 15 min (GABAAR␥2) with 4%
paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Fixing time
was increased to 30 min for other antibodies. Cultures were perme-
abilized with a pretreatment of 0.25% Triton X-100 (15 min) to allow
better access to intracellular epitopes and then blocked with 10%
normal horse serum (NHS) for 30 min to reduce nonspecific staining.
Cells were incubated for 1 h with pair combinations of the following
primary antibodies: mouse monoclonal antibody (MmAb) against
gephyrin (mAb7a; 1:100; Boehringer-Mannheim, Indianapolis, IN),
rabbit polyclonal antibody (RpAb) against the ␣1 and ␣2 subunit of
the GlyR (1:10; Calbiochem, La Jolla, CA), guinea pig polyclonal
antibody (GpAb) against the ␥2 subunit of the GABAAR (1:1,000;
kindly provided by Dr. J. M. Fritschy, University of Zurich), MmAb
against synaptophysin (1:100; Oncogene, San Diego, CA), RpAb
against synaptophysin (1:500; Zymed Laboratories Inc., San Fran-
cisco, CA), MmAb against SV2 (1:200; kindly provided by K. M.
Buckley, Harvard Medical School, Boston, MA), and RpAb against
synapsin I (1:1,000; Calbiochem). We tested the following combina-
tions of antibodies: gephyrin/synapsin I, synaptophysin/SV2, synap-
tophysin/synapsin I, GlyR/gephyrin, GABAAR␥2/gephyrin, GlyR/
SV2, GABAAR␥2/SV2, and GABAAR␥2/gephyrin/synapsin I. Im-
munoreactive (IR) sites were visualized after incubation for 1 h with
appropriate secondary antibodies raised in donkey and conjugated to
FITC or Cy3 (1:50; Jackson ImmunoResearch Laboratories, West
Grove, PA). With every combination, we used a MmAb mixed with
a RpAb or GpAb. Presynaptic markers were usually labeled with
FITC-conjugated secondary antibodies and postsynaptic gephyrin and
GABAAR␥2 antibodies with Cy3-conjugated antibodies. For the tri-
ple immunolocalization, together with the normal GABAAR␥2/
gephyrin double immunolabeling, synapsin I was labeled with a
biotin-SI-conjugated RpAb (1:50) and stained with Cy5-conjugated
streptavidin (1:250; Jackson ImmunoResearch Laboratories). The
cells were coverslipped using Vectashield (Vector Laboratories, Bur-
lingame, CA).
Image visualization and sampling
For quantitative analysis of immunocytochemical data, samples of
spinal neurons (10 –15 cells from 2 separate experiments of paired
control and colchicine-treated cells) were chosen randomly for imag-
ing using confocal microscopy (Olympus Fluoview; 100⫻ oil immer-
sion objective, N.A. 1.35, digitally zoomed 3 times, pixel size ⫽ 0.07
␮m). Stacks of optical sections separated by 0.5 ␮m in the z axis were
acquired throughout whole cells. Dual color immunofluorescent im-
ages were captured in simultaneous two-channel mode. Antibody
dilutions were chosen for adequate immunofluorescent intensity to
minimize cross-talk between the channels. If even minimal cross-talk
between FITC and Cy3 fluorescence was observed (FITC usually
labeled the presynaptic marker and resulted in brighter fluorescence),
this was abolished by collecting Cy3 fluorescence above 610 nm. The
number of synaptic gephyrin and GlyR clusters was determined as the
number of clusters apposed/co-localized to punctate synapsin I or SV2
immunoreactivity. Co-localization was studied by superimposing both
color channels. For triple immunolocalizations, neurons were imaged
using a Leica TCS confocal microscope using multitracking imaging
of each channel independently and optical and confocal conditions
similar to those previously used and described above.
Analysis of cluster size
In contrast to previous confocal quantitative studies on gephyrin
cluster sizes done in tissue sections (Geiman et al. 2000; Lim et al.
1999; Oleskevich et al. 1999; Triller et al. 1990), the majority of
synapses in our cultures were positioned on the lateral sides of the
cells, and relatively few crossed over the top of neurons. Therefore
www.jn.org
1038 VAN ZUNDERT ET AL.
there were only few examples of gephyrin clusters viewed “en face”
where surface areas could be resolved accurately. Hence, we mea-
sured cluster lengths. Frequently, individual clusters were imaged in
more than one serial optical section. The optical section that contained
the largest and brightest immunofluorescence for each individual
cluster was selected for measurement. For cluster size measurements,
the stacks of confocal optical sections were analyzed using ImagePro
software (ver. 4.1; Media Cybernetics, Silver Spring, MD). Individual
clusters were detected in single optical sections by thresholding the
image from the maximum arbitrary gray level (AGL) pixel intensity
(4095) to 25% of this value. These image segmentation parameters
allowed us to detect even the fainter clusters while outlining intensely
fluorescent clusters inside their diffraction halo. Using this method,
individual clusters were automatically segmented and outlined, and
their maximum length was measured. For each experiment, imaging
conditions were kept constant, and no postcapture modifications were
done in images used for quantitative analysis. The data were compiled
in Microsoft Excel, analyzed in Statview, and plotted using Origin.
Importantly, our measurements of gephyrin cluster size gave similar
results to previous studies in spinal cord cultures (Meier et al. 2000;
Rosenberg et al. 2001) and spinal cord sections visualized with either
confocal microscopy (Oleskevich et al. 1999), conventional fluores-
cence microscopy, or three-dimensional reconstruction with electron
microscopy (Alvarez et al. 1997). For illustration, the neuron was
reconstructed from the stack of optical sections, and further figure
composition and labeling was done in CorelDraw 3.0, CorelDraw 8.0,
or SigmaPlot 4.0.
RT-PCR: Total RNA extraction and cDNA synthesis
To extract total RNA from the spinal cord cultures, cells were lysed
with 1 ml Trizol (5 min, 20°C, GIBCO), and 0.2 ml of chloroform was
added to each sample. After vigorous shaking, the mixture was
centrifuged (12,000g, 15 min, 4°C), and RNA was precipitated by
mixing the aqueous phase with isopropyl alcohol (0.5 ml, 10 min,
20°C) and centrifuged at 12,000g (15 min). Finally, the RNA was
washed two times with ethyl alcohol (75%) and spun down at 7,500g,
dried under the hood (5 min, 20°C), and dissolved in sterile RNAse
free H2O. The integrity and purity of each RNA sample was evaluated
by agarose gel electrophoresis (1–2%). The RNA concentration was
determined by: [RNA] ⫽ 40 ␮g/ml ⫻ A260 ⫻ Fd, where A260 is the
absorbance (260 nm) of the sample and Fd represents the dilution
factor. cDNA synthesis employed 12 ␮l of total RNA, 1 ␮l of random
hexamer primers (50 ng, Promega), 1 ␮l of 10 mM dNTPs, 4 ␮l of 5⫻
reaction buffer, 2 ␮l of DTT (0.1 M), 1 ␮l of RNAsin (40 U/␮l), and
1 ␮l of RT-Superscript II (200 U). The RT reactions (42°C) were
stopped after 1.5 h by cooling to ⫺20°C. cDNA was determined in
each case: [cDNA] ⫽ 33 ␮g /ml ⫻ A260 ⫻ Fd.
cDNA amplification and temporal subunit expression
PCR mixtures (25 ␮l final) contained 2.5 ␮l 10⫻ Taq polymerase
buffer, 0.5 ␮l dNTPs mix (10 mM), 0.75 ␮l MgCl2 (50 mM), 0.75 ␮l
sense (10 ␮M) and 0.75 ␮l antisense primers (10 ␮M), 3 ␮l cDNA
template (50 ng), 16.5 ␮l H2O, and 0.25 ␮l Taq polymerase (5 U/␮l).
A Biorad thermocycler was used with PCR conditions set to 94°C (3
min) for primary denaturation, 30 cycles of amplification (94°C de-
naturation, 30 s; 45–51°C annealing, 30 s; 72°C elongation, 30 s), and
10 min at 72°C for final elongation. The predicted sizes of ␣1, ␣2, and
␣3 amplicons were 529, 384, and 470 bp, respectively, and the ␤
amplicon was 183 bp. The PCR amplified products were resolved by
agarose gel electrophoresis (1–2%), photographed (Polaroid films),
and digitized (Cannon device) using Adobe Photoshop (4.0). The
following primers were used to amplify mouse GlyR subunit cDNA:
␣1 (sense: 5⬘-aggcccaacttcaaaggtcc-3⬘; anti-sense: 5⬘-ctgtgttgtagtgct-
tggtgc-3⬘); ␣2 (sense: 5⬘-ggtacaccatgaatgacctg-3⬘; anti-sense: 5⬘-
ccatccagatgtcaattgcctt-3⬘); ␣3 (sense: 5⬘-gctgacattaacactctcttgtcc-3⬘;
J Neurophysiol • VOL 91 • FEBRUARY 2004 •
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
anti-sense: 5⬘-ccatccagatgtcaattgcctt-3⬘); ␤ (sense: 5⬘-ggaattcgggatg-
gagacgtcc-3⬘; anti-sense: 5⬘-gctctcaagttgcattttgc-3⬘). The sequence of
all the primers used as the amplified fragments (sequence, restriction
maps) has been previously described (Kirchhoff et al. 1996). Negative
(omitting RNA, template sense primers) and positive (adult and em-
bryonic spinal cord cDNAs) controls were routinely included. At least
three independent experiments per condition were considered.
Data analysis
Data are expressed as means ⫾ SE. Statistical comparisons were
performed using Student’s t-test or ANOVA. Cumulative probability
distributions of mIPSC amplitudes were compared with Kolmogorov-
Smirnov test. P ⬍ 0.05 was considered significant.
RESULTS
Colchicine decreases spontaneous glycinergic mIPSCs in a
developmentally regulated manner
In agreement with a previous study (van Zundert et al.
2002), intracellular dialysis with colchicine decreased glycin-
ergic transmission in young (⬍12 DIV) cultured spinal neu-
rons. The amplitude of control glycinergic mIPSCs, isolated in
the presence of CNQX, bicuculline, and TTX, was rather stable
over a recording period of 25 min (Fig. 1A). In contrast,
neurons dialyzed with 20 ␮M colchicine in the patch pipette
displayed a large change in the amplitude histogram distribu-
tion after 25 min of recording (Fig. 1B, left). This decrease in
glycinergic activity resulted in a shift toward smaller current
amplitudes as shown in the cumulative probability amplitude
distributions (P ⬍ 0.001, Fig. 1B, right). Colchicine also
significantly reduced mean glycinergic mIPSC amplitude and
frequency by 29 ⫾ 4% (P ⬍ 0.001) and 69 ⫾ 8% (P ⬍ 0.001),
respectively.
We investigated whether the colchicine-induced alterations
of glycinergic mIPSCs was dependent on developmental stage.
To facilitate analysis, the spinal neurons were grouped accord-
ing to their stage of development as immature (5–7 DIV),
intermediate (10 –12 DIV), and mature (15–17 DIV) neurons.
It was found that the mean amplitude of glycinergic mIPSCs
increased from 33 ⫾ 4 pA in immature neurons to 56 ⫾ 8 pA
in mature neurons (Fig. 1C1). At the same time, the frequency
increased from 0.84 ⫾ 0.22 Hz in immature neurons to 1.64 ⫾
0.56 Hz in mature neurons (Fig. 1D1). Interestingly, we found
that colchicine dialysis reduced both the amplitude (37 ⫾ 5%
reduction, P ⬍ 0.001) and frequency (72 ⫾ 15% reduction,
P ⬍ 0.01) in immature neurons (n ⫽ 5), while having no effect
in mature neurons (n ⫽ 8; Fig. 1, C2 and D2). Smaller
colchicine effects on both the amplitude (18 ⫾ 3% reduction;
P ⬍ 0.01) and the frequency (61 ⫾ 7% reduction; P ⬍ 0.01)
were found in neurons of intermediate stage of development
(n ⫽ 7).
Colchicine-induced reductions of glycinergic mIPSC activity
are due to postsynaptic microtubule depolymerization
A summary of the effects produced by different agents
acting on microtubules on the normalized mean mIPSC am-
plitude is shown in Fig. 2A. The amplitude of glycinergic
mIPSCs remained highly stable (1.07 ⫾ 0.07 presented as the
amplitude ratio 25 min/1 min, from 34.7 ⫾ 7 pA at 1 min)
when the neurons were dialyzed with normal internal solution
www.jn.org
 COLCHICINE AFFECTS SYNAPTIC GLYRS IN IMMATURE NEURONS
for 25 min (n ⫽ 7). On the other hand, dialysis of sister neurons
with the microtubule disrupter colchicine (20 ␮M, n ⫽ 10) or
nocodazole (20 ␮M, n ⫽ 5) reduced the normalized mIPSC
amplitude to 0.71 ⫾ 0.04 (P ⬍ 0.001) and 0.88 ⫾ 0.01 (P ⬍
0.05), respectively. When dialyzed with the inactive analog of
colchicine, ␥-lumicolchicine (20 ␮M, n ⫽ 5), the amplitude
was not significantly changed (0.93 ⫾ 0.09, P ⬎ 0.05). Taxol
(20 ␮M, n ⫽ 4), a cytotoxin that increases the stabilization of
tubulin dimers (Nogales 2000), was unable to change the
amplitude of the glycinergic transmission (0.98 ⫾ 0.04, P ⬎
0.05). Dialysis with taxol for even 60 min (data not shown) was
unable to affect the glycinergic transmission, and extracellular
application of this alkaloid (20 ␮M; 3 h to the media) did not
alter the number and morphology of gephyrin clusters (data not
shown; Kirsch and Betz 1995), suggesting that stabilization of
actin filaments and actin dynamics did not affect the integrity
of postsynaptic gephyrin/GlyRs clusters.
It was previously shown in Xenopus oocytes that extracel-
lular colchicine inhibited overexpressed ␣2 and ␣1 GlyR sub-
J Neurophysiol • VOL
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
1039
FIG. 1. Glycinergic miniature postsynaptic currents (mIPSCs)
become insensitive to colchicine with maturation of spinal
neurons. A: no differences were found in synaptic glycinergic
mIPSCs after 25 min of dialysis with normal (control) intracel-
lular solution. Amplitude histograms, obtained from 5–12 DIV
neurons (n ⫽ 7), at 1 and 25 min, dialyzed with normal
intracellular solution, are shown at left (bin size, 1 pA). Bath
solution contained CNQX (2 ␮M), bicuculline (2 ␮M), MgCl2
(2 mM), and TTX (100 nM). Examples of current traces at these
2 time points are shown above the corresponding histograms.
Cumulative amplitude distributions (n ⫽ 7 neurons) are shown
at right. B: current traces and amplitude histograms generated
during intracellular application of 20 ␮M colchicine (n ⫽ 10)
showed significant differences. After 25 min colchicine dialy-
sis, glycinergic mIPSC activity is strongly reduced, and the
cumulative probability distribution (right) is shifted toward
smaller amplitudes (P ⬍ 0.001). C and D: mean peak-amplitude
(C1) and frequency (D1) of glycinergic mIPSCs increased
during maturation. Inhibitory action of colchicine on both am-
plitude (C2) and frequency (D2) decreased with development.
Each symbol represents means ⫾ SE obtained from 5–10
neurons. **P ⬍ 0.01, ***P ⬍ 0.001. Asterisks indicate a
statistically significant action of colchicine compared with con-
trol.
units with IC50s of approximately 64 and 324 ␮M, respectively
(Machu 1998). We obtained four lines of experimental evi-
dence indicating that our results with 20 ␮M intracellular
colchicine cannot be explained by this competitive inhibition.
First, analysis with extracellular colchicine (1–1,000 ␮M)
showed that the whole cell glycine-activated current (30 ␮M)
was inhibited with very similar affinities in immature and
mature neurons (IC50 of 143 ⫾ 28 and 187 ⫾ 13 ␮M, respec-
tively; P ⬎ 0.05, Fig. 2C). Second, internal nocodazole, which
was reported to be devoid of antagonistic properties on the
GlyR when applied extracellularly (Machu 1998), was still
able to reduce the mIPSC amplitude (Fig. 2A). Third, the
application of 20 ␮M external colchicine for 20 min did not
affect the average mIPSC amplitude (0.93 ⫾ 0.03, n ⫽ 8, P ⬎
0.05, Fig. 2A) or the cumulative probability amplitude distri-
butions (P ⬎ 0.05, Fig. 2B). Finally, intracellular GTP (500
␮M), a guanine nucleotide with low membrane permeability
and known for its capacity to stabilize microtubules (Nogales
2000), blocked the colchicine-induced reduction of glycinergic
91 • FEBRUARY 2004 • www.jn.org
1040 VAN ZUNDERT ET AL.
mIPSCs amplitude (1.0 ⫾ 0.08, n ⫽ 4, Fig. 2A). Control
experiments showed that GTP had no effect when added alone
to the internal solution (0.98 ⫾ 0.04, n ⫽ 4, Fig. 2A).
Effect of colchicine cannot be explained by a presynaptic
site of action
We also analyzed the number of mIPSC events and found
that the frequency was decreased when the neurons were
dialyzed with either colchicine (0.31 ⫾ 0.08 presented as the
frequency ratio 25 min/1 min, P ⬍ 0.001) or nocodazole
(0.49 ⫾ 0.11, P ⬍ 0.05). On the other hand, normal internal
solution (0.84 ⫾ 0.15, P ⬎ 0.05) or addition of ␥-lumicolchi-
cine (0.85 ⫾ 0.17, P ⬎ 0.05) to the patch pipette was unable
to significantly alter mIPSC frequency. A decrease in mIPSC
J Neurophysiol • VOL
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
FIG. 2. Colchicine inhibits mIPSC by affecting
postsynaptic microtubules. A: effects of different agents
that are able to interact with microtubules on the normal-
ized mean glycinergic mIPSC amplitude. Note that only
intracellular colchicine and nocodazole reduced the
mIPSC amplitude, while external colchicine (also 20 ␮M
for 20 min) had no effect. To avoid variability due to
different levels of spontaneous activity in different neu-
rons that could mask changes induced by the drugs, all
data at 20 –25 min after intracellular dialysis of drugs or
control recording solution were normalized with respect to
activity measured in the same cell at 1 min (control ⫽
34.7 ⫾ 7 pA). Symbols represent means ⫾ SE, *P ⬍ 0.05,
***P ⬍ 0.001. B: cumulative probability distribution is
similar after 25 min of 20 ␮M external colchicine appli-
cation. C: effect of increasing concentrations of colchicine
in immature (E) and mature (F) neurons on 30 ␮M glycine.
IC50 values were 143 ⫾ 28 and 187 ⫾ 13 ␮M in immature
and mature neurons, respectively. D and E: absence of
autapses in cultured neurons. D: current response with a
2-ms test pulse (from ⫺80 to 0 mV). This pulse activated
a large “unclamped” Na⫹ current, which was followed by
a 2nd downward reflection corresponding to a combina-
tion of capacitative and Na⫹ tail currents. Lack of effect
with strychnine (100 nM) indicates the absence of a gly-
cinergic autaptic current (dashed line). Repetitive stimu-
lation (5 Hz) was unable to increase the IPSC frequency
(inset). E: voltage responses from the same neuron stim-
ulated with a 0.6-nA depolarizing current pulse (5 ms).
Dashed line shows that strychnine did not change the
shape of the voltage response. Inset: the neuron exhibited
spontaneous glycinergic synaptic activity.
frequency can be interpreted as a reduction in the probability of
neurotransmitter release (Walmsley et al. 1998). Intracellular
dialysis of membrane permeable colchicine could therefore
have altered presynaptic cytoskeleton structures associated
with neurotransmitter release after diffusion into the presynap-
tic terminal. However, our results with extracellular colchicine
and GTP do not support this idea.
Alternatively, functional autaptic synapses could form under
our culture conditions (Bekkers and Stevens 1991), and col-
chicine action could be partly due to alterations in autaptic
transmission. To investigate this possibility, we examined
whether the neurons used in our studies showed evidence of
glycinergic autaptic transmission. Under the low Cl⫺ gradient
used, GlyR activation should be associated with a negative sign
current. Figure 2D illustrates the effect of applying an 80-mV
91 • FEBRUARY 2004 • www.jn.org
 COLCHICINE AFFECTS SYNAPTIC GLYRS IN IMMATURE NEURONS
depolarizing voltage step into a neuron held at ⫺80 mV. This
depolarizing pulse was able to activate a large somatic “un-
clamped” Na⫹ inward current (approximately 2 nA) but no
autaptic glycinergic postsynaptic current was detected (Fig.
2D). In addition, the frequency of spontaneous glycinergic
mIPSCs in the same neuron was unchanged even after a
sustained depolarization elicited by a high-frequency (5 Hz)
train of depolarizing pulses (Fig. 2D, inset). Additional exper-
iments using current-clamp recordings showed that soma-elic-
ited action potentials were not able to induce strychnine-sen-
sitive autaptic synaptic potentials (Fig. 2E). From the 11 neu-
rons examined, only 1 displayed an autaptic response,
suggesting that it is unlikely that the colchicine effect is pro-
duced by alterations in autaptic transmission.
The above experiments ruled out the possibility that a pre-
synaptic mechanism was associated to the action of colchicine
on the glycinergic mIPSC frequency. Therefore we can argue
that colchicine decreased the frequency of mIPSCs by reducing
the current amplitude in such a way that a number of synaptic
events fall under the threshold of detection (approximately 12
pA). To determine whether a change in mIPSC peak amplitude
might account for the decrease in frequency, the mIPSC am-
plitude was reduced 27 ⫾ 2% (37 ⫾ 4 to 27 ⫾ 2 pA, n ⫽ 5)
by lowering the Cl⫺ driving force. This closely simulates the
colchicine-induced reduction in mIPSC amplitude (shown in
J Neurophysiol • VOL
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
1041
FIG. 3. Colchicine altered the properties of
gephyrin clusters in immature neurons. A1–D1:
confocal images of synapsin I-IR terminals (green)
in the soma and processes of spinal interneurons.
A2–D2: gephyrin clusters (red) in the same cells
from A1–D1. A3–D3: superimposed images of
gephyrin and synapsin I immunofluorescence. Syn-
aptic gephyrin clusters are found apposed to or
co-localized (yellow) with synapsin I-IR. A: im-
ages obtained from an immature control neuron.
Note that only approximately 60% of the gephyrin
clusters are apposed/co-localized with synapsin I,
suggesting that many nonsynaptic gephyrin clus-
ters are present in immature neurons. B: application
of colchicine (20 ␮M for 3 h) caused a reduction in
both size and intensity of synaptic gephyrin clus-
ters in immature neurons (B3). C: mature neurons
show more synaptic terminals and gephyrin clus-
ters compared with immature neurons. Size of the
gephyrin clusters is also increased, and the major-
ity (⬎95%) are localized at synapses. D: colchicine
does not affect morphology of gephyrin clusters in
mature neurons. Images were reconstructed from a
stack of optical sections. Insets: magnified views of
boxed areas showing examples of synaptic and
nonsynaptic gephyrin clusters. Scale bar: 10 ␮m.
Fig. 2A). As found with colchicine, the decrease in mIPSC
quantal size was accompanied by a large (54 ⫾ 5%) reduction
in frequency, supporting the idea that the reduction in fre-
quency after 25 min of colchicine treatment is best explained
by an alteration in current amplitude.
Colchicine treatment decreased the size and intensity of
gephyrin/GlyR clusters in immature spinal neurons but not
in mature neurons
We next investigated whether the developmentally regulated
action of colchicine might be correlated with morphological
changes in postsynaptic gephyrin clusters on microtubule dis-
ruption (Kirsch and Betz 1995). Immature, intermediate, and
mature neurons were treated for 3 h with 20 ␮M colchicine
(Kirsch and Betz 1995; van Zundert et al. 2002). After fixation,
gephyrin clusters were immunolabeled with monoclonal anti-
body 7a, and the size (maximal length), density (luminosity),
and numbers of synaptic and extrasynaptic gephyrin clusters
were analyzed. Synaptic gephyrin clusters were detected by
combining gephyrin-IR with a presynaptic marker (see METH-
ODS). We considered gephyrin clusters synaptic when they
were opposite to immunolabeled boutons and extrasynaptic if
they were not. We compared synapsin I to other presynaptic
markers like synaptophysin or SV2 and found that these three
91 • FEBRUARY 2004 • www.jn.org
1042 VAN ZUNDERT ET AL.
presynaptic markers labeled a similar population of boutons.
Synaptophysin and synapsin I showed 78 ⫾ 9% co-localization
(n ⫽ 4 neurons), whereas synaptophysin and SV2 were co-
localized in ⬎90% of the boutons (n ⫽ 4 neurons). We found
that synapsin I was the brighter marker and labeled a few more
varicosities and thus was chosen for further studies. Analysis
of synapsin I-IR showed that colchicine treatment was unable
to significantly alter the bouton size in both immature (0.93 ⫾
0.04 ␮m in control vs. 0.89 ⫾ 0.05 ␮m in treated) and mature
neurons (0.80 ⫾ 0.03 ␮m in control vs. 0.77 ⫾ 0.04 ␮m in
treated).
Immature control neurons showed gephyrin-IR clusters at
the periphery of the neuronal soma and along proximal neurites
(Fig. 3A). Not all gephyrin clusters were apposed or co-local-
ized (yellow) with synapsin I (Fig. 3A3), indicating the exis-
tence of extrasynaptic gephyrin clusters at this developmental
stage. Synapsin I immunoreactivity was very robust in all
cultures; however, we cannot completely rule out the possibil-
ity that some newly formed synaptic varicosities might contain
low synapsin I antigenicity, below our detection threshold.
Nevertheless, previous ultrastructural analysis has demon-
strated the existence of extrasynaptic gephyrin clusters at early
stages of development in spinal cord cultures (Colin et al.
1996). In addition, the proportion of extrasynaptic to synaptic
gephyrin clusters are well matched to another study of cultured
spinal cord neurons that used a different presynaptic marker
(Dumoulin et al. 2000). During development, the number of
synaptic gephyrin clusters per cell increased from 14.8 ⫾ 1.6
in immature neurons (n ⫽ 14) to 29.3 ⫾ 3.5 in intermediate
(n ⫽ 14) and 52.1 ⫾ 4.7 in mature neurons (n ⫽ 14). The
number of extrasynaptic gephyrin clusters per cell decreased
from 9.6 ⫾ 2.2 in immature neurons to 4.9 ⫾ 0.9 and 1.9 ⫾ 0.4
in intermediate and mature neurons, respectively. Colchicine
treatment did not change the number of synaptic gephyrin
clusters per cell at any developmental stage; however, the
J Neurophysiol • VOL
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
number of extrasynaptic clusters was reduced to 4.4 ⫾ 1.1 in
immature neurons (P ⬍ 0.05).
Interestingly, it was found that the size and immunofluores-
cent intensity of synaptic gephyrin clusters were altered after
treating immature neurons with colchicine (Figs. 3B and 4, A
and B). Thus colchicine treatment resulted in a significant
decrease (77 ⫾ 5% of control) in the average length of gephy-
rin clusters in immature neurons from 0.39 ⫾ 0.02 ␮m in
control to 0.30 ⫾ 0.02 ␮m after treatment (P ⬍ 0.01, Fig. 4A).
In addition, analysis of cumulative probability cluster size
distribution revealed an apparent shift toward the left (P ⬍
0.001; data not shown), indicating that large clusters were more
sensitive to the colchicine treatment than smaller clusters. On
colchicine application, the immunofluorescent intensity was
also reduced from 916 ⫾ 37 to 746 ⫾ 21 arbitrary gray level
units (Fig. 4B, P ⬍ 0.001). At intermediate developmental
stages, a smaller but significant decrease in gephyrin cluster
size was detected after colchicine treatment (85 ⫾ 5% of
control; P ⬍ 0.05; from 0.39 ⫾ 0.01 ␮m in control to 0.33 ⫾
0.02 ␮m after treatment), but the immunofluorescent intensity
was unchanged (P ⬎ 0.05).
During in vitro development, more synaptic interactions
among the neurons are formed as indicated by an increased
number of synapsin I contacts in mature neurons (Fig. 3C1).
Correspondingly, mature neurons expressed approximately
three times more gephyrin clusters, and these were always
juxtaposed to synapsin I (Fig. 3C3). Synaptic gephyrin clusters
grew bigger in size to a mean length of 0.48 ⫾ 0.01 ␮m (Fig.
3C2). Extrasynaptic clusters were of similar size (0.33 ⫾ 0.01
␮m) compared with the previous developmental stages (0.31 ⫾
0.02 ␮m in both immature and intermediate neurons). Consis-
tent with our electrophysiological data, colchicine did not alter
synaptic or extrasynaptic gephyrin cluster number, size, or
immunofluorescent intensity in mature neurons (Figs. 3D and
4, A and B).
FIG. 4. Size and luminosity of gephyrin clusters were af-
fected in a maturation-dependent manner by colchicine. Imma-
ture, intermediate, and mature spinal neurons were treated with
colchicine and stained with antibodies against gephyrin and
synapsin I to identify synaptic gephyrin clusters. A: colchicine
decreased the mean synaptic gephyrin cluster size of immature
and intermediate neurons. Size of gephyrin clusters in mature
neurons was not significantly affected by colchicine. Note the
increase in cluster size with time of development in vitro. B:
luminosity of synaptic gephyrin clusters expressed in arbitrary
gray levels (AGL) is decreased following colchicine treatment
in immature neurons but not in intermediate or mature neurons.
Each symbol represents means ⫾ SE obtained from 14 neurons.
Asterisks indicate a statistically significant action of colchicine
compared with control. *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001.
C and D: mean gephyrin cluster size plotted as a function of
mean glycinergic mIPSC amplitude at the 3 developmental
stages in control (open symbols) and colchicine-treated (closed
symbols) neurons. High correlations were found with intracel-
lular (C) and extracellular colchicine treatments (D).
91 • FEBRUARY 2004 • www.jn.org
 COLCHICINE AFFECTS SYNAPTIC GLYRS IN IMMATURE NEURONS
These results, as well as previous studies (Lim et al. 1999;
Oleskevich et al. 1999), suggest a positive correlation between
gephyrin/GlyR cluster size and glycinergic mIPSC amplitude.
Next, we performed a correlative analysis between the average
mean peak amplitude of glycinergic mIPSCs and gephyrin
cluster size in control and after colchicine application either by
dialysis into the neuron via the patch pipette or to the media for
3 h, similar to the protocol used to obtain the structural data
(see Fig. 3). As shown in Fig. 4, C and D, both colchicine
treatments gave a high positive correlation which ranged from
0.92 to 0.97. Similar results were found when the quantal
amplitude was correlated with the gephyrin cluster intensity
(r ⫽ 0.88). The results support the conclusion that colchicine
action on glycinergic activity is produced by a modification in
postsynaptic receptor clusters.
The data in Fig. 3 was obtained using an antibody against
gephyrin. Gephyrin immunolabeling constitutes a good method
for performing high-resolution morphological analysis of
J Neurophysiol • VOL
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
1043
postsynaptic cluster size and structure due to its favorable
fixation tolerance and epitope display characteristics (Alvarez
et al. 1997; Kirsch and Betz 1995; Triller et al. 1985, 1990).
Additional experiments were performed with an antibody that
targets GlyR ␣1/␣2 subunits, which co-localized with 75 ⫾ 2%
of gephyrin clusters (Fig. 5A). Similar to the action on gephyrin
clusters, colchicine (3 h to the media) decreased the size of
synaptic GlyR clusters in immature neurons (71 ⫾ 6% of
control, P ⬍ 0.01; Fig. 5, C and D), and the effect became less
evident in intermediate neurons (90 ⫾ 7% of control, P ⬎
0.05). Mature neurons were not analyzed because colchicine
had no action on either glycinergic mIPSCs or gephyrin cluster
size/intensity.
Previous studies have indicated that gephyrin is also asso-
ciated to GABAARs (Essrich et al. 1999; Kneussel et al. 1999),
and we found that 48 ⫾ 4% of ␥2-containing GABAARs
clusters co-localized with gephyrin in immature spinal neurons
(Fig. 5B). Therefore it is possible that colchicine treatment
FIG. 5. Colchicine affects the morphology of
synaptic gephyrin/glycine receptor (GlyR) clusters,
but not synaptic GABAAR␥2 clusters. A: images
show that GlyR (green) and gephyrin (red) clusters
are highly (80%) co-localized (yellow, arrows).
Few isolated gephyrin and GlyR clusters (double
arrowheads) are found in these neurons. B:
GABAAR␥2 (red) and gephyrin clusters (green)
are found to co-localize at approximately 50% (yel-
low, arrows). Isolated GABAAR␥2 (double arrow-
heads) and gephyrin clusters (arrow heads) are
frequently found in these neurons. C: images of
GlyR clusters alone (green, C1) and superimposed
with SV2 (red, C2) indicate the existence of syn-
aptic GlyR clusters (yellow, arrows). Note that not
all GlyR clusters are apposed/co-localized with
SV2 (arrowheads). D: application of colchicine (20
␮M for 3 h) reduced the size of synaptic GlyR
clusters. E: similar colchicine treatment did not
alter the morphology of GABAAR␥2 clusters. F:
triple immunolocalization shows that GABAAR␥2
(red) gephyrin (green) clusters are apposed (blue)
and co-localized with synapsin I (white), suggest-
ing the synaptic localization of gephyrin/
GABAAR␥2 cluster complexes. Images are recon-
structed from a stack of optical sections. All con-
focal images were obtained from immature spinal
interneurons. Insets: magnified views of boxed ar-
eas demonstrating examples of synaptic and non-
synaptic GlyR clusters. Scale bar: 10 ␮m.
91 • FEBRUARY 2004 • www.jn.org
1044 VAN ZUNDERT ET AL.
might also affect the properties of synaptic GABAAR clusters.
Contrary to this idea, our results showed that colchicine did not
alter the size of synaptic GABAAR␥2 clusters in immature
(108 ⫾ 3% of control) and intermediate (106 ⫾ 3% of control)
neurons (Fig. 5E). This lack of effect is in agreement with a
previous study from our laboratory that showed that colchicine
was unable to alter GABAergic synaptic currents in these
spinal neurons (van Zundert et al. 2002).
Immature cultured spinal cord neurons express ␣2␤ and
␣1␤ GlyRs in their synapses
During the course of our study, we noted that colchicine
treatment not only caused a reduction in the amplitude and
frequency of glycinergic mIPSCs, but it also accelerated its
kinetics in immature neurons (Fig. 6A). Interestingly, the de-
cay-phase of mIPSCs became approximately three times faster
with neuronal maturation, and colchicine was no longer able to
accelerate these rapid currents (Fig. 6, B and C). For instance,
while glycinergic mIPSCs in control immature neurons dis-
played a mean decay-time constant of 13.1 ⫾ 1.8 ms (n ⫽ 6
cells), this value was 8.3 ⫾ 1.1 ms in colchicine-treated neu-
rons (n ⫽ 5 cells, P ⬍ 0.05). In intermediate (7.9 ⫾ 1.4 ms,
n ⫽ 5) and mature neurons (4.3 ⫾ 0.4 ms, n ⫽ 5 cells), the
glycinergic currents displayed a faster decay-time constant and
colchicine did not significantly modify these values (Fig. 6, B
and C). The mean rise-time of mIPSCs, on the other hand,
slightly decreased during maturation (15% from 2.6 ⫾ 0.3,
P ⬎ 0.05) and was unaffected by colchicine. The properties of
developing mIPSCs in spinal neurons are in agreement with
those reported in developing brain stem motoneurons (Singer
et al. 1998).
FIG. 6. Acceleration of mIPSCs decay-phase kinetics with colchicine and
during spinal maturation. A: traces represent glycinergic currents recorded in
an immature neuron at 1 min and after 25 min of dialysis with colchicine. Note
that colchicine affects not only the amplitude, but also accelerates the decay-
phase. B: mIPSCs recorded from a mature neuron show fast decay-kinetics that
are unaffected by colchicine dialysis. C: mean decay-time constant of mIPSCs
at different times of development in control conditions (䡺) and in the presence
of colchicine (f). Top traces are typical averaged control responses obtained in
immature (␶1) and mature (␶3) neurons. Bottom traces are averaged responses
obtained from immature (␶2) and mature (␶4) colchicine-treated neurons. Thin
dotted line in the top control traces corresponds to the superimposed bottom
traces obtained in the presence of colchicine and shows acceleration of decay
induced by treatment. Subscript for ␶ represents the number indicated in the
graph. Each symbol represents means ⫾ SE obtained from 5– 6 cells. *P ⬍
0.05.
J Neurophysiol • VOL 91 • FEBRUARY 2004 •
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
Previous reports have proposed that acceleration of mIPSC
kinetics with synaptic development results from a switch be-
tween neonate ␣2␤ GlyRs (slow) to adult ␣1␤ GlyRs (fast)
(Ali et al. 2000; Krupp et al. 1994; Singer et al. 1998; Taka-
hashi et al. 1992). Two independent experiments support the
idea that these changes are recapitulated in cultured mouse
spinal cord neurons. First, using RT-PCR, we found that ␣1
and ␤-subunit mRNA levels were high at all three develop-
mental stages, while the ␣2-subunit expression was strongly
down-regulated in mature neurons (Fig. 7B). No mRNA for the
␣3-subunit was detected during any of the three developmental
stages. Similar expression patterns were previously described
during the development of cultured rat spinal neurons
(Bechade et al. 1996) and brain stem motoneurons (Singer et
al. 1998). Second, we found that 100 ␮M picrotoxin, a toxin
known to differentially affect ␣2␤ (IC50 ⫽ 0.3 mM) and ␣1␤
(IC50 ⫽ 1 mM) GlyRs (Pribilla et al. 1992), significantly
decreased the amplitude and decay-time of glycinergic mIPSCs
in immature neurons (Fig. 7C), but not in mature neurons (Fig.
7D). Supporting the idea that the ␤-subunit is required to
cluster the GlyR in the postsynaptic membrane (Meyer et al.
1995), we found that continuous application of 10 ␮M picro-
toxin, a concentration known to selectively inhibit ␣ homo-
meric (IC50 ⫽ 7 ␮M; Pribilla et al. 1992), was unable to affect
glycinergic mIPSCs (data not shown). Taken together, these
data suggest the presence of both ␣2␤ and ␣1␤ GlyRs in the
postsynaptic membrane of immature neurons and predomi-
nantly ␣1␤ receptor in mature synapses.
Colchicine and picrotoxin target the same population of
postsynaptic GlyRs
The previous results suggest that colchicine and 100 ␮M
picrotoxin could affect the same population of slow immature
␣2␤ GlyRs. Therefore, in the next series of experiments, we
analyzed whether glycinergic activity of colchicine treated
neurons (3 h in the media; 20 ␮M) displayed a different
sensitivity to picrotoxin. We reasoned that if colchicine pro-
voked the reduction of ␣2␤ GlyRs, this would reduce picro-
toxin sensitivity of the synaptic currents. The open circles in
the graph of Fig. 8A summarize data from five immature
control neurons. In the absence of picrotoxin, glycinergic
mIPSC amplitudes ranged from 15 to 150 pA (mean, 42 ⫾ 7
pA), and the decay-time constant varied from 2 to 35 ms
(mean, 12.5 ⫾ 2.4 ms). Picrotoxin caused a strong reduction in
glycinergic events with a large amplitude and slow decay-
phase (Fig. 8A, F). After treatment of sister cultures with
colchicine, we found that the average amplitude and decay-
time constant of mIPSCs was reduced to 32 ⫾ 4 pA and 7.7 ⫾
1.4 ms, respectively (Fig. 8B, E). Interestingly, most glycin-
ergic mIPSCs in these colchicine-treated neurons (n ⫽ 4) were
resistant to picrotoxin application (Fig. 8B, F).
A correlation (r ⫽ 0.65) between the decay-time constant and
the amplitude of mIPSCs was found in control and treated neurons
(Fig. 8, A and B, lines). This correlation is unlikely to be due to
dendritic cable properties because no correlation was detected
between decay-time constant and rise-time (r ⫽ 0.35; Fig. 8, C
and D, lines). The mean rise-time under control conditions was
2.9 ⫾ 0.4 ms and remained similar after treatment with colchicine
and picrotoxin (P ⬎ 0.05, data not shown).
In summary, these results suggest that glycinergic mIPSCs
www.jn.org
 COLCHICINE AFFECTS SYNAPTIC GLYRS IN IMMATURE NEURONS
become less sensitive to picrotoxin after colchicine treatment, and
support the hypothesis that colchicine predominantly affects neo-
natal ␣2␤ GlyRs, leaving the adult ␣1␤ GlyRs unaffected.
DISCUSSION
Disruption of postsynaptic clusters affects glycinergic
transmission
The data presented here and in a previous report (van
Zundert et al. 2002) strongly suggest that colchicine actions on
glycinergic currents are due to structural alterations of postsyn-
aptic receptor clusters consequent to microtubule depolymer-
ization. We found no evidence supporting the idea that intra-
cellular dialysis of colchicine in the postsynaptic neurons
might affect neurotransmitter release from presynaptic termi-
nals. For instance, although glycinergic transmission was re-
duced by intracellular dialysis of colchicine, the synaptic trans-
missions mediated by GABAARs and AMPARs remained
largely unmodified (van Zundert et al. 2002). In cultured spinal
neurons, GlyRs and GABAARs frequently co-localize in
postsynaptic gephyrin-containing clusters opposite to presyn-
aptic boutons containing inhibitory amino acids (Dumoulin et
al. 2000). Hence, it is not likely that alterations in neurotrans-
J Neurophysiol • VOL
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
1045
FIG. 7. Immature neurons express ␣2, ␣1,
and ␤-GlyR subunits, while mature neurons
down-regulated ␣2.A: bars represent GlyR tran-
scripts derived from murine ␣ and ␤ gene struc-
ture (closed regions are the signal peptides).
Selected primers for amplification of GlyR ␣-
and ␤-subunit isoforms are indicated by hori-
zontal arrows. cDNA fragment size for each
subunit is shown. B: profiles of GlyR ␣1 (529
bp), ␣2 (385 bp), and ␤ (183 bp) subunits
obtained from cultures with different stages of
development are visualized by agarose gel elec-
trophoresis. Note that immature neurons, but
not mature cells, displayed high levels of ␣2
GlyR cDNA. C and D: picrotoxin (PTX)
changed the properties of mIPSCs in immature
neurons but not in mature neurons. C: top
traces show glycinergic mIPSCs recorded from
an immature spinal neuron before and during
the application of 100 ␮M PTX. Bottom cur-
rent traces are typical averaged responses. Thin
dotted line in the left trace corresponds to su-
perimposed right trace obtained in the presence
of PTX and shows an acceleration of decay
phase. Bars illustrate the inhibitory effect of
PTX on the amplitude and decay time constant
in immature neurons. D: effect of PTX on
events recorded from mature neurons. Super-
imposed trace obtained in the presence of PTX
shows a small effect of the toxin on decay.
Each bar shows means ⫾ SE obtained from
5– 6 neurons. Normalized data in the presence
of PTX was obtained using the response at 1
min as 100%. *P ⬍ 0.05, **P ⬍ 0.01.
mitter release would affect glycinergic and leave GABAAergic
transmission unaffected. In addition, application of extracellu-
lar colchicine (20 min) did not reduce the frequency or ampli-
tude of glycinergic mIPSCs. Further evidence supporting a
postsynaptic action of colchicine was obtained during this
study. First, intracellular application of the microtubule stabi-
lizer GTP completely blocked the colchicine-induced reduction
in glycinergic activity. Second, the rare occurrence of autapses
in these cultured spinal neurons indicate that the effect of
colchicine cannot be explained by depolymerization of vesicle-
associated microtubules in presynaptic autaptic terminals. Fi-
nally, we found that the reduction in event frequency with
colchicine could be well reproduced by changing the Cl⫺
driving force to obtain mIPSCs with smaller amplitudes.
Colchicine has been shown to competitively inhibit ␣2 and
␣1 GlyR subunits in Xenopus oocytes with IC50s of approxi-
mately 64 and 324 ␮M, respectively (Machu 1998). Analysis
the whole cell glycine-activated current during in vitro devel-
opment of spinal cord cultures demonstrated that immature and
mature neurons display a similar sensitivity to colchicine with
IC50s of 143 ⫾ 28 and 187 ⫾ 13 ␮M, respectively. In addition,
extracellular application of 20 ␮M colchicine had no effect on
glycinergic mIPSCs. Taken together, the effects observed with
91 • FEBRUARY 2004 • www.jn.org
1046 VAN ZUNDERT ET AL.
FIG. 8. Colchicine inhibits the effect of picrotoxin on mIPSCs. A: correla-
tion between decay-time constant and mIPSC amplitude before (E) and during
(F) application of 100 ␮M PTX in immature neurons. B: lack of PTX effect (F)
on colchicine-treated neurons (20 ␮M for 3 h). Note that amplitudes and
decay-time constants are already reduced after colchicine treatment (E). r ⬍
0.7 in all treatments. Plots of decay-time constant vs. rise-time before (䡺) and
during (f) application of 100 ␮M PTX in untreated (C) and colchicine-treated
neurons (D). No significant correlation was found (r ⫽ 0.35). Data points are
obtained from 4 –5 neurons.
internal dialysis of 20 ␮M colchicine in immature and mature
neurons are best explained by a postsynaptic mechanism con-
sequent to microtubule disruption (see van Zundert et al. 2002).
In addition, our results provide good evidence supporting the
idea that microtubules are important to maintain gephyrin/
GlyRs clustered in the postsynaptic membrane of immature
neurons, since we found that colchicine efficiently reduced the
size and luminosity of postsynaptic gephyrin/GlyR clusters.
Previous studies (Lim et al. 1999; Oleskevich et al. 1999), in
addition to our present data, suggest that the amplitude of
glycine-mediated mIPSCs and the size of gephyrin clusters are
positively correlated. Colchicine reduced both parameters in
immature, but not in mature neurons. In conclusion, the reduc-
tion in the amplitude of the glycinergic mIPSCs after micro-
tubule disruption can be best explained by a structural alter-
ation of the postsynaptic density that results in decreased
numbers of functional GlyRs in the synapse.
Microtubule disruption preferentially affects immature
␣2␤-GlyRs
While the amplitude of the mIPSC appears to reflect the
number of postsynaptic receptors, its decay is dictated by the
kinetic properties of single channels. For example, in overex-
pression studies, ␣1 and ␣2 subunits assemble homomeric
GlyRs with mean open times of 2 and 174 ms, respectively
(Takahashi et al. 1992). In parallel with the known subunits
switching from ␣2 to ␣1 subunits during postnatal develop-
ment (Becker et al. 1988), patch-clamp analysis in spinal cord
slices demonstrated that the open time of native GlyR channels
decreased from 40 ms at E20 to 6 ms at P22 (Takahashi et al.
1992). Therefore it is believed that the differences in the
J Neurophysiol • VOL 91 • FEBRUARY 2004 •
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
decay-phase of glycinergic mIPSCs during development de-
pends on a transition between slow ␣2 and fast ␣1 subunits
(Krupp et al. 1994; Legendre 2001; Singer et al. 1998; Taka-
hashi et al. 1992). We also found that the time-course of the
glycinergic mIPSCs markedly accelerated with maturation of
spinal neurons in culture. Analysis of mIPSCs properties
showed that immature slow events were more sensitive to
picrotoxin than mature faster events. However, our data sug-
gest that immature glycinergic currents are primarily associ-
ated to ␣2␤ subunits since these slow decay mIPSCs were
affected by higher picrotoxin concentrations than those used to
inhibit (IC50 ⫽ 7 ␮M) homomeric ␣ receptors (Legendre 1997;
Pribilla et al. 1992; Tapia and Aguayo 1998; Ye 2000). This
conclusion is in agreement with other previous studies in
immature spinal neurons showing the presence of a 48 pS
channel, typical of heteromeric ␣2␤ receptors, and not a ⬎85
pS associated to homomeric receptors (Bormann et al. 1987,
1993; Takahashi and Momiyama 1991; Twyman and Mac-
donald 1991). Similar to results obtained in newborn brain
motoneurons (Singer et al. 1998), our results indicate that
immature neurons express GlyRs that are formed by ␣2 and ␤
subunits. During maturation, the mRNA level for the ␣2-
subunit decreased, shifting the balance of mRNA expression
toward ␣1 and ␤-subunits. In addition, analysis of the decay-
time constant and the sensitivity to picrotoxin suggests that
mature neurons express primarily ␣1␤ GlyRs. This finding is
in agreement with previous studies (Krupp et al. 1994; Singer
et al. 1998; Takahashi et al. 1992).
These data suggest that mature and immature GlyRs are
composed of ␤-subunits and that they are linked to underlying
postsynaptic microtubules. However, disruption of microtu-
bules in immature neurons selectively affected a population of
glycinergic currents displaying slow decay-times, thus leaving
faster events unaffected. These results indicate that microtu-
bules regulate the immature ␣2␤ GlyR, but not the adult ␣1␤
receptor. Two lines of evidence support this idea. First, the
picrotoxin-sensitive population of mIPSCs was no longer evi-
dent after disruption of microtubules with colchicine, suggest-
ing they represent the same population of receptors. Second,
mature neurons with fast ␣1␤ GlyRs were insensitive to col-
chicine.
Based on the current understanding regarding functional and
structural properties of synaptic GlyRs, we postulate a model
in which immature neurons express both ␣2␤ and ␣1␤ GlyRs
at the postsynaptic membrane, while mature neurons contain
mainly ␣1␤ GlyRs at the synapse (Fig. 9). Microtubules, via
gephyrin, anchor both types of GlyRs in the postsynaptic
membrane. On microtubule depolymerization, the molecular
complex made up by gephyrin and ␣2␤ GlyRs is liberated and
diffuses to the extrasynaptic membrane, reducing glycinergic
mIPSCs and gephyrin/GlyR cluster size and density. In con-
trast, colchicine treatment does not affect ␣1␤ GlyR/gephyrin
clusters in immature and mature neurons (Fig. 9). This can be
explained by the linkage of GlyR/gephyrin complexes to mi-
crotubules that are stabilized by posttranslational modifica-
tions, such as acetylation and/or the binding of molecules such
as MAPs (Nogales 2000). In addition, the interaction of GlyRs
with subsynaptic microtubules could depend on the type of ␣
subunit present in the receptor rather than to differences in
cytoskeleton properties. Interestingly, more than 10 potential
www.jn.org
 COLCHICINE AFFECTS SYNAPTIC GLYRS IN IMMATURE NEURONS
gephyrin isoforms can be generated by alternative splicing,
allowing ␣1␤- and ␣2␤-GlyRs to associate with gephyrin
variants that have distinct affinities for cytoskeletal elements.
Thus the developmental dependent change of the GlyR molec-
ular composition could affect the manner in which the receptor
interacts with the gephyrin scaffold and subsynaptic microtu-
bules.
Another possibility is that, after enough ␣1␤ GlyR/gephyrin
complexes have accumulated in the synapse during maturation,
gephyrin forms stable hexagonal scaffolds in the PSD, which
maintain ␣1␤ GlyRs anchored at the postsynaptic membrane
and independent of microtubules (Fig. 9; see also Kneussel and
Betz 2000; Liu et al. 2000). Thus as proposed for the role of
actin filaments in excitatory hippocampal synapses (Allison et
al. 2000; Zhang and Benson 2001), the state of microtubules
could play a role in the development and maintenance of
predominantly immature glycinergic synapses, which need to
be highly plastic to shape efficient synaptic transmission. In
agreement with this idea, electron microscopy studies have not
consistently found microtubules within the 20-to 30-nm region
beneath adult symmetric (inhibitory) or gephyrin-containing
synapses (Alvarez et al. 1997; Peters et al. 1991; Triller et al.
1985, 1987). It is possible that postsynaptic microtubules are
not well preserved in routine or immunocytochemical electron
microscopy preparations as previously shown for presynaptic
microtubules (Gray 1975). Accordingly, some microtubules
were shown in close relationship to immature and mature
postsynaptic densities (not necessarily inhibitory) in tissue
sections or cultures pretreated with taxol, cryosubstitution
techniques, or other procedures aimed to stabilize cytoskeletal
components (Bird 1989; Ichimura and Hashimoto 1988; Le-
Beux and Willemot 1975; Westrum and Gray 1977). However,
the most convincing ultrastructural evidence available on mi-
crotubule presence in the PSD was obtained in neonatal syn-
apses (Westrum and Gray 1977). To our knowledge, no sys-
tematic ultrastructural analysis of the spatial relationships be-
tween microtubules and the postsynaptic density of inhibitory
synapses is available.
J Neurophysiol • VOL
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
1047
FIG. 9. Proposed mechanisms for anchoring ␣2␤ and ␣1␤
GlyRs in glycinergic synapses. Microtubules, via gephyrin,
anchor ␣2␤- and ␣1␤-containing GlyRs in the postsynaptic
membrane. While immature synapses are enriched with ␣2
GlyRs, these are down-regulated at mature synapses that are
composed almost exclusively of ␣1-containing GlyRs. Depo-
lymerization of microtubules by colchicine causes the release of
␣2␤ GlyR/gephyrin complexes and diffusion to the extrasyn-
aptic membrane. In contrast, gephyrin-coupled ␣1␤ GlyRs are
linked to colchicine insensitive microtubules, which could be
stabilized by MAPs or acetylation (Ac). Alternatively, stabili-
zation of ␣1␤ GlyR/gephyrin clusters can be achieved by
assembling gephyrin into hexagonal lattices. Therefore the in-
sensitivity of mature receptors to cytoskeleton disruption can be
also associated to the presence of ␣1␤ GlyR/gephyrin com-
plexes rather than to microtubule-dependent mechanisms. Ver-
tical lines indicate the location of synaptic region.
In conclusion, this study shows that disruption of microtu-
bules by colchicine reduced both postsynaptic glycinergic cur-
rents and the size and density of synaptic gephyrin clusters in
immature spinal neurons. Thus this study expands those on
regulation of n-methyl-D-aspartic acid receptors (NMDARs),
AMPARs, and nicotinic acetylcholine receptors (nAChRs) by
the state of the cytoskeleton (Allison et al. 1998, 2000; Sattler
et al. 2000; Shoop et al. 2000). Moreover, our data suggest that
microtubule disruption affects immature synapses rather than
mature synapses. This property could explain previous contro-
versial findings in which disruption of microtubules altered the
number and morphology of gephyrin clusters in 10 –12 DIV
spinal neurons (Kirsch and Betz 1995), while gephyrin distri-
bution in ⬎21 DIV hippocampal neurons was not affected by
microtubule depolymerization (Allison et al. 2000).
ACKNOWLEDGMENTS
We thank Drs. P. Legendre and B. Walmsley for reading and suggestions on
this manuscript. The authors thank Dr. J. M. Fritschy for making the
GABAAR␥2 antibody available and L. J. Aguayo for expert technical assis-
tance.
GRANTS
Research support was provided by Fondo Nacional de Ciencia y Tecnologia
(FONDECYT) 2000135 and Programa de Mejoramiento de la Calidad y
Equidad de la Educación Superior del Ministerio de Educaión UCH9903 to B.
van Zundert, FONDECYT 1980106, 1020475, and GIA-DIUC to L. G.
Aquayo, FONDECYT 2990063 to J. C. Tapia, the National Science Founda-
tion 9984441 to F. J. Alvarez, and National Institute of Neurological Disorders
and Stroke Grants NS-24830 and NS-41489 to H. H. Yeh.
REFERENCES
Aguayo LG and Pancetti FC. Ethanol modulation of the gamma-aminobu-
tyric acidA- and glycine-activated Cl⫺ current in cultured mouse neurons.
J Pharmacol Exp Ther 270: 61– 69, 1994.
Ali DW, Drapeau P, and Legendre P. Development of spontaneous glycin-
ergic currents in the Mauthner neuron of the zebrafish embryo. J Neuro-
physiol 84: 1726 –1736, 2000.
Allison DW, Chervin AS, Gelfand VI, and Craig AM. Postsynaptic scaf-
folds of excitatory and inhibitory synapses in hippocampal neurons: main-
91 • FEBRUARY 2004 • www.jn.org
1048 VAN ZUNDERT ET AL.
tenance of core components independent of actin filaments and microtu-
bules. J Neurosci 20: 4545– 4554, 2000.
Allison DW, Gelfand VI, Spector I, and Craig AM. Role of actin in
anchoring postsynaptic receptors in cultured hippocampal neurons: differ-
ential attachment of NMDA versus AMPA receptors. J Neurosci 18: 2423–
2436, 1998.
Alvarez FJ, Dewey DE, Harrington DA, and Fyffe RE. Cell-type specific
organization of glycine receptor clusters in the mammalian spinal cord.
J Comp Neurol 379: 150 –170, 1997.
Bechade C, Colin I, Kirsch J, Betz H, and Triller A. Expression of glycine
receptor alpha subunits and gephyrin in cultured spinal neurons. Eur J Neu-
rosci 8: 429 – 435, 1996.
Becker D-M, Hich W, and Betz H. Glycine receptor heterogeneity in rat
spinal cord during postnatal development. EMBO J 7: 3717–3726, 1988.
Bekkers JM and Stevens CF. Excitatory and inhibitory autaptic currents in
isolated hippocampal neurons maintained in cell culture. Proc Natl Acad Sci
USA 88: 7834 –7838, 1991.
Bird MM. Microtubules and their relationships with other cytoskeletal com-
ponents at cholinergic tectal synapses in culture. J Anat 166: 1– 6, 1989.
Bormann J, Hamill OP, and Sakmann B. Mechanism of anion permeation
through channels gated by glycine and gamma-aminobutyric acid in mouse
cultured spinal neurones. J Physiol 385: 243–286, 1987.
Bormann J, Rundstrom N, Betz H, and Langosch D. Residues within
transmembrane segment M2 determine chloride conductance of glycine
receptor homo- and hetero-oligomers. EMBO J 12: 3729 –3737, 1993.
Colin I, Rostaing P, and Triller A. Gephyrin accumulates at specific plas-
malemma loci during neuronal maturation in vitro. J Comp Neurol 374:
467– 479, 1996.
Cotman CW, Banker G, Churchill L, and Taylor D. Isolation of postsyn-
aptic densities form rat brain. J Cell Biol 63: 441– 455, 1974.
Dumoulin A, Levi S, Riveau B, Gasnier B, and Triller A. Formation of
mixed glycine and GABAergic synapses in cultured spinal cord neurons.
Eur J Neurosci 12: 3883–3892, 2000.
Essrich C, Lorez M, Benson JA, Fritschy JM, and Luscher B. Postsynaptic
clustering of major GABAA receptor subtypes requires the gamma 2 subunit
and gephyrin. Nat Neurosci 1: 563–571, 1999.
Feng G, Tintrup H, Kirsch J, Nichol MC, Kuhse J, Betz H, and Sanes JR.
Dual requirement for gephyrin in glycine receptor clustering and molyb-
doenzyme activity. Science 282: 1321–1324, 1998.
Geiman EJ, Knox MC, and Alvarez FJ. Postnatal maturation of gephyrin/
glycine receptor clusters on developing Renshaw cells. J Comp Neurol 426:
130 –142, 2000.
Gray EG. Presynaptic microtubules and their association with synaptic vesi-
cles. Proc R Soc Lond Biol Sci 190: 367–372, 1975.
Ichimura T and Hashimoto PH. Structural components in the synaptic clefts
captured by freeze substitution and deep etching of directly frozen cerebellar
cortex. J Neurocytol 17: 3–12, 1988.
Kelly PT and Cotman CW. Synaptic proteins. Characterization of tubulin and
actin and identification of a distinct postsynaptic density polypeptide. J Cell
Biol 79: 173–183, 1978.
Kins S, Betz H, and Kirsch J. Collybistin, a newly identified brain-specific
GEF, induces submembrane clustering of gephyrin. Nat Neurosci 3: 22–29,
2000.
Kirchhoff F, Mulhardt C, Pastor A, Becker CM, and Kettenmann H.
Expression of glycine receptor subunits in glial cells of the rat spinal cord.
J Neurochem 66: 1383–1390, 1996.
Kirsch J. Assembly of signaling machinery at the postsynaptic membrane.
Curr Opin Neurobiol 9: 329 –335, 1999.
Kirsch J and Betz H. The postsynaptic localization of the glycine receptor-
associated protein gephyrin is regulated by the cytoskeleton. J Neurosci 15:
4148 – 4156, 1995.
Kirsch J, Langosch D, Prior P, Littauer UZ, Schmitt B, and Betz H. The
93-kDa glycine receptor-associated protein binds to tubulin. J Biol Chem
266: 22242–22245, 1991.
Kirsch J, Wolters I, Triller A, and Betz H. Gephyrin antisense oligonucle-
otides prevent glycine receptor clustering in spinal neurons. Nature 366:
745–748, 1993.
Kneussel M and Betz H. Clustering of inhibitory neurotransmitter receptors
at developing postsynaptic sites: the membrane activation model. Trends
Neurosci 23: 429 – 435, 2000.
Kneussel M, Brandstatter JH, Laube B, Stahl S, Muller U, and Betz H.
Loss of postsynaptic GABA(A) receptor clustering in gephyrin-deficient
mice. J Neurosci 19: 9289 –9297, 1999.
J Neurophysiol • VOL 91 • FEBRUARY 2004 •
Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.
Krupp J, Larmet Y, and Feltz P. Postnatal change of glycinergic IPSC decay
in sympathetic preganglionic neurons. Neuroreport 5: 2437–2440, 1994.
LeBeux YJ and Willemot J. An ultrastructural study of the microfilaments in
rat barin by means of E-PTA staining and heavy meromyosin labeling. II.
The synapses. Cell Tissue Res 160: 37– 68, 1975.
Legendre P. Pharmacological evidence for two types of postsynaptic glycin-
ergic receptors on the Mauthner cell of 52-h-old zebrafish larvae. J Neuro-
physiol 77: 2400 –2415, 1997.
Legendre P. The glycinergic inhibitory synapse. Cell Mol Life Sci 58: 760 –
793, 2001.
Lim R, Alvarez FJ, and Walmsley B. Quantal size is correlated with receptor
cluster area at glycinergic synapses in the rat brainstem. J Physiol 516:
505–512, 1999.
Liu MT, Wuebbens MM, Rajagopalan KV, and Schindelin H. Crystal
structure of the gephyrin-related molybdenum cofactor biosynthesis protein
MogA from Escherichia coli. J Biol Chem 275: 1814 –1822, 2000.
Machu T. Colchicine competitively antagonizes glycine receptors expressed
in Xenopus oocytes. Neuropharmacology 37: 391–396, 1998.
Mammoto A, Sasaki T, Asakura T, Hotta I, Imamura H, Takahashi K,
Matsuura Y, Shirao T, and Takai Y. Interactions of drebrin and gephyrin
with profilin. Biochem Biophys Res Commun 243: 86 – 89, 1998.
Matus AI and Taff-Jones DH. Morphology and molecular composition of
isolated postsynaptic junctional structures. Proc R Soc Lond B Biol Sci 203:
135–151, 1978.
Meier J, Meunier-Durmort C, Forest C, Triller A, and Vannier C. For-
mation of glycine receptor clusters and their accumulation at synapses.
J Cell Sci 113: 2783–2795, 2000.
Meyer G, Kirsch J, Betz H, and Langosch D. Identification of a gephyrin
binding motif on the glycine receptor beta subunit. Neuron 15: 563–572,
1995.
Nogales E. A structural view of microtubule dynamics. Cell Mol Life Sci 56:
133–142, 2000.
Oleskevich S, Alvarez FJ, and Walmsley B. Glycinergic miniature synaptic
currents and receptor cluster sizes differ between spinal cord interneurons.
J Neurophysiol 82: 312–319, 1999.
Peters A, Palay SL, and Webster H. The Fine Structure of the Nervous
System: The Neurons and Supporting Cells. Philadelphia, PA: W. B. Saun-
ders, 1991.
Pribilla I, Takagi T, Langosch D, Bormann J, and Betz H. The atypical M2
segment of the beta subunit confers picrotoxinin resistance to inhibitory
glycine receptor channels. EMBO J 11: 4305– 4311, 1992.
Rosenberg M, Meier J, Triller A, and Vannier C. Dynamics of glycine
receptor insertion in the neuronal plasma membrane. J Neurosci 21: 5036 –
5044, 2001.
Rosenmund C and Westbrook GL. Calcium-induced actin depolymerization
reduces NMDA channel activity. Neuron 10: 805– 814, 1993.
Sattler R, Xiong Z, Lu WY, MacDonald JF, and Tymianski M. Distinct
roles of synaptic and extrasynaptic NMDA receptors in excitotoxicity.
J Neurosci 20: 22–33, 2000.
Sheng M and Pak DT. Ligand-gated ion channel interactions with cytoskel-
etal and signaling proteins. Annu Rev Physiol 62: 755–778, 2000.
Shoop RD, Yamada N, and Berg DK. Cytoskeletal links of neuronal ace-
tylcholine receptors containing alpha 7 subunits. J Neurosci 20: 4021– 4029,
2000.
Singer JH, Talley EM, Bayliss DA, and Berger AJ. Development of gly-
cinergic synaptic transmission to rat brain stem motoneurons. J Neuro-
physiol 80: 2608 –2620, 1998.
Takahashi T and Momiyama A. Single-channel currents underlying glycin-
ergic inhibitory postsynaptic responses in spinal neurons. Neuron 7: 965–
969, 1991.
Takahashi T, Momiyama A, Hirai K, Hishinuma F, and Akagi H. Func-
tional correlation of fetal and adult forms of glycine receptors with devel-
opmental changes in inhibitory synaptic receptor channels. Neuron 9: 1155–
1161, 1992.
Tapia JC and Aguayo LG. Changes in the properties of developing glycine
receptors in cultured mouse spinal neurons. Synapse 28: 185–194, 1998.
Triller A, Cluzeaud F, and Korn H. Gamma-aminobutyric acid-containing
terminals can be apposed to glycine receptors at central synapses. J Cell Biol
104: 947–956, 1987.
Triller A, Cluzeaud F, Pfeiffer F, Betz H, and Korn H. Distribution of
glycine receptors at central synapses: an immunoelectron microscopy study.
J Cell Biol 101: 683– 688, 1985.
Triller A, Seitanidou T, Franksson O, and Korn H. Size and shape of
www.jn.org
 COLCHICINE AFFECTS SYNAPTIC GLYRS IN IMMATURE NEURONS 1049

glycine receptor clusters in a central neuron exhibit a somato-dendritic
gradient. New Biol 2: 637– 641, 1990.
Twyman RE and Macdonald RL. Kinetic properties of the glycine receptor
main- and sub-conductance states of mouse spinal cord neurones in culture.
J Physiol 435: 303–331, 1991.
van Zundert B, Alvarez FJ, and Aguayo LG. Colchicine affects the ampli-
tude of glycinergic synaptic currents and the size of glycine receptor clusters
in the postsynaptic membrane. Soc Neurosci Abstr 27: 728, 2001.
van Zundert B, Alvarez FJ, Yevenes GE, Cárcamo JG, Vera JC, and
Aguayo LG. Glycine receptors involved in synaptic transmission are selec-
tively regulated by the cytoskeleton in mouse spinal neurons. J Neurophysiol
87: 640 – 644, 2002.
Walmsley B, Alvarez FJ, and Fyffe RE. Diversity of structure and function
at mammalian central synapses. Trends Neurosci 21: 81– 88, 1998.
Westrum LE and Gray EG. Microtubules associated with postsynaptic
thickenings. J Neurocytol 6: 505–518, 1977.
Ye J. Physiology and pharmacology of native glycine receptors in developing
rat ventral tegmental area neurons. Brain Res 862: 74 – 82, 2000.
Zhang W and Benson DL. Stages of synapse development defined by
dependence on F-actin. J Neurosci 21: 5169 –5181, 2001.

J Neurophysiol • VOL 91 • FEBRUARY 2004 • www.jn.org

Downloaded from www.physiology.org/journal/jn (131.000.054.235) on April 12, 2019.