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Developmental-Dependent Action of Microtubule Depolymerization on the Function and Structure of Synaptic Glycine Receptor Clusters in Spinal Neurons

¹Laboratory 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

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Microtubules have been proposed to interact with gephyrin/glycine receptors (GlyRs) in synaptic aggregates. 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), disruption 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 reduced the average length and immunofluorescence intensity of synaptic 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. Finally, RT-PCR, kinetic analysis and picrotoxin blockade of glycinergic 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

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Postsynaptic densities (PSD) harbor neurotransmitter receptors that are anchored in the membrane at precise locations opposite to presynaptic active zones releasing neurotransmitters. 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 Cotman 1978; Matus and Taff-Jones 1978), and several bridging proteins have been proposed to link membrane macromolecules 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 GABA_A receptors (GABA_ARs) 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 GABA_ARs 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 intermediate 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; Kneussel 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 number, structure, and intensity of gephyrin/GlyR postsynaptic clusters (Kirsch and Betz 1995). However, microtubule disruption in hippocampal cultures failed to alter gephyrin/GABA_A 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 inhibitory 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 extrasynaptic receptors, was changed in cultured spinal cord neurons dialyzed with the microtubule depolymerization agent colchicine (van Zundert et al. 2002). In contrast, the function of synaptic -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) and gamma amino butyric acid type A receptors (GABA_ARs) 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. GABA_ARs postsynaptic clusters were unaltered at any developmental stage. Preliminary results were presented in abstract form (van Zundert et al. 2001).

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
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 µM) 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 configuration, 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 configuration was established, the capacitance of the cell and the series resistance were compensated using the amplifier (>80%). Spontaneous glycinergic miniature postsynaptic currents (mIPSCs) were isolated by the addition of CNQX (2 µM, RBI), bicuculline (2 µM, RBI), TTX (0.1 µM, 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 currents 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.

Immunocytochemistry

Neurons were fixed for 5 (GlyR) or 15 min (GABA_AR2) with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Fixing time was increased to 30 min for other antibodies. Cultures were permeabilized 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 GABA_AR (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 Francisco, 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 combinations of antibodies: gephyrin/synapsin I, synaptophysin/SV2, synaptophysin/synapsin I, GlyR/gephyrin, GABA_AR2/gephyrin, GlyR/SV2, GABA_AR2/SV2, and GABA_AR2/gephyrin/synapsin I. Immunoreactive (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 GABA_AR2 antibodies with Cy3-conjugated antibodies. For the triple immunolocalization, together with the normal GABA_AR2/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, Burlingame, 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 imaging using confocal microscopy (Olympus Fluoview; 100x oil immersion objective, N.A. 1.35, digitally zoomed 3 times, pixel size = 0.07 µm). Stacks of optical sections separated by 0.5 µm in the z axis were acquired throughout whole cells. Dual color immunofluorescent images 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 there were only few examples of gephyrin clusters viewed "en face" where surface areas could be resolved accurately. Hence, we measured 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 fluorescence 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 µg/ml x A₂₆₀ x Fd, where A₂₆₀ is the absorbance (260 nm) of the sample and Fd represents the dilution factor. cDNA synthesis employed 12 µl of total RNA, 1 µl of random hexamer primers (50 ng, Promega), 1 µl of 10 mM dNTPs, 4 µl of 5x reaction buffer, 2 µl of DTT (0.1 M), 1 µl of RNAsin (40 U/µl), and 1 µl 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 µg /ml x A₂₆₀ x Fd.

cDNA amplification and temporal subunit expression

PCR mixtures (25 µl final) contained 2.5 µl 10x Taq polymerase buffer, 0.5 µl dNTPs mix (10 mM), 0.75 µl MgCl₂ (50 mM), 0.75 µl sense (10 µM) and 0.75 µl antisense primers (10 µM), 3 µl cDNA template (50 ng), 16.5 µl H₂O, and 0.25 µl Taq polymerase (5 U/µl). A Biorad thermocycler was used with PCR conditions set to 94°C (3 min) for primary denaturation, 30 cycles of amplification (94°C denaturation, 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'-ctgtgttgtagtgcttggtgc-3'); 2 (sense: 5'-ggtacaccatgaatgacctg-3'; anti-sense: 5'-ccatccagatgtcaattgcctt-3'); 3 (sense: 5'-gctgacattaacactctcttgtcc-3'; anti-sense: 5'-ccatccagatgtcaattgcctt-3'); (sense: 5'-ggaattcgggatggagacgtcc-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 embryonic 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

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
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 glycinergic transmission in young (<12 DIV) cultured spinal neurons. 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 µM colchicine in the patch pipette displayed a large change in the amplitude histogram distribution 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.

View larger version (29K): [in this window] [in a new window]   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) intracellular 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 µM), bicuculline (2 µM), 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 µM colchicine (n = 10) showed significant differences. After 25 min colchicine dialysis, 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 amplitude (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 control.

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 amplitude 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 for 25 min (n = 7). On the other hand, dialysis of sister neurons with the microtubule disrupter colchicine (20 µM, n = 10) or nocodazole (20 µM, 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 µM, n = 5), the amplitude was not significantly changed (0.93 ± 0.09, P > 0.05). Taxol (20 µM, 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 µM; 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.

View larger version (25K): [in this window] [in a new window]   FIG. 2. Colchicine inhibits mIPSC by affecting postsynaptic microtubules. A: effects of different agents that are able to interact with microtubules on the normalized mean glycinergic mIPSC amplitude. Note that only intracellular colchicine and nocodazole reduced the mIPSC amplitude, while external colchicine (also 20 µM for 20 min) had no effect. To avoid variability due to different levels of spontaneous activity in different neurons 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 µM external colchicine application. C: effect of increasing concentrations of colchicine in immature () and mature () neurons on 30 µM glycine. IC50 values were 143 ± 28 and 187 ± 13 µM 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 combination of capacitative and Na+ tail currents. Lack of effect with strychnine (100 nM) indicates the absence of a glycinergic autaptic current (dashed line). Repetitive stimulation (5 Hz) was unable to increase the IPSC frequency (inset). E: voltage responses from the same neuron stimulated 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.

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 -lumicolchicine (0.85 ± 0.17, P > 0.05) to the patch pipette was unable to significantly alter mIPSC frequency. A decrease in mIPSC 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 presynaptic 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 colchicine 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 depolarizing voltage step into a neuron held at -80 mV. This depolarizing pulse was able to activate a large somatic "unclamped" 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 experiments using current-clamp recordings showed that soma-elicited action potentials were not able to induce strychnine-sensitive autaptic synaptic potentials (Fig. 2E). From the 11 neurons examined, only 1 displayed an autaptic response, suggesting that it is unlikely that the colchicine effect is produced by alterations in autaptic transmission.

The above experiments ruled out the possibility that a presynaptic 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 amplitude 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 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 frequency 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 disruption (Kirsch and Betz 1995). Immature, intermediate, and mature neurons were treated for 3 h with 20 µM colchicine (Kirsch and Betz 1995; van Zundert et al. 2002). After fixation, gephyrin clusters were immunolabeled with monoclonal antibody 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 METHODS). 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 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 µm in control vs. 0.89 ± 0.05 µm in treated) and mature neurons (0.80 ± 0.03 µm in control vs. 0.77 ± 0.04 µm 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-localized (yellow) with synapsin I (Fig. 3A3), indicating the existence of extrasynaptic gephyrin clusters at this developmental stage. Synapsin I immunoreactivity was very robust in all cultures; however, we cannot completely rule out the possibility that some newly formed synaptic varicosities might contain low synapsin I antigenicity, below our detection threshold. Nevertheless, previous ultrastructural analysis has demonstrated 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 number of extrasynaptic clusters was reduced to 4.4 ± 1.1 in immature neurons (P < 0.05).

View larger version (54K): [in this window] [in a new window]   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. Synaptic gephyrin clusters are found apposed to or co-localized (yellow) with synapsin I-IR. A: images 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 clusters are present in immature neurons. B: application of colchicine (20 µM for 3 h) caused a reduction in both size and intensity of synaptic gephyrin clusters in immature neurons (B3). C: mature neurons show more synaptic terminals and gephyrin clusters compared with immature neurons. Size of the gephyrin clusters is also increased, and the majority (>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 µm.

View larger version (33K): [in this window] [in a new window]   FIG. 4. Size and luminosity of gephyrin clusters were affected in a maturation-dependent manner by colchicine. Immature, 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 intracellular (C) and extracellular colchicine treatments (D).

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 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.

View larger version (37K): [in this window] [in a new window]   FIG. 5. Colchicine affects the morphology of synaptic gephyrin/glycine receptor (GlyR) clusters, but not synaptic GABAAR2 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: GABAAR2 (red) and gephyrin clusters (green) are found to co-localize at approximately 50% (yellow, arrows). Isolated GABAAR2 (double arrowheads) 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 synaptic GlyR clusters (yellow, arrows). Note that not all GlyR clusters are apposed/co-localized with SV2 (arrowheads). D: application of colchicine (20 µM for 3 h) reduced the size of synaptic GlyR clusters. E: similar colchicine treatment did not alter the morphology of GABAAR2 clusters. F: triple immunolocalization shows that GABAAR2 (red) gephyrin (green) clusters are apposed (blue) and co-localized with synapsin I (white), suggesting the synaptic localization of gephyrin/GABAAR2 cluster complexes. Images are reconstructed from a stack of optical sections. All confocal images were obtained from immature spinal interneurons. Insets: magnified views of boxed areas demonstrating examples of synaptic and nonsynaptic GlyR clusters. Scale bar: 10 µm.

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 decay-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 displayed 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 neurons (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).

View larger version (24K): [in this window] [in a new window]   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 () and in the presence of colchicine (). 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.

View larger version (43K): [in this window] [in a new window]   FIG. 7. Immature neurons express 2, 1, and -GlyR subunits, while mature neurons down-regulated 2.A: bars represent GlyR transcripts derived from murine and gene structure (closed regions are the signal peptides). Selected primers for amplification of GlyR - and -subunit isoforms are indicated by horizontal 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 electrophoresis. 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 µM PTX. Bottom current traces are typical averaged responses. Thin dotted line in the left trace corresponds to superimposed 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. Superimposed 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.

The previous results suggest that colchicine and 100 µM 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 µM) displayed a different sensitivity to picrotoxin. We reasoned that if colchicine provoked the reduction of 2 GlyRs, this would reduce picrotoxin 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, ). 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, ). Interestingly, most glycinergic mIPSCs in these colchicine-treated neurons (n = 4) were resistant to picrotoxin application (Fig. 8B, ).

View larger version (41K): [in this window] [in a new window]   FIG. 8. Colchicine inhibits the effect of picrotoxin on mIPSCs. A: correlation between decay-time constant and mIPSC amplitude before () and during () application of 100 µM PTX in immature neurons. B: lack of PTX effect () on colchicine-treated neurons (20 µM for 3 h). Note that amplitudes and decay-time constants are already reduced after colchicine treatment (). r < 0.7 in all treatments. Plots of decay-time constant vs. rise-time before () and during () application of 100 µM 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.

In summary, these results suggest that glycinergic mIPSCs become less sensitive to picrotoxin after colchicine treatment, and support the hypothesis that colchicine predominantly affects neonatal 2 GlyRs, leaving the adult 1 GlyRs unaffected.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
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 postsynaptic receptor clusters consequent to microtubule depolymerization. We found no evidence supporting the idea that intracellular dialysis of colchicine in the postsynaptic neurons might affect neurotransmitter release from presynaptic terminals. For instance, although glycinergic transmission was reduced by intracellular dialysis of colchicine, the synaptic transmissions mediated by GABA_ARs and AMPARs remained largely unmodified (van Zundert et al. 2002). In cultured spinal neurons, GlyRs and GABA_ARs frequently co-localize in postsynaptic gephyrin-containing clusters opposite to presynaptic boutons containing inhibitory amino acids (Dumoulin et al. 2000). Hence, it is not likely that alterations in neurotransmitter release would affect glycinergic and leave GABA_Aergic transmission unaffected. In addition, application of extracellular colchicine (20 min) did not reduce the frequency or amplitude of glycinergic mIPSCs. Further evidence supporting a postsynaptic action of colchicine was obtained during this study. First, intracellular application of the microtubule stabilizer 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. Finally, 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 approximately 64 and 324 µM, respectively (Machu 1998). Analysis the whole cell glycine-activated current during in vitro development of spinal cord cultures demonstrated that immature and mature neurons display a similar sensitivity to colchicine with IC₅₀s of 143 ± 28 and 187 ± 13 µM, respectively. In addition, extracellular application of 20 µM colchicine had no effect on glycinergic mIPSCs. Taken together, the effects observed with internal dialysis of 20 µM colchicine in immature and mature neurons are best explained by a postsynaptic mechanism consequent 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 reduction in the amplitude of the glycinergic mIPSCs after microtubule disruption can be best explained by a structural alteration 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 overexpression 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 development (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 decay-phase of glycinergic mIPSCs during development depends on a transition between slow 2 and fast 1 subunits (Krupp et al. 1994; Legendre 2001; Singer et al. 1998; Takahashi 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 suggest that immature glycinergic currents are primarily associated to 2 subunits since these slow decay mIPSCs were affected by higher picrotoxin concentrations than those used to inhibit (IC₅₀ = 7 µM) 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 Macdonald 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 microtubules in immature neurons selectively affected a population of glycinergic currents displaying slow decay-times, thus leaving faster events unaffected. These results indicate that microtubules 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 evident after disruption of microtubules with colchicine, suggesting they represent the same population of receptors. Second, mature neurons with fast 1 GlyRs were insensitive to colchicine.

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 contrast, 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 microtubules that are stabilized by posttranslational modifications, 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 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 molecular composition could affect the manner in which the receptor interacts with the gephyrin scaffold and subsynaptic microtubules.

View larger version (38K): [in this window] [in a new window]   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. Depolymerization of microtubules by colchicine causes the release of 2 GlyR/gephyrin complexes and diffusion to the extrasynaptic membrane. In contrast, gephyrin-coupled 1 GlyRs are linked to colchicine insensitive microtubules, which could be stabilized by MAPs or acetylation (Ac). Alternatively, stabilization of 1 GlyR/gephyrin clusters can be achieved by assembling gephyrin into hexagonal lattices. Therefore the insensitivity of mature receptors to cytoskeleton disruption can be also associated to the presence of 1 GlyR/gephyrin complexes rather than to microtubule-dependent mechanisms. Vertical lines indicate the location of synaptic region.

In conclusion, this study shows that disruption of microtubules by colchicine reduced both postsynaptic glycinergic currents 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 controversial findings in which disruption of microtubules altered the number and morphology of gephyrin clusters in 10-12 DIV spinal neurons (Kirsch and Betz 1