Article |
Address correspondence to Kazuhito Tomizawa, Department of Physiology, Okayama University Graduate School of Medicine and Dentistry, Shikata-cho 2-5-1, Okayama 700-8558, Japan. Tel.: 81-86-235-7109. Fax: 81-86-235-7111. email: tomikt{at}md.okayama-u.ac.jp
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Abstract |
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Key Words: endocytic protein; p35; cyclin-dependent kinase; presynapse; synaptosome
Abbreviations used in this paper: DLS, dynamic light scattering; ESI, electrospray ionization; LC, liquid chromatography; MALDI-MS, matrix assisted laser desorption/ionization mass spectrometry; PRD, proline-rich domain; SH3, Src homology 3; VDCC, voltage-dependent calcium channel.
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Introduction |
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The interactions among the various endocytic proteins are essential for the progression and maturation of clathrin-mediated endocytosis. For example, disruption of the amphiphysin Idynamin I interaction results in the inhibition of synaptic vesicle endocytosis (Marks and McMahon, 1998). It has also been suggested that phosphorylation and dephosphorylation of the endocytic proteins regulate their interactions, resulting in the regulation of synaptic vesicle endocytosis (Marks and McMahon, 1998; Cousin and Robinson, 2001). Previous results showing that endocytic proteins undergo dephosphorylation during the maturation of clathrin-mediated endocytosis strongly support this suggestion (Bauerfeind et al., 1997; Slepnev et al., 1998). A Ca2+/calmodulin-dependent phosphatase, calcineurin, plays an important role in the dephosphorylation (Slepnev et al., 1998). The switching from the phosphorylated state of the endocytic proteins to the dephosphorylated state after nerve terminal depolarization may trigger the clathrin-mediated endocytosis.
Cdk5 is a serine/threonine kinase with close structural homology to the cdc2 family (Dhavan and Tsai, 2001). Cdk5 forms a heterodimer with its neuron-specific activators, p35 or p39, and the association is essential for the kinase activation in neurons (Dhavan and Tsai, 2001). Cdk5 has multiple functions in neurons, implicating it in the regulation of a range of cellular processes from adhesion and motility to synaptic plasticity and drug addiction (Bibb et al., 2001; Dhavan and Tsai, 2001).
Cdk5 is abundant in presynaptic terminals in mature neurons (Tomizawa et al., 2002). Previous studies have identified many presynaptic proteins, such as Munc 18 (nSec-1) (Shuang et al., 1998), synapsin I (Matsubara et al., 1996), P/Q-type voltage-dependent calcium channel (Tomizawa et al., 2002), and amphiphysin I (Floyd et al., 2001), as substrates of Cdk5. These results suggest that Cdk5 is one of the most important kinases in the regulation of neurotransmitter release. Moreover, a very recent paper showed that Cdk5 phosphorylates dynamin I but not amphiphysin I, and the phosphorylation enhances synaptic vesicle endocytosis (Tan et al., 2003). However, this result is contradictory to the hypothesis of the trigger of synaptic vesicle endocytosis by calcineurin-mediated dephosphorylation of the endocytic proteins. Here, we show that Cdk5 phosphorylates both amphiphysin I and dynamin I in vitro and in vivo. The simultaneous phosphorylation of both these proteins inhibits synaptic vesicle endocytosis through inhibition of the association of these proteins with their partner proteins.
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Results |
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The effect of olomoucine on vesicle exocytosis was also examined using a previously reported protocol with some modifications (Di Paolo et al., 2002). The rate of exocytosis was not affected by olomoucine when compared with the control group (Fig. 1 B). Thus, inhibition of Cdk5 seems to specifically enhance the endocytic process rather than the exocytic process.
The effects of inhibition of Cdk5 on the kinetics of vesicle endocytosis were then investigated. The vesicle recycling was initiated by depolarization, and FM1-43 was added after 30-, 60-, and 90-s delay times (Fig. 1 C, inset). Thus, only the vesicles that underwent endocytosis after the delay time were labeled, whereas those that underwent endocytosis during the delay period were not labeled. The active components of FM1-43 uptake at each delay time were normalized by that labeled at delay time 0 s (Fig. 1 C). When the delay time was longer, fewer endocytosed vesicles were stained. At each delay time point, the fraction of labeled vesicles was reduced by olomoucine treatment. These results suggest that more vesicles were endocytosed during the initial 30 s in the olomoucine-treated terminals. Thus, inhibition of Cdk5 increased the endocytic process.
In addition, the changes in total recycling pool size were also investigated. The total recycling pools were labeled by depolarizing the neurons with 90 mM KCl. Olomoucine increased the KCl-induced FM1-43 uptake (134 ± 6%, P < 0.05; Fig. 1 D). Taken together, these findings indicate that the inhibition of Cdk5 promoted the endocytic process and increased the total recycling pool size.
Vesicle endocytosis in p35-deficient mice
To further confirm the role of Cdk5 in endocytosis, the vesicle recycling in hippocampal neuronal cultures prepared from p35 knockout and wild-type embryonic mice was examined. Olomoucine increased the FM1-43 uptake in wild-type neurons (159 ± 10%; Fig. 1 E). In p35 knockout neurons, however, olomoucine did not produce any changes (96 ± 7%, P < 0.05, compared with p35 wild-type neurons). This result suggests that olomoucine enhanced the vesicle endocytic process by specific inhibition of Cdk5 activity.
The kinetics of vesicle endocytosis in p35 knockout neurons were also examined. The ratio of endocytosed vesicles after a 3090-s delay time to that without a delay time in p35 knockout neurons was significantly reduced compared with that in p35 wild-type neurons (Fig. 1 F). This result is consistent with the changes in endocytic kinetics induced by olomoucine treatment in normal neuronal cultures (Fig. 1 C). Taken together, these findings reveal that Cdk5 plays an inhibitory role in vesicle endocytosis, and the suppression of endocytosis by Cdk5 is relieved in p35 knockout neurons.
Cdk5/p35 phosphorylates amphiphysin I and dynamin I in vitro
The specific substrates of Cdk5 in the synaptosomes were next identified. Solubilized synaptosomes from mouse brains were incubated with recombinant Cdk5/p35. Mass spectrometry showed the phosphorylation of both amphiphysin I and dynamin I from the synaptosomes (unpublished data). An in vitro kinase assay also revealed that both amphiphysin I and dynamin I were phosphorylated by Cdk5/p35 (Fig. 2 A). However, stoichiometric analysis showed that the number of phosphorylated residues differed between amphiphysin I and dynamin I. The maximum phosphate incorporation was 1.06 ± 0.03 mol of phosphate/mol of dynamin I (Fig. 2 B). Subsequent addition of fresh Cdk5/p35 at 60 min did not result in any further increase. In contrast, there were clearly multiple substrate sites suitable for Cdk5 in amphiphysin I. The maximum phosphate incorporation was 4.61 ± 0.1 mol of phosphate/mol of amphiphysin I (Fig. 2 B).
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Effect of Cdk5-dependent phosphorylation of amphiphysin I and dynamin I on the interaction with those binding proteins
The present results showed that Cdk5/p35 phosphorylated the PRD of both amphiphysin I and dynamin I (Fig. 2 F). PRD of dynamin I interacts with the Src homology 3 (SH3) domain of amphiphysin I (Owen et al., 1998; Wigge and McMahon, 1998). On the other hand, PRD of amphiphysin I binds to the heavy chain of clathrin and the clathrin adaptor protein AP-2 (- and ß-adaptin; Wang et al., 1995; McMahon et al., 1997; Slepnev et al., 1998). Therefore, whether Cdk5-dependent phosphorylation of amphiphysin I and dynamin I affected the interaction with those binding proteins in vitro was examined. Phosphorylation of amphiphysin I by Cdk5 had no effect on the interaction with dynamin I (Fig. 4 A). However, the phosphorylation significantly inhibited its binding to ß-adaptin (Fig. 4 B). Phosphorylation of dynamin I by Cdk5 inhibited the interaction with amphiphysin I (Fig. 4 C). As PRD of dynamin I interacts with the SH3 domain of amphiphysin I, the effect of the phosphorylation of dynamin I on the binding to the GST-SH3 domain of amphiphysin I was examined. The phosphorylation reduced the ability to interact with the GST-SH3 domain (Fig. 4 D). These results suggest that Cdk5-dependent phosphorylation of dynamin I and amphiphysin I regulates the interaction with their partner proteins.
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The increase in intracellular Ca2+ in presynaptic terminals induces calcineurin activity, resulting in the triggering of synaptic vesicle endocytosis (Cousin and Robinson, 2001). Next, whether electric stimulation (10 Hz, 1 min) changed the expression of phospho-dynamin I in hippocampal slices was examined. The stimulation reduced the level of expression of phospho-dynamin I (Fig. 7, C and D), and olomoucine enhanced the effect of electric stimulation on the reduction in the phospho-dynamin I expression level. Preincubation with FK506 (1 µM) inhibited the effect of electric stimulation on the reduction in phospho-dynamin I expression (Fig. 7, C and D). To examine whether this phenomenon occurred in presynaptic terminals, we applied specific inhibitors of N-type and P/Q-type voltage-dependent calcium channels (VDCCs) and then electrically stimulated the hippocampal slices using a protocol of 10 Hz for 1 min. N- and P/Q-type VDCCs predominantly mediate excitatory neurotransmitter release in the hippocampus (Wheeler et al., 1996). Coapplication of -CgTxGVIA and
-AgaIVA, which are specific inhibitors of N-type and P/Q-type VDCCs, respectively, abrogated the effect of the electrical stimulation on the inhibition of expression level of phospho-dynamin I (Fig. 7, C and D). These results suggest that the phosphorylation of both amphiphysin I and dynamin I is regulated by Cdk5/p35 and calcineurin in the presynaptic terminals of neurons.
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Discussion |
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Regarding the relationship between Cdk5/p35 and amphiphysin I, a previous study showed that a complex of Cdk5 and p25, a truncated form of p35, is associated with amphiphysin I in bovine brain extract (Rosales et al., 2000). Amphiphysin I interacts with p35 and is phosphorylated by Cdk5 (Floyd et al., 2001). However, it has been unclear whether Cdk5 regulates the endocytosis of synaptic vesicles through interaction with and phosphorylation of amphiphysin I. Our present results show for the first time that Cdk5-dependent phosphorylation of amphiphysin I has a critical role in the regulation of synaptic vesicle endocytosis.
Most recently, a concomitantly conducted study by Tan et al. (2003) also found that Cdk5 regulates synaptic vesicle endocytosis through the phosphorylation of dynamin I. However, their results showed that Cdk5 enhances the endocytosis similarly to calcineurin, and the phosphorylation sites they identified differ from the site we have identified here. It is hard to explain the discrepancy between these two studies. One possibility is that Tan et al. (2003) used bacterially expressed GST-Cdk5/p25 fusion protein, while the present study used the more nearly physiological form of Cdk5/p35 derived from eukaryotic expression. This form is more than 100-fold more active than the bacterial form (unpublished data). The difference of recombinant Cdk5 and its activator may have caused the discrepancy of the identified phosphorylation sites of dynamin I by Cdk5 (Figs. 2 and 3). Another possibility is the pharmacological specificity of Cdk5 inhibitors. In the present study, we applied 10 µM roscovitine in the hippocampal neurons, as well as olomoucine, and confirmed the occurrence of consistent effects. We also showed that synaptic vesicle endocytosis was enhanced in the neurons of p35-deficient mice. Moreover, we confirmed the importance of the phosphorylation of threonine 780 of dynamin I in vesicle recycling by FM dye experiments in GFP-dynamin I mutantoverexpressing neurons (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200308110/DC1). Our results agree with the theory of calcineurin-dependent regulation of the clathrin-mediated endocytosis.
Seven proteins (dynamin I, amphiphysin I and II, synaptojanin, epsin, AP180, and Eps15) have been identified as targets of calcineurin (Cousin and Robinson, 2001). These proteins act at different stages of synaptic vesicle endocytosis. Epsin, AP180, and Eps15 regulate the nucleation stage of endocytosis (Cousin and Robinson, 2001). In contrast, both amphiphysin I and dynamin I mediate invagination and vesicle fission of synaptic vesicles. The question arises of whether Cdk5/p35 phosphorylates all these proteins and regulates synaptic vesicle endocytosis at all stages. In the present study, we identified the PRDs of both amphiphysin I and dynamin I as Cdk5-dependent phosphorylation sites. Cdk5 is one of the proline-directed kinases and phosphorylates the SP and TP motifs (Dhavan and Tsai, 2001). These motifs are abundant in PRD. Synaptojanin, EPS15, and amphiphysin II also have PRDs (Cousin and Robinson, 2001). Moreover, one previous study showed that cdc2 kinase phosphorylates epsin and Eps15, and the phosphorylation inhibits the interaction with AP2 (Chen et al., 1999). However, cdc2 is expressed in mitotic cells, and the expression is very low in mature neurons. Cdk5 is a member of the cdc2 kinase family, and its phosphorylation motif closely resembles that of cdc2 (Dhavan and Tsai, 2001). Therefore, Cdk5/p35 may physiologically phosphorylate epsin and Eps15 and may regulate synaptic vesicle endocytosis through the phosphorylation of all these proteins at all stages.
The mechanism of the regulation of synaptic vesicle endocytosis by phosphorylation and dephosphorylation remains controversial. Two possibilities have been proposed for the mechanism as follows: (1) regulation of proteinprotein interactions and subcellular localization; and (2) regulation of the enzyme activity of dynamin I. At the fission stage, dynamin I assembles into rings around the neck of invaginated vesicles to form a collar (Hinshaw, 2000). The liposome-ring formation activity of dynamin I is enhanced by the addition of amphiphysin I (Takei et al., 1999). It has been unclear whether the effect of amphiphysin I is due to enhanced recruitment of dynamin I to the vesicle neck or stimulation of the GTPase activity of dynamin I. The present results showed that Cdk5-dependent phosphorylation of amphiphysin I and dynamin I inhibited the interaction with the partner proteins. Furthermore, cophosphorylation of amphiphysin I and dynamin I completely disrupted the vesicle formation from liposomes. These data provide strong evidence that phosphorylation and dephosphorylation of endocytic proteins regulate endocytosis by altering proteinprotein interactions.
In conclusion, the findings of the present study suggest that cophosphorylation of both amphiphysin I and dynamin I by Cdk5 is critical for the inhibition of synaptic vesicle endocytosis. The switching from the phosphorylated form of these proteins to the dephosphorylated form after membrane depolarization results in the reconstitution of proteinprotein interaction and induction of synaptic vesicle endocytosis.
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Materials and methods |
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Dynamin 1 was purified from bovine brain essentially as described previously (Liu et al., 1994). Human dynamin I cDNA encoding the PRD domain was subcloned into pGEX-6P, and the PRD domain mutant construct was made using QuickChange site-directed mutagenesis kit (Stratagene). Recombinant Cdk5/p35 was prepared using a baculovirus expression system as described previously (Saito et al., 2003).
In vitro phosphorylation and binding assay
The phosphorylation reaction and binding assay were performed as described previously (Tomizawa et al., 2000, 2002). In brief, phosphorylation reactions were performed in kinase buffer containing 20 mM MOPS, pH 7.4, 5 mM MgCl2, 100 µM ATP, [-32P]ATP (300 dpm/pmol), and 1 mM DTT. Purified dynamin I and amphiphysin I were incubated with each of the tested concentrations of His-tagged Cdk5/p35 at 32°C. The reactions were terminated by the addition of boiled SDS sample buffer. After electrophoresis of the samples on SDS-PAGE and staining with Coomassie blue, the relevant gel slices were excised and Cerenkov counted to determine the total 32P incorporation.
For binding assays for dynamin I and amphiphysin I, dynamin I or amphiphysin I was phosphorylated by Cdk5/p35 in kinase buffer without [-32P]ATP for 1 h at 32°C. To remove His-tagged Cdk5/p35 complex and ATP, the phospho-dynamin I and -amphiphysin I were incubated with ProBond nickel-chelating resin (Invitrogen) in PBS with 0.5% Triton X-100 for 1 h at 4°C. After centrifugation, the supernatants were dialyzed against PBS overnight at 4°C (phospho-dynamin I or -amphiphysin I). Either phospho- (5 µg) or dephospho-amphiphysin I (5 µg) was incubated with phospho- (4 µg) or dephospho-dynamin I (4 µg) in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.4% NP-40) for 30 min at 4°C, and 1 µg of antidynamin I polyclonal antibody (C-16; Santa Cruz Biotechnology), antiamphiphysin I monoclonal antibody (Transduction Laboratories), mouse IgG, or rabbit IgG was then added for 1 h at 4°C. After the addition of 40 µl of protein GSepharose (Amersham Biosciences), the complex was further incubated for 1 h at 4°C. The beads were washed three times with RIPA buffer, and bound proteins were analyzed by Western blotting analysis. For the binding assay of amphiphysin I to clathrin, phospho- or dephospho-amphiphysin I was incubated with 1 mg of adult rat brain lysate in RIPA buffer with protease and phosphatase inhibitors for 1 h at 4°C. Amphiphysin I complexes were then immunoprecipitated with 1 µg of antiamphiphysin I antibody as described above, and ß-adaptin binding was then analyzed using antiß-adaptin antibody (Transduction Laboratories). The quantification of binding proteins was performed by scanning x-ray films and analyzing the scanned images with the NIH image program.
Mass spectrometry
Sample solution (0.5 µl) was loaded onto a MALDI-MS sample plate with 0.5 µl of matrix solution (60 g/liter) of 2,5-dihydroxybenzoic acid in ACN/water (1/2, vol/vol) containing 0.1% TFA. The samplematrix solution was allowed to air dry at room temperature. MALDI-MS measurements were obtained using a PerSeptive Voyager Linear DE model (Applied Biosystems). The remaining 90% (4.5 µl) of samples were analyzed by means of electrospray ionization (ESI) MS combined with HPLC. HPLC separations were made with a C18 silica gel column (Magicms C18 50 mm x 0.1 mm, 200 Å, 5 µm; Michrom BioResources, Inc.) at a flow-rate of 1 µl/min. The eluent from the HPLC column was connected directly to the ESI source. Mobile phase A was ACN/aqueous 0.2% acetic acid (5:95, vol/vol), and mobile phase B was ACN/ aqueous 0.2% acetic acid (95:5). A linear gradient program was run from 0 to 50% B over a period of 60 min. LC-MS and LC-MS/MS analyses were performed with an ESI ion trap mass spectrometer (LCQ; ThermoFinnigan) operated in a mode that alternated single MS scans (m/z 6002000) with MS/MS scans (data-dependent scan mode in which the most intense ion peak in the previous MS scan was isolated and subjected to collision-induced dissociation).
In vitro small vesicle formation
Large unilamellar liposomes composed of 80% (wt/wt) brain extract and 20% cholesterol (1 mg/ml) were prepared as described previously (Takei et al., 2001). The liposomes (final concentration 100 µg/ml) were incubated in 1 ml of "cytosolic buffer" (25 mM Hepes-KOH, pH 7.2, 25 mM KCl, 2.5 mM magnesium acetate, 100 mM potassium glutamate) for 15 min at 37°C with proteins and GTP at the various combinations indicated. The final concentrations of proteins and nucleotides were as follows: phosphorylated or dephosphorylated dynamin, 1 or 20 µg/ml; phosphorylated or dephosphorylated amphiphysin, 1 or 50 µg/ml; GTP, 1 mM.
DLS assay
The sizes of the lipid vesicles and relative distribution in numbers of each size of lipid vesicle were measured by DLS using a DLS-7000 AR-III spectrophotometer (Otsuka Electronics Co.) as described previously (Kinuta et al., 2002).
EM
For negative staining, samples were adsorbed onto freshly glow-discharged Formvar- and carbon-coated EM grids, stained with 2% uranyl acetate in ddH2O for 1 min, blotted, and air dried. The grids were examined using a Hitachi H-7100 transmission EM (Hitachi Ltd.) at the Central Research Laboratory at Okayama University Medical School.
Hippocampal neuronal culture and FM1-43 experimental conditions
Dissociated neuronal culture was performed using a modification of a previously described procedure (Matsushita et al., 2001). p35 mutant hippocampal neuronal cultures were prepared from E1718 of hetero p35 mutant mice (Ohshima et al., 2001). The hippocampal cells from each embryo were plated onto the coated glass-bottom dishes separately. The genotype of neuronal cultures was identified from each mouse embryo.
Cultures at day 914 after plating were used in fluorescence experiments. The culture was superfused with normal saline solution at room temperature. The normal saline solution contained 119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM Hepes (pH 7.4), and 20 mM glucose. To load FM1-43 dye into the vesicles, field stimulation at 20 Hz, 30 s, duration 1.0 ms, and intensity 15 V was delivered to the culture through a parallel platinumiridium electrode immersed into the perfusion chamber. Kynurenic acid (5 mM; Sigma-Aldrich) and D-2-amino-5-phosphonopentanoic acid (25 µM; Sigma-Aldrich) were applied during loading and unloading stimulation to prevent recurrent activity. FM1-43 dye (15 µM; Molecular Probes) was present in the superfusing solution from 45 s before stimulation to 45 s after stimulation. After FM1-43 loading, the culture was rinsed with dye-free superfusing solution for 10 min, and fluorescence imaging was performed. An unloading stimulation (10 Hz, 45 s or 20 Hz, 30 s) was delivered to the culture in the dye-free solution to unload the previously loaded dye. To label the total recycling vesicle pools, FM1-43 was loaded and unloaded by depolarization with 90 mM KCl solution. To measure the kinetics of vesicle endocytosis, FM1-43 was loaded after a delay time after the cessation of depolarization.
Fluorescence imaging and data analysis
Optical imaging experiments were performed with a Carl Zeiss MicroImaging, Inc. Axiovert 200 inverted microscope. FM1-43 fluorescence was excited at 488 nm with a Xenon lamp. The illumination and exposure to the CCD camera (Hamamatsu Photonics) were controlled and synchronized using AquaCosmos software to minimize photobleaching of the dye. A Carl Zeiss MicroImaging, Inc. 63x/1.4 NA (Plan Apochromat) was used to view the cells, and a narrow bandpass FITC filter was used when capturing the fluorescence images.
The active boutons were measured by subtracting the unloading image from the loading image. The resulting image was filtered by thresholding at a level of mean plus four standard deviations of the Gaussian background intensity. The remaining puncta were taken as activity-dependent boutons. These boutons showed uptake of the FM1-43 dye during vesicle recycling, thus reflecting endocytosis.
Production of phosphospecific antibodies for dynamin I and immunoblot analysis
A peptide corresponding to residues 772784 of rat dynamin I was chemically phosphorylated at residue Thr 780 and employed to generate rabbit polyclonal antibodies that specifically detected phospho-dynamin I as described previously (Czernik et al., 1991). Western blot analysis was performed at high stringency, essentially as described previously (Tomizawa et al., 2002).
Generation of p35 knockout mice and preparation of synaptosomes
p35 mutant mice were generated and maintained in a 129/Sv x C57BL/6J background as described previously (Ohshima et al., 2001). Synaptosomes were prepared from wild-type and p35 knockout mice as described previously (Tomizawa et al., 2002). Stimulation (depolarization) of synaptosomes was performed as described previously (Bauerfeind et al., 1997). FK506 (1 µM) was incubated with synaptosomes for 5 min in control buffer. Incubations were terminated by adding SDS-PAGE buffer, and the samples were used for Western blotting analysis.
Preparation of hippocampal slices and electric stimulation
Preparation of hippocampal slices and electric stimulation were performed as described previously (Tomizawa et al., 2002). In brief, the hippocampus of male C57BL/6 mice aged 78 wk was dissected, and 400-µm transverse slices were prepared. A bipolar stimulating electrode was placed along the Schaffer collateral fibers, and a glass micropipette filled with artificial cerebrospinal fluid was placed in the stratum radiatum of the CA1 region to record field excitatory postsynaptic potentials (EPSPs) and to adjust the intensity of the stimulation. The intensity of the stimulation was adjusted to produce an EPSP with a slope of 50% of the maximum.
-Conotoxin GVIA (
-CgTX GVIA; RBI),
-Agatoxin IVA (
-Aga IVA; RBI), and FK506 (Fujisawa Pharmaceutical) were added to the perfusion medium. The final concentration of
-CgTX GVIA and FK506 was 1 µM, and the final concentration of
-Aga IVA was 0.5 µM. After preincubation with these drugs for 30 min, hippocampal slices were electrically stimulated with 10 Hz for 1 min, and the slices were then sonicated in boiled 1% SDS.
Statistical analysis
Data were analyzed using either the t test to compare the two conditions or ANOVA followed by planned comparisons of the multiple conditions, and P < 0.05 was considered to be significant.
Online supplemental material
The supplemental material (Fig. S1) is available online at http://www.jcb.org/cgi/content/full/jcb.200308110/DC1. Fig. S1 shows enhancement of vesicle endocytosis in hippocampal neurons overexpressing dynamin I mutant at Thr 780.
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Acknowledgments |
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This work was supported by Industrial Technology Research Grant Program in 2002 from New Energy and Industrial Technology Development Organization, Japan (K. Tomizawa) and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (K. Tomizawa, S. Hisanaga, M. Kinuta, M. Matsushita, K. Takei, and H. Matsui). Some of the vesicle endocytosis experiments were performed in Dr. Alan Everett's lab at the Department of Physiology, University of Western Australia (UWA) (Crawley, Australia) with support from a UWA postdoctoral fellowship (Y.-F. Lu).
Submitted: 20 August 2003
Accepted: 7 October 2003
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