A novel function of synapsin II in neurotransmitter release

Molecular Brain Research 85 (2000) 133–143 www.elsevier.com / locate / bres

Research report

A novel function of synapsin II in neurotransmitter release Takashi Sugiyama a , Toru Shinoe a , Yoko Ito a , Hidemi Misawa b , Takuro Tojima c , Etsuro Ito c , Tohru Yoshioka a,d , * a

Department of Molecular Neurobiology, School of Human Sciences, Waseda University, Tokorozawa, Saitama 359 -1192, Japan b Department of Neurology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Tokyo 183 -8526, Japan c Laboratory of Animal Behavior and Intelligence, Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Hokkaido 060 -0810, Japan d Advanced Research Institute for Science and Engineering, Waseda University, Shinjuku, Tokyo 169 -8555, Japan Accepted 12 September 2000

Abstract Although synapsin has been localized to presynaptic structures, its function remains poorly understood. In the present study, we investigated the presynaptic function of synapsin II using a synaptic vesicle recycling process using synapsin-II-overexpressing NG108-15 cells. Western blot analysis with antibodies for synaptic-vesicle-associated protein indicated that the number of synaptic vesicles was approximately doubled in synapsin II transfectants as reported previously. In differentiated synapsin-II-overexpressing and control cells, the application of high potassium induced strong intracellular calcium elevation along neurites and varicosities after differentiation and a weak calcium rise in the cell bodies. The uptake and release of the fluorescent dye FM1-43 revealed that synaptic vesicle recycling in synapsin-II-transfected cells occurred with the same kinetics in the cell body and neuritic varicosities. Furthermore, the area labeled with FM1-43 fluorescence in the synapsin-II-transfected cells was approximately twice as much as in control cells after stimulation, and ATP released after synaptic vesicle fusion with the plasma membrane in synapsin-II-expressing cells was significantly elevated relative to controls. The number of synaptic vesicles paralleled the amount of transmitter released from the cells leading to the conclusion that the number of releasable synaptic vesicles were increased by synapsin II transfection into NG108-15 cells, suggesting that synapsin II may have a role in the regulation of synaptic vesicle number in presynapse-like structures in NG108-15 cells.  2000 Elsevier Science B.V. All rights reserved. Theme: Excitable membranes and synaptic transmission Topic: Mechanisms of neurotransmitter release Keywords: Synapsin; NG108-15; Ca 21 imaging; FM1-43; Synaptic vesicle recycling; ATP release

1. Introduction The synapsin family of proteins (Ia, Ib, IIa, and IIb) are associated with synaptic vesicles and implicated in the short-term regulation of neurotransmitter release [3,6,12,15,29]. Previous studies have shown that the total amount of synapsin I in nervous tissue is highly regulated [20–23], but its function is poorly understood. Han et al. reported that the number of neuritic varicosities, small clear vesicles, and large dense core vesicles are highly increased by overexpression of synapsin IIb in the clonal

*Corresponding author. Tel. / fax: 181-33-205-6419. E-mail address: [email protected] (T. Yoshioka).

cell line NG108-15 [8]. Based on these results they suggested that synapsin IIb is involved in the regulation of synapse formation in long-term neuronal signaling. In contrast, Rosahl et al. observed that synapsin-II-deficient mice, but not synapsin-I-deficient mice, exhibit decreased posttetanic potentiation and significant synaptic depression with repetitive stimulation [24]. In addition, they found that intrinsic synaptic vesicle membrane proteins are slightly decreased in synapsin-I- or synapsin-II-deficient mice and more severely reduced in synapsin-I- and synapsin-II-deficient mice, both effects associated with a decrease in the number of synaptic vesicles. Taken together, these data suggest that synapsin functions to maintain a stable number of releasable synaptic vesicles, and may increase the stability of the synaptic vesicles themselves,

0169-328X / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 00 )00231-X

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which is required for the normal short-term regulation of exocytotic transmitter release. In the present study, we tested the hypothesis that synapsin II has a significant physiological role in the regulation of the number of synaptic vesicles by examining membrane recycling by imaging uptake and release of FM1-43, a fluorescent dye, and measurement of ATP, which is released with acetylcholine, in synapsin-II-overexpressing NG108-15 cells. Intracellular calcium (Ca 21 ) elevation induced by high potassium (K 1 ) stimulation caused a 2-fold increase in the number of functional synaptic vesicles that exhibited membrane fusion (recycling) and extracellular release of ATP in synapsin-IItransfected cells compared with controls. Interestingly, other proteins associated with synaptic vesicles were also increased in the synapsin-II-transfected cell line. These data suggest that synapsin II functions to maintain synaptic vesicle recycling in NG108-15 cells.

2. Materials and methods

2.1. Materials The pSV2bsr vector kit was from Funakoshi (Tokyo, Japan). The Hybond-ECL filter, Multiprime DNA labeling system, and the ECL Western blotting detection reagent were from Amersham (Buckinghamshire, UK). The TimeSaverE cDNA synthesis kit was from Pharmacia (Uppsala, Sweden). Peroxidase-goat anti-rabbit IgG was from Zymed (San Francisco, CA, USA). Fura-2 and FM143 were from Molecular Probes (Eugene, OR, USA).

screened using plaque hybridization with a 207-bp fragment (nucleotides 458–665) of rat synapsin II cDNA [29]. The cDNA fragment was amplified using polymerase chain reaction (PCR) and was 32 P-labeled using a Multiprime DNA labeling system. Prehybridization and hybridization were performed as described by Kengaku et al. [13]. Positive clones were digested with EcoRI, and DNA inserts were subcloned in a pBluescript SK(2) vector. DNA sequences were determined from both strands using the dideoxy method [27].

2.4. Construction and transfection of the expression vector ( pEFSYNIIa and pEFSYNIIb) To construct pEFSYNIIa and pEFSYNIIb, cDNA fragments with the whole protein coding sequence of rat synapsin IIa or IIb were inserted into the EcoRI site of pEF321A, which contains the promoter region for the human elongation factor 1a [14]. Orientation of the cDNA insert was confirmed using DNA sequencing. NG108-15 cells were plated at a density of 5310 5 cells per 60-mm dish and cultured for 24 h. Transfection of the cells was performed using the modified calcium phosphate precipitation method [5] with 10 mg of pEFSYNIIa or pEFSYNIIb and 2 mg of pSV2bsr, which contains the blasticidin-S-resistance gene under the control of the SV40 early promoter [10]. After 3 days, blasticidin S hydrochloride (5 mg / ml) was added to the medium. Stable transfected cell clones were isolated after 2 weeks of selection. Mock-transfected cells were established with pEF321A (10 mg) and pSV2bsr (2 mg) without the synapsin II inserts.

2.5. Antibody preparation 2.2. Cell culture An NG108-15 cell line was grown at 378C in a 7% CO 2 , 93% air atmosphere in Dulbecco’s modified Eagle’s medium (DMEM) containing penicillin (10 5 units / L), streptomycin sulfate (100 mg / l), 13HAT (100 mM hypoxanthine, 0.4 mM aminopterine, 16 mM thymidine), and 10% fetal bovine serum (FBS). To induce neuronal differentiation, dibutyryl cyclic AMP (dBcAMP; 1 mM) was added to the medium for 3 days or more, and the concentration of FBS was reduced to 1%.

2.3. Cloning of synapsin IIa and IIb cDNAs Total RNA was extracted from rat brain, and poly(A)1 RNA was selected using oligo(dT)-cellulose column chromatography [2]. An oligo(dT)-primed cDNA library was constructed in lgt10 using the TimeSaverE cDNA synthesis kit with 5 mg of poly(A)1 RNA, according to the manufacturer’s protocol. The rat brain cDNA library was

A 17-amino-acid peptide (CTQQPQSGTLKEPDSSK; amino acids 433–448), corresponding to the region common to synapsin IIa and IIb [29], was synthesized and conjugated to maleimide-activated keyhole limpet hemocyanin (KLH) via the amino-terminal cysteine residue by the Fujiya BioScience Laboratory (Kanagawa, Japan). Two New Zealand white rabbits were injected intradermally with 1 mg of the peptide-KLH conjugate emulsified in Freund’s complete adjuvant. The animals were boosted 4 times with 0.5 mg of the antigen in Freund’s incomplete adjuvant at 2 week intervals. The sera were collected and tested for their titer using an indirect enzyme-linked immunosorbent assay [1].

2.6. Immunoblot analysis Cell homogenates were prepared in 50 mM Tris–HCl, pH 7.2, containing 2 mM EDTA and 2 mM dithiothreitol, and proteins were separated (50 mg of protein / lane) on a

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7.5% polyacrylamide sodium dodecyl sulfate (SDS) gel using electrophoresis, and transferred to a nitrocellulose membrane (Hybond-ECL). The membrane was preincubated with 10% skim milk in Tris-buffered saline containing 0.1% Tween–20, pH 7.4 (TBS–T), and then incubated with anti-synapsin II, anti-synaptotagmin I, antisynaptotagmin II, or anti-VAMP2 / synaptobrevin antibodies (1:10 5 dilutions; a gift from Dr. M. Takahashi at the Life Science Institute, Mitsubishi Chemical Co.) for 1 h at room temperature (23–258C). The membrane was washed 5 times with TBS–T, incubated with peroxidase-goat antirabbit IgG at a 1:2000 dilution for 1 h at room temperature, and washed 5 times with TBS–T. Immunopositive bands were detected with ECL Western blotting detection reagent. The membrane was scanned using a Densitograph (AE-6920V-05, ATTO), and the area under each peak was integrated using a Lane & Spot Analyzer (AE-6920WLSA, ATTO).

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from an initial image after dye loading with high K 1 stimulation [16,25]. The subtracted image was pseudocolored for illustrative purposes.

2.9. Measurement of released ATP Cells were cultured in growth medium until they reached confluency. To induce differentiation, dBcAMP was added to the medium (1 mM final concentration). After washing, the cells were incubated in a buffer consisting of 5.6 mM glucose, 125 mM NaCl, 4.8 mM KCl, 1.2 mM potassium phosphate, 1.3 mM CaCl 2 , 1.2

2.7. Ca 21 imaging Undifferentiated or differentiated cells were cultured on glass coverslips, and treated with 5 mM fura-2 acetoxymethylester for 30 min at 378C. Cells were observed using a fluorescence microscope (Axiovert 405 M, Zeiss) equipped with an intensified charge-coupled detector video camera (Hamamatsu Photonics, C 2400–84, Hamamatsu, Japan). A band-pass filter (340 nm and 380 nm; Nikon, Tokyo) was placed in the excitation path, and the emitted light was filtered with both a 395 nm dichroic mirror and a 397 nm cut-off filter. Image acquisition and analysis were performed using an Argus-50 / CA (Hamamatsu Photonics). For stimulation, the incubation medium was abruptly changed to basal salt solution (BSS; 130 mM NaCl, 5.4 mM KCl, 5.5 mM D-glucose, 20 mM HEPES, 2 mM CaCl 2 , and 1 mM MgSO 4 , pH 7.3) containing 50 mM KCl. After acquisition, the mean background level was subtracted for each image series.

2.8. Uptake of FM1 -43 into cells Cells were differentiated using 1 mM dBcAMP for more than 5 days, and then were loaded with FM1-43 (10 mM) in a high K 1 solution (50 mM) for 3 min. After washing with BSS for 30 min, the cells were examined using a fluorescence microscope equipped with a CCD camera. A band-pass filter (450–490 nm) was placed in the excitation path, and a dichroic mirror (510 nm) and a cut-off filter (520 nm) were placed in the emission path. Images were acquired and analyzed using an imaging system (Argus-50 / CA, Hamamatsu Photonics) and software (NIH image). To distinguish nonspecific labeling from dye labeling dependent on synaptic vesicle recycling, an image of the residual fluorescence without high K 1 stimulation was subtracted

Fig. 1. Immunoblot analysis of synapsin-IIa- and synapsin-IIb-transfected NG108-15 cells. (A) Homogenates of mock-transfected cells, synapsinIIa-transfected cells, synapsin-IIb-transfected cells, and rat brain (50 mg / lane) were run on SDS–PAGE and blotted onto nitrocellulose membranes. Synapsin II was detected using anti-synapsin-II antibody. (B) Blotted membranes were incubated with antibodies to synaptotagmin I, synaptotagmin II, and VAMP/ synaptobrevin, followed by HRP-conjugated secondary antibody. Protein size markers are indicated on the left (kDa).

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mM MgSO 4 , and 25 mM HEPES (pH 7.2). To stimulate the cells, the buffer was changed to a buffer containing 50 mM KCl. The ATP content in the buffer was determined using a luciferin–luciferase luminescence system [31].

3. Results

3.1. Expression of synapsin II and other synapticvesicle-associated proteins in transfected NG108 -15 cells Expression vectors containing either synapsin IIa or IIb cDNA were introduced into NG108-15 cells along with a blasticidin-S-resistance gene (pSV2bsr), as a drug-resistance marker [10]. The expression of synapsin II proteins in independent clones was analyzed using immunoblotting with an anti-synapsin-II antiserum that recognized the common region of synapsin IIa and IIb. Synapsin IIa and IIb were detected at 70 kDa and 56 kDa, respectively. Increased amounts of synapsin IIa (20-fold) or IIb (15fold) were detected in the respective transfectants (Fig. 1 A). Previously, synapsin-IIb-overexpressing NG108-15 cells were shown to have both more varicosities along their neurites and more vesicles per varicosity than did mocktransfected cells following differentiation [8]. In addition, these cells had increased amounts of other synaptic-vesicle-associated proteins, such as synapsin I and synaptophysin. For further study, we performed immunoblotting analyses using anti-synaptotagmin I, anti-synaptotagmin II, and anti-VAMP2 / synaptobrevin antibodies, and found that the expression levels of these proteins were also significantly increased in synapsin-IIb-transfected cells compared to mock-transfected cells (3.2-fold for synaptotagmin I, 4.7-fold for synaptotagmin II, and 2.4-fold for VAMP2 / synaptobrevin; Fig. 1 B). Levels of these synaptic-vesicle-associated proteins were similar for mock-transfected and nontransfected NG108-15 cells. Northern blot analysis revealed that only the mRNA for synapsin II increased in the synapsin-II-transfected cells, while other synaptic-vesicle-associated proteins increased (data not shown). These results suggested that synapsin II protein slows down the metabolic rate of synaptic vesicles.

3.2. Imaging of intracellular Ca 21 mobilization in the transfected cells Using the fura-2 staining method, elevations in Ca 21

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level were not detected in undifferentiated cells following high K 1 stimulation (Fig. 2 A), which was likely due to the lack of voltage-sensitive Ca 21 channels in these cells [11]. After the cells differentiated, intracellular Ca 21 levels increased markedly in the neurites of the cells after high K 1 stimulation in both synapsin-II-overexpressing cells (Fig. 2 B) and control cells. These data suggest that after differentiation, voltage-sensitive Ca 21 channel expression increases in the neurites. The amount of Ca 21 evoked by high K 1 stimulation increased with increasing number of days of dBcAMP treatment up to day 5 (Fig. 2 C).

3.3. Visualization of synaptic vesicle recycling using FM1 -43 To examine the recycling of vesicles, we introduced a fluorescent dye, FM1-43 (10 mM), into the exracellular fluid and treated the cells with high K 1 for 3 min (Fig. 3). The dye is ‘captured’ in newly formed vesicles during the process of endocytosis, and the area of FM1-43 labeling is proportional to the amount of endocytosis [4,16,19,25,26]. Both mock (Fig. 3 B) and synapsin-II-transfected cell lines (Fig. 3D) exhibited FM1-43 labeling to some extent after high K 1 stimulation following differentiation, but did not exhibit any labeling without differentiation treatment (Fig. 3F). Both synapsin-II- and mock-transfected cells were labeled along the contours of the cell body, whereas synapsin-II-transfected cells also exhibited labeling in the varicosities (Fig. 3D). Differentiated synapsin-II-transfected cells were labeled more intensely (Fig. 4A). The area of FM1-43 labeling in synapsin-II-transfected cells was twice that of mock-transfected cells (Fig. 4A; 105.9643.0 pixels / mock-transfected cell, 197.2690.9 pixels / synapsinII-transfected cell). To determine whether the intense labeling in synapsin-II-transfected cells indicated an increasing number of vesicles or an increase in volume of new vesicles, the intensity of the fluorescence of the spots was measured and the distribution shown in Fig. 4B. The distribution of the fluorescence intensity was similar for mock-transfected cells (Fig. 4Ba) and synapsin-II-transfected cells (Fig. 4Bb), while the number of fluorescent spots per cell was greater in synapsin-II-transfected cells. The similar distribution of the fluorescence intensity in mock-transfected cells (mean value512.361.3) and synapsin-II-transfected cells (mean value510.360.7) provides evidence that the vesicle volume was unchanged by the transfection, since the fluorescence intensity is proportional to vesicle volume. However, an increase in the number of fluorescence spots suggests that the number of the vesicles

Fig. 2. Imaging of Ca 21 mobilization in synapsin-II-transfected NG108-15 cells. Undifferentiated cells (A) and differentiated cells (B) loaded with the acetoxymethyl ester form of fura-2 were stimulated with 50 mM high K 1 . Bar550 mm (C) Ca 21 response to 50 mM high K 1 stimulation increased with increasing number of days treated with dBcAMP up to 5 days (mean6S.E.M.). Cells (1310 5 / 35-mm dish; n57) were treated daily with dBcAMP and each dish was examined independently.

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Fig. 5. Effect of extracellular Ca 21 concentration on FM1-43 uptake in transfected NG108-15 cells. The differentiated mock-transfected cells were incubated in BSS containing FM1-43 under various Ca 21 concentrations as indicated. After high K 1 stimulation, the FM1-43-labeled area was determined using an imaging system. The concentration of Ca 21 in the normal extracellular medium was 2 mM.

mately 2-fold in cells overexpressing synapsin II, and these vesicles were recycled in a Ca 21 -dependent manner.

Fig. 4. Changes in the area labeled by FM1-43 induced by cell differentiation and transfection. (A) Averaged FM1-43-labeled area of the mock(open bar) and synapsin-II-transfected cells (filled bar). The FM1-43labeled area was expressed as the number of fluorescent pixels per cell using NIH image software. Bar graphs indicate mean6S.E.M. (3 fields of each dish, 4 dishes per condition). Undiff, undifferentiated cells; Diff, differentiated cells. (B) Distribution of fluorescence intensity in the transfected cells after stimulation. Distribution of FM1-43 fluorescence in mock-transfected NG108-15 cells (A) and synapsin-II-transfected cells (B).

labeled with FM1-43 was increased in synapsin-II-transfected cells. Because the depolarization-induced synaptic vesicle recycling is dependent on the influx of extracellular Ca 21 , the Ca 21 concentration in the stimulating medium was changed to test the Ca 21 dependency of the event. As shown in Fig. 5, there was little or no FM1-43 labeling in the absence of extracellular Ca 21 , while the amount of high-K 1 -induced FM1-43 labeling increased with increasing extracellular Ca 21 concentration. Therefore, it can be concluded that the number of vesicles increased approxi-

3.4. Kinetics of exocytosis at varicosities and the cell body surface in transfected NG108 -15 cells Synapsin-II-transfected NG108-15 cells exhibited FM143 labeling primarily in the varicosities, while synaptic vesicle recycling at the terminal boutons around the cell bodies occurred infrequently (Fig. 3D). We measured and compared the kinetics of membrane fusion in the varicosities and the cell body. After FM1-43 labeling, synapsin-II-transfected cells were stimulated with high K 1 to induce FM1-43 release and the time course of the change in fluorescence intensity was recorded from varicosities (Fig. 6Aa and 6Ab) and terminal boutons (Fig. 6Ac and 6Ad). Although many boutons could not show apparent fluorescence changes in synapsin-II-transfected cells (Fig. 6Bd), the destaining of fluorescence was rarely observed around the cell body (Fig. 6Bc). In this case, the destaining time course was similar for varicosities and the terminal boutons. The control cells showed a similar destaining time course as shown in Fig. 6Bd. These small decreases in fluorescence may be due to the lack of staining varicosity in the control cells (Fig. 3B). This indicates that synaptic vesicles located either in varicosities or the cell body have the different characteristics in the secretion.

Fig. 3. FM1-43 labeling in transfected NG108-15 cells. Differentiated mock- (A, B) and synapsin-II-transfected cells (C, D) and undifferentiated synapsin-II-transfected cells (E, F) were stimulated by high K 1 in the medium containing FM1-43 and the fluorescent images were obtained before (A, C, E) and after (B, D, F) stimulation. The images were pseudocolored to illustrate fluorescence intensity as indicated by the bar to the right. Differentiated cells exhibited FM1-43 labeling in the cell body and varicosities, but little labeling was seen in undifferentiated cells. Arrowheads indicate neuritic varicosities. Bar550 mm.

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dent manner and the amount of ACh released is not enhanced by high K 1 stimulation [18]. In contrast, ATP does not leak across the plasma membrane in these cells [28,32], therefore, the exocytotic release of ACh from NG108-15 cells may be better estimated by measuring extracellularly released ATP [32]. Both synapsin-IIa- and synapsin-IIb-transfected cells released more ATP in response to high K 1 stimulation after differentiation than before differentiation (Fig. 7A). After differentiation, extracellular ATP levels in response to stimulation of synapsin-II-transfected cells were significantly greater than that of mock-transfected cells (mock-transfected cell, 0.6160.03%; synapsin-IIa-transfected cell, 2.4860.09%; synapsin-IIb-transfected cell, 2.5860.04% of intracellular ATP, P,0.001). The mock-transfected cells exhibited a slight enhancement in stimulation-induced ATP release. Furthermore, in response to stimulation, the time courses of ATP release from differentiated and undifferentiated synapsin-IIb-transfected cells were compared (Fig. 7B). In undifferentiated cells, high K 1 stimulation had very little effect on ATP release, while following differentiation, stimulation induced a slow release of ATP. These data indicate that synapsin-II-transfected cells contain more than twice the number of functional synaptic vesicles than do mock-transfected cells.

4. Discussion

Fig. 7. ATP released from synapsin-II-transfected and mock-transfected cells. (A) Undifferentiated (Undiff) and differentiated (Diff) cells were stimulated with 50 mM high K 1 for 5 min. Amounts of released ATP from mock- (open bar), synapsin-IIa- (striped bar), or synapsin-IIbtransfected NG108-15 cells (filled bar) were determined using a fluorescent luciferin–luciferase-based detection system, and were expressed as a percentage of the total ATP present in the cells (mean6S.E.M.; n510 dishes per condition) ** P,0.001 (synapsin-II-transfected versus mocktransfected cells; Student’s t-test). (B) ATP released into the extracellular medium was measured at 1-min intervals. Undifferentiated (open circle) and differentiated (solid circle) synapsin-IIb-expressing NG108-15 cells were stimulated with high K 1 . Solid bar indicates time of stimulation. Amount of ATP released is expressed as a percentage of the total ATP present in the cells (mean6S.E.M.; n510 dishes per condition).

3.5. Stimulation-induced release of ATP It has been shown that cholinergic synaptic vesicles contain both ATP and acetylcholine (ACh) [32]. In NG108-15 cells, ACh can be released in a Ca 21 -indepen-

The results of the present study confirm the effectiveness of FM1-43 labeling [16,25] for the study of synaptic vesicle recycling and demonstrate that there was an increased number of releasable synaptic vesicles in both varicosities and the cell body of synapsin-II-transfected NG108-15 cells. Intracellular Ca 21 elevation induced by high K 1 stimulation caused a 2-fold increase in the number of functional synaptic vesicles that exhibited membrane fusion (recycling) and a 3-fold increase in extracellular release of ATP in synapsin-II-transfected cells compared with controls. This suggests that synapsin II has a role in the regulation of synaptic vesicle number. Consistent with the results of the present study, an increase in the number of synaptic vesicles in synapsin-IIbexpressing NG108-15 cells has been reported previously [8,30]. However, the synaptic vesicles generated in these cells were not demonstrated to be functional. In the present study, we observed an increase in several synaptic-vesicleassociated proteins (i.e., synaptotagmin I, synaptotagmin II, and VAMP/ synaptobrevin), consistent with the idea that the newly generated synaptic vesicles were functional. To

Fig. 6. Characteristics of exocytosis at varicosities and cell body synapses (terminal boutons) by FM1-43 in transfected cells. (A) Fluorescent image of syapsin-II-transfected cells loaded with FM1-43 after high K 1 stimulation. Both the varicosity (a, b) and the cell body (c, d) were labeled after stimulation. Bar525 mm. (B) The FM1-43-labeled cells were destained by application of high K 1 . Kinetics of the change in the intensity of the fluorescence of the varicosity (a, b) were similar to one point of the cell body (c, d), but totally diffent from others of the cell body. Bar indicates period of application of high K 1.

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examine this possibility further, we studied synaptic vesicle recycling using FM1-43. The area of FM1-43 labeling in synapsin-II-transfected cells was twice that in control cells and included labeling of varicosities in addition to the cell body. These results suggest that both membrane fusion and the number of recycled synaptic vesicles may be increased in synapsin-II-transfected cells. Finally, there was a significant increase of synaptic vesicle exocytosis in synapsin-II-transfected cells, as measured by the destaining of FM1-43 labeling at the varicosities and the release of ATP after high K 1 stimulation. The experimental accuracy in the measurement of ATP is even greater than that in the measurement of FM1-43, thus it is reasonable to assume that the increase in synaptic vesicles generated by the overexpression of synapsin II underlies the increase in functional synaptic vesicles in these cells (4-fold by ATP measures and 2-fold by FM1-43 measures). These discrepancies between these two measurements may be due to the deficiency of destaining process at the surface of cell body. ATP molecules released into the synaptic cleft between the terminal bouton and the cell body surface can easily diffused into the external solution, while FM1-43 fluorescence molecules cannot diffused completely, because the molecules will be captured in the cell membrane due to its weak hydrophobicity [9]. In synapsin-II-depleted neurons in the hippocampus, a selective decrease in synaptic-vesicle-associated proteins, such as, synapsin I, synaptotagmin, synaptophysin, and syntaxin, has been reported [7]. The authors suggested that suppression of synapsin II disrupts early neuronal development. Rosahl et al. suggested that synapsin I may have dual inhibitory and facilitory functions in accelerating synaptic vesicle traffic, whereas synapsin II may be only facilitory [24]. Recently, it was suggested that synapsin I and II function on synaptic vesicles as ATP donors for the phosphorylation of synaptic-vesicle-associated proteins, and that ATP binding is differentially regulated by calcium [17]. Although the ATP binding ability of synapsin I was shown to be facilitated by Ca 21 in this previous study, synapsin II did not exhibit Ca 21 dependent ATP binding, and the authors suggested that synapsin II may be constitutively active, whereas synapsin I may only be activated upon increases in Ca 21 . These suggestions support our previous findings that the synaptic vesicles generated by the overexpression of synapsin II are already releasable and fuse with the cell membrane soon after transient Ca 21 rise. The results of the present study indicate that synapsin II transfection increased the number of functional synaptic vesicles. It is likely, therefore, that synapsin II regulates the number of mature synaptic vesicles recycled after neurotransmitter release in NG108-15 cells.

Acknowledgements This work was supported in part by a Grant-in-Aid

(0727910) for Scientific Research on Priority Areas on ‘Functional Development of Neural Circuits’ from the Ministry of Education, Science, Sports, and Culture of Japan, and also by Research for the Future Program 96L00310 from the Japan Society for Promotion of Science.

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