Synaptotagmin I Regulates Patterned Spontaneous Activity in the Developing Rat Retina via Calcium Binding to the C2AB Domains

Background In neonatal binocular animals, the developing retina displays patterned spontaneous activity termed retinal waves, which are initiated by a single class of interneurons (starburst amacrine cells, SACs) that release neurotransmitters. Although SACs are shown to regulate wave dynamics, little is known regarding how altering the proteins involved in neurotransmitter release may affect wave dynamics. Synaptotagmin (Syt) family harbors two Ca2+-binding domains (C2A and C2B) which serve as Ca2+ sensors in neurotransmitter release. However, it remains unclear whether SACs express any specific Syt isoform mediating retinal waves. Moreover, it is unknown how Ca2+ binding to C2A and C2B of Syt affects wave dynamics. Here, we investigated the expression of Syt I in the neonatal rat retina and examined the roles of C2A and C2B in regulating wave dynamics. Methodology/Principal Findings Immunostaining and confocal microscopy showed that Syt I was expressed in neonatal rat SACs and cholinergic synapses, consistent with its potential role as a Ca2+ sensor mediating retinal waves. By combining a horizontal electroporation strategy with the SAC-specific promoter, we specifically expressed Syt I mutants with weakened Ca2+-binding ability in C2A or C2B in SACs. Subsequent live Ca2+ imaging was used to monitor the effects of these molecular perturbations on wave-associated spontaneous Ca2+ transients. We found that targeted expression of Syt I C2A or C2B mutants in SACs significantly reduced the frequency, duration, and amplitude of wave-associated Ca2+ transients, suggesting that both C2 domains regulate wave temporal properties. In contrast, these C2 mutants had relatively minor effects on pairwise correlations over distance for wave-associated Ca2+ transients. Conclusions/Significance Through Ca2+ binding to C2A or C2B, the Ca2+ sensor Syt I in SACs may regulate patterned spontaneous activity to shape network activity during development. Hence, modulating the releasing machinery in presynaptic neurons (SACs) alters wave dynamics.


Introduction
Binocular visual circuit refinement requires a robust patterned spontaneous activity in the developing retina (i.e. retinal waves). Retinal waves are spontaneous, correlated action potentials propagating through the retinal ganglion cell (RGC) layer during a developmental critical period prior to visual experience. In particular, retinal waves are mediated via synaptic transmission with a periodicity on the order of minutes. In the developing mammalian retina, three different stages of retinal waves have been classified, of which the stage-II waves are the best studied [1,2,3]. The stage-II waves during postnatal days (P0-P9) are initiated by a subset of starburst amacrine cells (SACs) releasing neurotransmitters acetylcholine (ACh) and c-aminobutyric acid (GABA) onto neighboring SACs and RGCs, allowing depolarizations to propagate across the RGC layer [4,5,6,7]. Correlated depolarizations further induce Ca 2+ influx. Accordingly, the intracellular Ca 2+ concentrations exhibit spontaneous and correlated increases, producing wave-like Ca 2+ oscillations across the RGC layer [8,9,10]. Imaging spontaneous and correlated Ca 2+ transients in RGCs has been widely used to elucidate the synaptic mechanisms underlying retinal waves [2,3].
Previous studies revealed that preferential Ca 2+ binding to distinct C2 domains in Syt controls the choice of exocytotic modes [18,19], regulates the kinetics of exocytosis, and modulates the dynamics of exocytotic fusion pores [20,21]. In particular, weakened Ca 2+ binding to C2A reduces full fusion by limiting fusion pore dilation and preventing release of some vesicle content. By contrast, weakened Ca 2+ binding to C2B abolishes both full fusion and kiss-and-run [18,19,21]. Detailed kinetic analysis has resolved two separate sequential steps in regulated exocytosis, fusion pore opening and dilation, which are differentially controlled by Ca 2+ binding to C2A or C2B in Syt I [21,22]. As a result, Ca 2+ binding to various C2 domains of Syt confers a repertoire of functions in controlling neurotransmitter release and synaptic transmission [17,18,21,23,24,25,26,27]. However, the impact of these molecular signaling processes on large-scale, repetitive synaptic circuit activity has not been investigated.
Many studies have characterized the role of SACs in regulating wave dynamics during the stage-II period. However, the release mechanism in SACs has not been well understood. Immunolabeling and confocal microscopy suggested that several SNARE proteins are expressed in SACs, such as syntaxin 1 and 2 [28], synaptobrevin 2 [29] and SNAP-25 [30]. However, their functional roles in SACs have not been characterized. Classic physiological experiments showed that perinatal rabbit SACs demonstrate oscillations of Ca 2+ spikes and Ca 2+ -dependent afterhypolarizations [6], suggesting release from SACs is Ca 2+dependent. Borges et al. reported that a small number of primed vesicles, very likely one per release site, can be quickly exocytosed Figure 1. Syt I is strongly expressed in neonatal SACs and IPL. Ai-Aiii. Merged images of Syt I (green) and ChAT (red) staining in retinal crosssections from P0-P2 rats. The colocalization of Syt I and ChAT labeling was found in the IPL (yellow). Scale bars, 25 mm. ChAT, choline acetyltransferase. NBL, neuroblast layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Bi-Biii. Immunofluorescence labeling of Syt I (green) in the IPL of retinal cross-sections from P0-P2 rats. Ci-Ciii. Immunofluorescence staining of ChAT (red) labeling SACs and the IPL in retinal cross-sections from P0-P2 rats. Di-Diii. Merged images of Syt I and ChAT staining in the same retinal cross-sections. The Syt I and ChAT labeling was colocalized to the SAC somata (yellow) as indicated by arrows. Scale bars for B-D, 50 mm. Ei-Eiii. The high magnification for merged images of Syt I and ChAT staining in the boxes of Di-Diii. Scale bars for E, 5 mm. doi:10.1371/journal.pone.0047465.g001 upon Ca 2+ influx into SACs [31]. These findings point out the possibility that oscillations of Ca 2+ influx into SACs may cause Ca 2+ binding to its sensor Syt to drive exocytosis during stage-II waves.
Although the human genome encodes more than seventeen Syt isoforms [32], the Syt isoform responsible for the initiation of stage-II waves remains completely unknown. Moreover, it is unclear how the C2AB domains of Syt regulate stage-II waves by controlling specific characteristics of retinal waves. Here we used immunofluorescence, molecular perturbation and live Ca 2+ imaging to investigate the expression of Syt I in SACs and determine the roles of C2AB domains in regulating stage-II waves during the development of neural circuits.

Syt I is Strongly Expressed in Cholinergic Synapses of the Developing Retina
Syt I is the major Syt isoform found in many parts of the nervous system [15]. However, the expression of Syt I in the developing SACs, which release neurotransmitters to initiate stage-II waves, remains to be determined. We thus examined if Syt I is expressed in SACs during stage-II waves. In P0-P2 rat retinal cross-sections, double immunolabeling for Syt I and choline acetyltransferase (ChAT, the SAC marker) showed that Syt I was mainly localized to the inner plexiform layer (IPL) surrounding the SACs (Fig. 1). The high-magnification image from the IPL indicated that Syt I was also localized to the somata of SACs ( Fig. 1Ei-iii, yellow), suggesting that Syt I is expressed in the cholinergic synapses originating from SACs. Thus, Syt I is located where it can serve as a Ca 2+ sensor in triggering neurotransmitter release during stage-II waves.

Targeted Molecular Perturbation to SACs
To manipulate Syt I molecules in the developing SACs, we developed a molecular perturbation method for the retinal explant culture. We first detected the effects of culture condition on retinal wave dynamics (Fig. S1). We used Ca 2+ imaging to measure waveassociated Ca 2+ transients in both acutely isolated retinas and retinal explants after 3-4 day in vitro culture. The characteristics of wave-associated Ca 2+ transients were not significantly changed after in vitro culture ( Fig. S1A-D). Moreover, both acutely isolated retinas and retinal explants demonstrated similar pairwise correlations over distance for spontaneous Ca 2+ transients, although acutely isolated retinas demonstrated more correlated waves over longer distances (.400 mm) (Fig. S1E). These data suggest that retinal networks may remain healthy and functional after 3-4 day in vitro culture.
We constructed platinum electrodes to electroporate genes of interest into P0-P2 whole-mount retinas ( Fig. 2A-B). This design achieved gene delivery within two parallel electrodes (Fig. 2C). Western blot analysis verified ectopic expression of HA-Syt I in the developing retina by this electroporation strategy (Fig. 2D). This method achieved homogenous expression across the entire retina ( Fig. 3ACDF) without altering the major characteristics of retinal Ai. The arrangement of the six slides for the (+) electrode base. The colors of slides represent the slide arrangement in different layers. The numbers in the circles indicate the corresponding sequence to arrange slides. Aii. The Pt foil (15615 mm with one extra 565 mmoverhanging square) was aligned to one side of the 7 th slide with the overhanging square left out. Epoxy glue was applied to attach the Pt foil onto the slide and connect all the slides together. Aiii. The wire was soldered to the edge of the overhanging Pt square. Aiv. The last (8 th ) slide was glued onto the (+) electrode base. Bi-ii. The arrangement of the (2) electrode. A pen tube (green) with a diameter of 10 mm was used to attach the same-sized Pt foil by epoxy glue. The electric wire was inserted through the pen tube and soldered onto the Pt foil. C. The setup for electroporation in a horizontal configuration. The retinal explant was placed in a well [with dimensions 12 (length) 612 (width) 63 (height) mm] on the (+) electrode. The (2) electrode made contact with solution above the well and covered the retinal explant. The distance between the (+) and (2) electrodes was adjusted by a micromanipulator that held the (2) electrode. D. The P1 rat retinas were transfected with pCMV-HA (vector) or pCMV-HA-Syt I (HA-Syt I) with this electroporation device. The retinal explants were incubated for 72 hr to allow gene expression. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with antibodies indicated on the right (HA or a-tubulin). Only the retinas transfected with HA-Syt I displayed the HA signal with a size of ,65 kD, which corresponded to the molecular weight of Syt I. Data shown were representative blots from 3 different experiments. doi:10.1371/journal.pone.0047465.g002 waves, such as the frequency, duration, and amplitude of waveassociated Ca 2+ transients (Table 1). In addition, within 4 days after transfection, retinal waves were reliably blocked by the nAChR antagonist, dihydro-b-erythroidine (DHbE) (10-20 mM) [33,34]. These results suggest that transfected retinas, which we cultured in vitro for 3-4 days after transfection, still generate stage-II waves mediated by cholinergic transmission with the same essential properties. Moreover, no significant differences in waveassociated Ca 2+ transients were observed in the retinas transfected either on P0 or P1/2 (Table 1). Therefore, in the subsequent Ca 2+ imaging experiments, we utilized P0-P2 transfected retinas with 3-4 day in vitro culture for studying the mechanisms mediating stage-II waves.
To manipulate neurotransmitter release from SACs during stage-II waves, we aimed to transfect SACs with Syt I mutants with the weakened Ca 2+ binding in the C2 domains. Since the metabotropic glutamate receptor type II (mGluR2) promoter has been shown to target SACs specifically [7,35,36,37], we placed our Syt constructs under the control of the mGluR2 promoter to manipulate Syt I specifically in SACs. To verify the effectiveness of our transfection strategy, we compared the specificity of the mGluR2 promoter with the ubiquitous cytomegalovirus (CMV) promoter by immnunofluorescence (Fig. 3). With the CMV promoter ( Fig. 3A-C), the HA/EGFP immunoreactivity was scarcely colocalized with ChAT (the SAC marker), but mostly appeared in relatively round and large retinal neurons (,20 mm), likely RGCs. In contrast, with the mGluR2 promoter ( Fig. 3D-F), some HA/EGFP immunoreactivity was colocalized with ChAT ( Fig. 3Fii, yellow), suggesting that gene expression was efficiently targeted to the SAC somata. The HA/EGFP immunoreactivity appeared in fibers originating from relatively small cells (,5 mm), similar to SACs (Fig. 3Dii). This pattern is consistent with the expression pattern of endogenous Syt I in the IPL (Fig. 1), justifying that the mGluR2 promoter can target gene expression to SACs and the IPL.
The immunoreactivities from retinal cross-sections were used to evaluate our transfection in SACs. The HA/EGFP immunoreactivity represented all transfected cells (the green cells in Fig. 3GH), whereas ChAT immunoreactivity (in red) indicated SACs. Therefore the transfected SACs would display yellow color (Fig.  3GH). We first determined our transfection efficiency in SACs, which was calculated as the number of cells with yellow signal divided by the number of cells with red signal (DAPI signal indicated nuclei for cell count). The transfection efficiency with the mGluR2 promoter was 57.1464.56% (mean 6 S.E.M., N = 6 sections), compared to 30.2563.45% (mean 6 S.E.M., N = 4 sections) with the CMV promoter.
We then calculated the promoter specificity, which was defined as the number of cells with yellow signal divided by the number of cells with green signal (DAPI signal indicated nuclei for cell count) (Fig. 3GH, right insets). With the mGluR2 promoter, the promoter specificity for SACs was 83.8761.61% (mean 6 S.E.M, N = 9 sections), compared to 8.7961.22% (mean 6 S.E.M.) with the CMV promoter (N = 6 sections). Thus, the mGluR2 promoter achieved a significantly higher SAC specificity compared to the CMV promoter (p,0.001, two-tailed unpaired t-test). This indicates that the mGluR2 promoter can be used to target SACs for molecular perturbation in the developing rat retina.
Ca 2+ Transient Frequency is Reduced by Weakened Ca 2+ Binding to C2A or C2B of Syt I To study the roles of Syt I C2 domains in retinal waves, we used this established electroporation strategy to introduce Syt I mutants with weakened Ca 2+ -binding C2 domains. With the aid of the mGluR2 promoter, transfected retinal explants reliably demonstrated EGFP fluorescence in small cells (,5 mm), compared to relatively large RGCs (,20 mm) stained by the Ca 2+ indicator fura-2 (Fig. 4A). Moreover, the mGluR2 promoter-driven EGFP was localized to the deeper region underneath the RGC layer. The EGFP expression pattern was essentially the same among all transfection groups, suggesting that transfection efficiency was comparable in all groups.
Weakened Ca 2+ binding to the C2A or C2B domain differentially controls the modes of exocytosis [18,21]. In particular, D230S confers a Ca 2+ -binding site mutation in the C2A domain of Syt I (designated Syt I-C2A*), whereas D363N confers a Ca 2+ -binding site mutation in the C2B domain of Syt I (designated Syt I-C2B*). Each mutated residue abolishes the first ligand of the third loop in the Ca 2+ -binding pocket of the respective Syt C2 domain [14]. To determine the effects of these Syt I mutants on the Ca 2+ transient frequency in the developing rat retina, live Ca 2+ imaging was conducted in the retinal explants after transfection. Spontaneous, correlated Ca 2+ transients in individual cells revealed retinal waves in the RGC layer of explants transfected with control vector, wild-type Syt I, Syt I-C2A* or Syt I-C2B* (Fig. 4B). To quantify the effects of different transfections on Ca 2+ transient frequency, we employed two independent measurements, including the change in Ca 2+ transient frequency ( Fig. 4CD) and the fraction of cells displaying Ca 2+ transients (Fig. 4E).
First, to detect the change in Ca 2+ transient frequency, the mean frequency was determined from a number of transfected retinas, thereby cancelling the possible transfection variance across retinas or cells. Wild-type Syt I left the mean frequency of Ca 2+ transients identical to that seen in control retinas (    Wave-Associated Ca 2+ Transients are Dampened by Weakened Ca 2+ Binding to C2A or C2B of Syt I Retinal waves occur with a periodicity on the order of minutes. Spontaneous Ca 2+ transients associated with waves can last for tens of seconds following relatively brief spontaneous depolarizations [3]. To examine if the wave patterns are changed by Syt I mutants, we measured the duration and amplitude of single Ca 2+ transients in an unbiased way (Fig. 5A, see Materials and Methods for details). The representative Ca 2+ transients were shown in Fig. 5B. Both Syt I-C2A* and Syt I-C2B* reduced Ca 2+ transient size compared to wild-type Syt I and control, including a decrease   in Ca 2+ transient duration and amplitude, and this impression was confirmed by quantitative analysis.
To quantify the effects of Syt I mutants on Ca 2+ transient duration, we compared the mean durations from different transfection groups. Both Syt I-C2A* and Syt I-C2B* reduced Ca 2+ transient duration compared to wild-type Syt I and control (Fig. 5C, mean 6 (Fig. 5D), and the differences in these values were consistent with the differences in Ca 2+ transient duration across retinas (Fig. 5C). The curves of cumulative probability for Ca 2+ transient duration were left-shifted in Syt I-C2A* and Syt I-C2B*, suggesting that the majority of Syt I-C2A* and Syt I-C2B* cells had briefer Ca 2+ transients compared to control and wild-type Syt I (Fig. 5D). Thus, weakened Ca 2+ binding to either the C2A or C2B domain may reduce the duration of wave-associated Ca 2+ transients.
To quantify the effects of Syt I mutants on Ca 2+ transient amplitude, we compared the mean amplitude from different transfection groups. Similar to the effects on Ca 2+ transient duration, wild-type Syt I did not change the Ca 2+ transient amplitude compared to control (p.0.05). Both Syt I-C2A* and Syt I-C2B* significantly reduced Ca 2+ transient amplitude compared to wild-type Syt I and control (Fig. 5E, (Fig. 5F), similar to the results of the mean Ca 2+ transient amplitude (Fig. 5E). The curves of cumulative probability for Ca 2+ transient amplitude were also left-shifted in Syt I-C2A* and Syt I-C2B*, suggesting that the majority of Syt I-C2A* and Syt I-C2B* cells had relatively small amplitude of Ca 2+ transients compared to control and wild-type Syt I (Fig. 5F). Together, these results show that weakened Ca 2+ binding to either the C2A or C2B domain in Syt I dampened the wave-associated Ca 2+ transients.

Spatial Correlation of Retinal Waves is not Altered by Weakened Ca 2+ Binding to C2AB of Syt I
Both Syt I-C2A* and Syt I-C2B* dampened wave-associated Ca 2+ transients, but it is unknown if these mutants also affect the spatial properties of stage-II waves (i.e. the correlated propagation over distance). To assess wave correlation structure across cells, we computed cross-correlograms over cell pairs [38,39] within imaged regions (3406460 mm). The representative cross-correlograms for control, Syt I, Syt I-C2A* and Syt I-C2B* were shown in Fig. 6A. The sharp peaks at t = 0 sec in all the correlograms for cell pairs ( Fig. 6A; Data summarized in Table 2) indicated that the waves were well correlated in the imaged region. Syt I-C2B* had more oscillatory behavior outside of t = 0 sec compared to all other groups. We thus suspected that waves in distant cell pairs lose their synchrony in the Syt I-C2B* transfected retina.
To further quantify the strength of the correlations, the correlation index (C.I.) [38,40] was computed and plotted as a function of intercellular distance (Fig. 6B). In all groups, the C.I. values were highest for nearby cells, and then decreased as a function of distance, consistent with the persistence of propagating waves. Although we suspected that Syt I-C2B* conferred the highest value of maximal C.I. and these C.I. values dropped off more quickly (Fig. 6B), the C.I. values at any given distance were not significantly different among all groups (P.0.05). Thus, weakened Ca 2+ binding to the C2AB domain of Syt I may not alter the spatial correlation structure of stage-II waves.

Discussion
In this study, we showed that Syt I is strongly expressed in cholinergic neurons (SACs) and synapses (the IPL) in retinas exhibiting stage-II waves. We further established a molecular perturbation method targeted to presynaptic SACs. By weakening Ca 2+ binding in the C2AB domains of Syt I, we tested the effects of presynaptic perturbation on wave-associated Ca 2+ transients. Our Ca 2+ imaging results provide the first demonstration that Ca 2+ binding to C2A or C2B of Syt I regulates the temporal properties of stage-II waves. In contrast, Ca 2+ binding to C2AB in Syt I has relatively minor effects on the spatial properties of stage-II retinal waves. These results suggest that the C2AB domains of Syt I regulate the dynamics of patterned spontaneous activity during the development of neural circuits.

Presynaptic Syt I in Regulating Patterned Spontaneous Activity
While the importance of Syt I in synaptic transmission has been recognized for a couple of years [15], its significance in regulating large-scale, repetitive synaptic circuit activity has yet been reported. In our study, we found that Syt I regulates the dynamics of wave-associated Ca 2+ transients via Ca 2+ binding to its C2AB domains. Together with the previous findings [5,31], our present study suggest that the Ca 2+ oscillations in SACs [5] may cause Ca 2+ binding to its sensor Syt I to trigger exocytosis [31] during stage-II waves. Hence, modulating fusion machinery in SACs alone may regulate wave dynamics.
Developing SACs corelease ACh and GABA in a Ca 2+dependent manner [5]. Both ACh and GABA are stored in synaptic vesicles, thereby sharing the common releasing mechanism. Note that GABA also exerts the excitatory effects during stage-II waves in rats [37]. Thus, advanced work is required to identify whether the effects we observed in this study may be due to the changes in ACh or GABA release, or both. Moreover, although it is a challenging work to directly measure exocytosis in SACs, how release from SACs is influenced by Ca 2+ binding to Syt I warrants further investigation, especially in the form of kiss-andrun or full fusion.
Manipulations that alter the spatiotemporal properties of stage-II waves result in defective retinogeniculate and retinocollicular projections [40,41,42,43,44]. Although the significance of retinal waves has gained recognition, previous studies of retinal wave generation focused only on ''postsynaptic'' loci, such as the nAChR during stage-II waves [45,46,47]. Our study first demonstrates that alterations in ''presynaptic'' fusion machinery can also regulate stage-II waves, thereby providing alternative targets for the manipulation of retinal waves. The previous studies have identified that stage-II waves are present from P0 until P9 in rats [33,37]. Our preliminary results showed that Syt I began to be expressed in the outer plexiform layer after P9 (unpublished data), implying that Syt I may also involve in regulating stage-III waves in rats. More work for the effects of various exocytotic molecules (including SNARE proteins and Syt isoforms) on the spatiotem-poral pattern of retinal waves will thus enhance our ability to control developmental transitions in the visual system.

The Roles of Syt I C2AB Domains in Shaping Patterned Spontaneous Activity
The roles of the C2AB domains in Syt I have been intensively investigated at the molecular and functional levels. Biochemical analysis showed that both Syt I-C2A* and Syt I-C2B* greatly reduce their binding to t-SNARE heterodimers (corresponding to target proteins on plasma membrane) and PS-containing liposomes (corresponding to vesicle membrane constituent) [15,19,21]. However, only Syt I-C2B* reduces binding to PIP2-containing liposomes (corresponding to vesicle and plasma membrane constituent) [15,19,21,48] and homo-oligomerization with other Syt I [49]. Moreover, studies of fusion pore kinetics revealed that Syt I-C2A* switches the exocytosis mode from full fusion to small kiss-and-run with no dilation step [18]. The long duration of these small kiss-and-run events has been proposed to make neurotransmitter release very slow, possibly elevating ambient extracellular neurotransmitter concentrations [18]. In contrast, Syt I-C2B* abolishes both exocytosis modes, thereby leaving brief kiss-and-run events which can hardly release neurotransmitter to support  Table 2 for statistics). The oscillatory behavior of the correlograms beyond | t | . synaptic responses [21]. All these molecular and functional findings suggest that the C2AB domains exert significant effects on exocytosis by engaging distinct effector interactions. Thus, our findings may be directly linked to the regulation of fusion machinery in SAC release.
Alternative interpretation for the effects of Syt I mutations on wave dynamics is that overexpression of Syt I-C2A* and Syt I-C2B* might alter cytoplasmic Ca 2+ dynamics in SACs and thus reduce exocytosis. However, several in vitro and in vivo studies have demonstrated that overexpression of Syt I with Ca 2+ -binding site mutations can not alter whole-cell Ca 2+ current or Ca 2+ dynamics [15,19,20,23,24], implying that the effects of presynaptic perturbations in the present study cannot be solely attributed to the changes in Ca 2+ channels or dynamics in SACs.
The spatial properties of stage-II waves arise from a single SAC that innervates multiple RGCs and other SACs [7], resulting in correlated Ca 2+ transients in nearby RGCs. We observed that Syt I-C2A* and Syt I-C2B* dampen the temporal properties of stage-II waves (Fig. 4-5), while these two mutants cannot alter the spatial properties of stage-II waves (Fig. 6). One explanation for this observation is that in transfected retinas, a few SAC terminals may still retain the capacity for ACh release contributed from endogenous wild-type Syt I to support the propagation of stage-II waves. Thus, the C2AB domain of Syt I cannot significantly influence the spatial correlation structure of retinal waves. Alternatively, the C2B domain of Syt I may slightly affect pairwise correlations, which is beyond the limitation of instrumentation.

Future Directions
A previous study revealed that Ca 2+ influx into RGCs during long-duration waves can induce a significant increase in cAMP/ PKA activity [33]. Thus, the RGCs with long-duration waves may lead to activity-dependent gene expression via the cAMP/PKA/ CREB pathway, which has been shown to be essential for visual circuit development [50]. In our study, both Syt I-C2A* and Syt I-C2B* significantly reduce Ca 2+ transient duration compared to Syt I (Fig. 5), suggesting that downstream gene activation in neighboring RGCs may be reduced by presynaptic Syt I-C2A* and Syt I-C2B*. How gene expression patterns in postsynaptic RGCs are impacted by expressing presynaptic Syt I, Syt I-C2A* and Syt I-C2B* is an important topic for future investigation.
Retinal waves propagate through the developing retina, inducing similar burst patterns in the thalamus and visual cortex to form sensory loops [3]. Although similar burst patterns occur in the synapses of these sensory loops, the identities and roles of the Syt isoforms in each synapse have yet to be determined. In particular, the etiology of schizophrenia and autism has been attributed to a developmental disorder of ''neural connectivity'' in the formation of sensory maps, especially in the thalamus [51,52,53,54]. Therefore, it is important to identify and characterize the roles of exocytotic molecules in developing neural circuits. This will provide new insight and contribute to the development of novel therapeutic methods for human psychiatric diseases.

Molecular Biology
The metabotropic glutamate receptor subtype II (mGluR2) promoter was used for SAC-specific expression [35,36,37]. First, the cDNAs encoding Syt I were subcloned into the pCMV-IRES2EGFP (Clontech #6029-1). The mutants Syt I-C2A* (D230S) and Syt I-C2B* (D363N) were prepared by the modified sequential PCR procedure [55] with the pCMV-IRES2EGFP encoding wild-type Syt I as template [18]. Second, the CMV promoter was replaced with the mGluR2 promoter. The resultant vector pmGluR2-IRES2EGFP allowed gene expression targeted to SACs under the control of the mGluR2 promoter. Enhanced green fluorescent protein (EGFP) was translated from the same strand of coding mRNA driven by the internal ribosome entry site (IRES), so the EGFP fluorescence was used to identify transfected SACs for Ca 2+ imaging (Fig. 4A). For Western analysis (Fig. 2D), the pCMV-HA-Syt I was prepared by subcloning the Syt I cDNA into pCMV-HA vector (Clontech #631604). For immunofluorescence (Fig. 3), the pCMV-HA-Syt I-IRES2EGFP and pmGluR2-HA-Syt I-IRES2EGFP were prepared by subcloning the IRE-S2EGFP fragment into pCMV-HA-Syt I and pmGluR2-HA-Syt I, respectively. The pmGluR2-HA-Syt I was previously acquired from pCMV-HA-Syt I by replacing the CMV with the mGluR2 promoter. All constructs were confirmed by sequencing. The DNA plasmids were amplified (Qiagen) and dissolved in Tris buffer (10 mM, pH 7.6) for transfection.

Ethics Statement and Animals
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol for all animal experiments was approved by the Institutional Animal Care and Use Committee of National Taiwan University (Permit Number: 97-27 and 99-82). Neonatal (P0-P2) Sprague-Dawley rats (BioLASCO, Taiwan) were used in the study. All rat pups were housed with their own mothers in the individually ventilated cages with well-controlled conditions (12:12 light/dark cycle with light on at 7 AM; 2061uC) and ad libitum access to food and water. Rat pups (P0-P2) were deeply anesthetized with ice before decapitation with all efforts to minimize suffering.  Retinal waves were measured from cultured whole-mount retinas expressing Syt I or its mutants (Syt I-C2A* and Syt I-C2B*) with the mGluR2 promoter. Control was transfected with pmGluR2-IRES2EGFP. Wave correlograms were constructed from 49 cell pairs in one imaged region and fitted to a Gaussian equation to yield the Gaussian mean and standard deviation (SD #I1882), 100 mg/mL Penicillin/100 mnits/mL Streptomycin (GIBCO #15140-122), and 6 mM Forskolin [33].

Transient Transfection
DNA plasmids (200 ng/mL in dissection buffer) encoding the proteins of interest were transiently transfected into cultured retinal explants. About 400 mL of DNA-containing buffer was used for transfection of each retinal explant (80 mg DNA per transfection). Before electroporation, the retinal explants were incubated in the DNA-containing buffer at room temperature (RT) for 10 min to enhance transfection efficiency. The homemade platinum electrodes with a horizontal configuration were connected to the electroporator (27 V; 50 ms of pulse duration; 2 pulses at 1 sec-interval; BTX ECM830 electroporator, Harvard Apparatus). The horizontal distance of the electrode gap was 4 mm to yield an electric strength of 6.75 V/mm. One retinal explant with the RGC layer facing up (toward the (2) electrode in Fig. 2C) was placed within the electrode gap filled with the DNAcontaining buffer. The electric pulses were applied with the parameters as mentioned above. Right after electroporation, the retinal explants were immediately placed in the cultures and maintained for 60-96 hr (3-4 days in vitro, DIV 3-4).
For retinal cross-section staining, transfected retinal explants were fixed with 4% PFA at 4uC overnight. After fixation, the explants were cryoprotected in 30% sucrose at 4uC for 1 day and preserved in Tissue-Tek optical cutting temperature (O.C.T.) gel (Sakura Finetech #4583). Retinal cross-sections (16 mm) were prepared with a cryostat (Leica CM1850), placed on poly-lysinecoated slides, washed with PBS, and blocked at RT for 1 hr in 3% DBS. The retinal sections were incubated with primary antibodies at 4uC overnight, washed with PBS, further incubated with secondary antibodies at RT for 1 hr, and washed with PBS again. The samples were finally stained with DAPI (Sigma #D9542) at RT for 10 min. The slides and immunofluorescence images were acquired as mentioned above. Quantification of colocalization was determined with MetaMorph software (Version 7.5, Molecular Devices).

Western Analysis
The P0-P2 rat retinal explants transfected with pCMV-HA or pCMV-HA-Syt I were spun down in dissection buffer at ,2,000 g at 4uC for 5 min, and stored at 280uC. The transfected retinal explants were lyzed with a homogenizer (Branson Sonifier S-250D) in the ice-cold RIPA lysis buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 1 mM sodium fluoride, and 50 mM HEPES, pH 7.4, supplemented with protease inhibitors). After sonication, samples were centrifuged at 18,000 g at 4uC for 10 min and protein concentration was determined by Bradford assay (Bio-Rad #500-0006). Protein in the cellular lysate (20 mg) was electrophoresed through standard 10% Laemmli SDS polyacrylamide gels, transferred to polyvinylidene fluoride membranes, and probed with appropriate antibodies. Membranes were blocked for 1 hr in 5% non-fat milk in TBST (100 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween-20) and then incubated with primary antibodies [mouse anti-HA (Covance #MMS-101P) and mouse anti-a-tubulin (Sigma #T5168)] at 4uC overnight. Membranes were washed three times with TBST and then incubated with secondary antibody in 5% milk in TBST at RT for 1 hr. Membranes were washed three times with TBST, and the signals were visualized by enhanced chemiluminescence followed by autoradiography.

Data Analysis
The Ca 2+ imaging data for each transfection group were acquired by the following procedures. First, 49 RGCs were randomly selected from the 767 divisions in one imaged region (3406460 mm) using MetaMorph. The wave data from single RGCs were acquired from the fluorescence changes across all the time frames, previously background subtracted for each frame. The fluorescence signals were further corrected for photobleaching by using Igor Pro to set the baseline. Second, to analyze wave characteristics (frequency/duration/amplitude) in an unbiased way, an Igor procedure was written in this laboratory to automatically pick the wave peaks, with their fluorescence intensity 2-fold greater than the RMS noise (,0.25% DF/F). The definitions of the duration and amplitude for individual waves are presented in Fig. 5A. Third, all wave data were averaged from each cell, averaged across 49 cells out of one imaged region, and then averaged from two imaged regions out of one retina. Finally, the mean wave data for each group were averaged from all retinas transfected with the same gene. Distributions of cumulative probability were constructed from single-cell wave data for the same transfection group.
Correlation of Ca 2+ waves between nearby cells was calculated from pairs of RGCs in the same imaged region separated by various distances (10-500 mm). The positions of individual cells were calculated using MetaMorph. The intercellular distance was taken as the shortest distance between selected cells. The lag times of the waves in these two nearby cells were acquired by individually subtracting from the presenting time of every wave in the paired cells. All the subtracted times were collected for each cell pair to construct cross-correlograms from one imaged region (Fig. 6A) [39]. In addition, the lag times of the waves in these two nearby cells were used to calculate the correlation index (C.I.) (Fig. 6B) [38,40]. The C.I. is a measure of the likelihood relative to chance of a pair of neurons firing together within a given interval (Dt = 3 sec), as given by the following equation: N AB is the wave number for which cell B fires with a time 6 Dt from cell A, N A and N B are the total numbers of waves fired by cells A and B, respectively, during the total recording time (both N A and N B $ 3 in 10 min), T is total recording time (600 sec), and Dt is the correlation time interval (3 sec). The cell pairs from one imaged region were grouped according to their intercellular distances with a bin width of 50 mm. The median C.I. values were computed from each distance group out of the same imaged region. The averaged C.I. values were computed from the same distance group out of many imaged regions for each transfection group, and then plotted against the upper limit of intercellular distance for each distance group (Fig. 6B).

Statistics
All data were presented as mean 6 S.E.M. Differences between means of different groups ($3 groups) were evaluated for statistical significance with One-way ANOVA followed by post-hoc Student-Newman-Keuls test for the parametric method, or Kruskal-Wallis method followed by post-hoc Dunn test for the nonparametric method (InStat 3, GraphPad). Figure S1 Comparison of wave-associated Ca 2+ transients in acutely isolated retinas (Acute) and retinal explants after 3-4 day in vitro culture (Culture). I. values as a function of intercellular distance. The methods to measure the properties of Ca 2+ transients were described in Fig. 4, 5, 6. Data were from 8 acutely isolated retinas (P2) and 7 retinal explants (dissected on P0-2 with DIV 3-4). (*p,0.05, unpaired ttest for comparison at a given distance; n.s., not significantly different from acutely isolated retinas) (TIF)