Calcineurin Aγ is a Functional Phosphatase That Modulates Synaptic Vesicle Endocytosis*

Variation in PPP3CC, the gene that encodes the γ isoform of the calcineurin catalytic subunit, has been reported to be associated with schizophrenia. Because of its low expression level in most tissues, there has been little research devoted to the specific function of the calcineurin Aγ (CNAγ) versus the calcineurin Aα (CNAα) and calcineurin Aβ (CNAβ) catalytic isoforms. Consequently, we have a limited understanding of the role of altered CNAγ function in psychiatric disease. In this study, we demonstrate that CNAγ is present in the rodent and human brain and dephosphorylates a presynaptic substrate of calcineurin. Through a combination of immunocytochemistry and immuno-EM, we further show that CNAγ is localized to presynaptic terminals in hippocampal neurons. Critically, we demonstrate that RNAi-mediated knockdown of CNAγ leads to a disruption of synaptic vesicle cycling in cultured rat hippocampal neurons. These data indicate that CNAγ regulates a critical aspect of synaptic vesicle cycling and suggest that variation in PPP3CC may contribute to psychiatric disease by altering presynaptic function.

Calcineurin is a Ca 2ϩ /calmodulin-activated phosphatase involved in a variety of critical neuronal processes (1). It functions as a heterodimer with a 61-kDa catalytic and 19-kDa regulatory subunit. There are three isoforms of the catalytic subunit, CNA␣, 3 ␤, and ␥, encoded by PPP3CA, PPP3CB, and PPP3CC, respectively (2). CNA␣ and ␤ are known to be abundant in the brain and to modulate neuronal processes (3)(4)(5)(6). In contrast, CNA␥ expression has been thought to be restricted to the testis (7). Recent evidence suggests that PPP3CC mRNA is indeed present in the human brain (8,9) and that CNA␥ forms a functional phosphatase (10). However, there is no reported evidence that the CNA␥ protein is expressed in the brain, and specific functions of CNA␥ have not been identified.
Several lines of evidence implicate calcineurin dysfunction in the etiology of schizophrenia. A mouse with forebrain-specific knockout of the calcineurin regulatory subunit, calcineurin B1 (CNB1), has a behavioral phenotype that recapitulates several of the endophenotypes characteristic of schizophrenia, including a selective disruption of working memory (11,12). Moreover, genetic and potential epigenetic variation in the PPP3CC gene has been reported to be associated with schizophrenia or its cognitive impairments in specific population-based or genome-wide methylation studies, respectively (9,(13)(14)(15)(16)(17)(18), although genetic association has not been reported from largescale, controlled genome-wide association studies performed to date. Finally, a reduction in PPP3CC mRNA levels was observed in the hippocampus of schizophrenia patients (8). Because the function of CNA␥ in the brain has not been examined, the potential role of the PPP3CC gene in schizophrenia has remained unclear.
In this study, we show that CNA␥ is expressed in adult rodent and human brains, dephosphorylates known presynaptic calcineurin substrates, and is present in presynaptic terminals in the hippocampus. We further show that RNAi-mediated knockdown of CNA␥ results in a slowing of synaptic vesicle endocytosis. These data suggest that PPP3CC risk alleles may contribute to schizophrenia pathogenesis by altering synaptic vesicle cycling, adding to evidence implicating presynaptic dysfunction in central nervous system disorders (19,20).
For antiserum specificity analysis, human and mouse CNA␣, CNA␤, and CNA␥ cDNAs were subcloned into the pcDNA-DEST53 mammalian expression vector (Invitrogen), which includes an N-terminal cycle 3 GFP tag. The rat CNA␥ construct was acquired in the pExpress-1 mammalian expression vector (Open Biosystems) without an epitope tag. Recombinant CNA proteins were generated by transfecting 293T cells in 6-well plates with the expression constructs using Lipofectamine 2000 (Invitrogen). Cells were lysed in radioimmune precipitation assay buffer (Sigma) and centrifuged at 15,000 ϫ g for 15 min. Supernatants were harvested, and protein concentrations were determined with a bicinchoninic acid kit (Pierce). Protein (30 g) was solubilized in NuPAGE running buffer and loaded onto 4 -12% NuPAGE gels (Invitrogen). Proteins were transferred to nitrocellulose membranes, blocked in 10% milk, and incubated in custom rabbit anti-mouse CNA␣, CNA␤, and CNA␥ antisera (1:5000) overnight and in secondary antibodies for 1 h in 5% milk/Tris-buffered saline plus 0.1% Triton X-100. The secondary antibody was HRP-conjugated anti-rabbit IgG (1:10,000, Santa Cruz Biotechnology). Blots were developed with chemiluminescence (PerkinElmer Life Sciences) and imaged on a Biochemi (UVP).
For tissue Western blot analyses, tissue protein samples from adult male BALB/c mice were obtained from a commercial supplier (Biochain) and solubilized in NuPAGE running buffer. Western blot analyses were performed using custom rabbit anti-mouse CNA␣, CNA␤, and CNA␥ antisera (1:5000) as described above. For brain tissue Western blot analysis of 2-week-old or younger animals, brain tissue from three Swiss-Webster mice of either gender was harvested. For samples from mice older than 2 weeks, brain tissue from three male Swiss-Webster mice was harvested. Western blotting was performed as described above.
Immunoprecipitation-Human cerebral cortex samples (Asterand) were homogenized in radioimmune precipitation assay buffer plus protease inhibitors (Roche) and centrifuged for 15 min at 15,000 ϫ g. Supernatants were incubated with affinity-purified antibody (5 g) overnight at 4°C with rocking. Protein A/G-agarose beads (20 l, Thermo Scientific) were added, and samples were rocked for 1 h. Beads were pelleted and washed in radioimmune precipitation assay buffer, and protein was solubilized with 20 l of NuPAGE sample buffer.
Co-transfection Assays-C terminus GFP-tagged constitutively active forms of human CNA␣, ␤, and ␥ were produced using PCR to generate sequences encoding the catalytic and CNB1 binding domains in pcDNA-DEST53 (Invitrogen). 293T cells in 6-well plates were co-transfected with individual constitutively active calcineurin constructs and a dynamin I cDNA using Lipofectamine 2000 (Invitrogen). After overnight incubation, protein was extracted from transfected cells using radioimmune precipitation assay buffer, and Western blotting was performed. Additional primary antibodies were anti-GAPDH Immunocytochemistry-Postnatal hippocampal cultures were generated as described previously (21). Neurons were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100, blocked with 5% BSA, and subsequently incubated with primary antibodies, purified rabbit anti-rat CNA␥ (1 g/ml) and mouse anti-vesicular glutamate transporter 1 (vGlut1, 1:1000, Synaptic Systems), overnight at 4°C. After washing to remove primary antibodies, Alexa Fluor 488 antimouse-or Alexa Fluor 546 anti-rabbit-conjugated secondary antibodies were applied (1:1000). Images were acquired using a ϫ63 (numerical aperture 1.34) objective lens on a Leica epifluorescence microscope with a CoolSNAP HQ charge-coupled device camera driven by Metamorph. To quantify presynaptic CNA␥ distribution, vGlut1-positive puncta were selected as a presynaptic marker, and the intensities of both vGlut1 and CNA␥ were quantified using ImageJ.
For post-embedding immuno-EM, sections were washed in phosphate buffer overnight and placed into sucrose solutions with increasing concentrations (0.5, 1, and 2 M sucrose for 0.5, 1, and 2 h, respectively). Sections were slammed onto copper blocks, cooled in liquid nitrogen, and, following low-temperature dehydration and freeze substitution, embedded in Lowicryl HM 20 resin (Chemische Werke Lowi). Post-embedding immunocytochemistry was performed on 60-nm serial sections on pioloform-coated nickel grids. Sections were incubated on drops of blocking solution for 1 h (20% BSA), followed by incubation on drops of anti-CNA␥ antisera overnight at room temperature. The primary and secondary antibodies (15 nm gold-conjugated anti-rabbit IgG, 1:20, Amersham Biosciences) were diluted in TBS plus 0.03% Triton X-100 and 2% human serum albumin (Sigma). Following washes in TBS, the sections were washed in water and contrasted with saturated aqueous uranyl acetate and lead citrate.
Presynaptic Functional Assays-Oligonucleotides of the rat Ppp3cc cDNA target sequence (CGGCTAACTTTGA-AGGAAGTA, Invitrogen) were annealed and ligated into pSU-PER. Constructs were transfected for 8 days, and experiments were performed 14 -21 days after plating. To quantify the efficiency of the knockdown, immunocytochemistry was performed on neurons co-transfected with the Ppp3cc shRNA vector and vGlut1-pHluorin (22) using GFP (Invitrogen) and CNA␥ antibodies and Alexa Fluor 488-or Alexa Fluor 546conjugated secondary antibodies. Cells were imaged using an epifluorescence microscope with an electron multiplying charge-coupled device camera. Fluorescence levels of CNA␥ were measured and corrected for background in surrounding regions at cell bodies in transfected knockdown neurons and in non-transfected control neurons, as described previously for related proteins (23). Transfected neurons were identified by vGlut1-pHluorin expression, which marks cell bodies when cells are fixed and permeabilized. For the shRNA-resistant construct, the rat Ppp3cc cDNA in the pExpress-1 expression vector was acquired (Open Biosystems). The shRNA target sequence was mutated using QuikChange site-directed mutagenesis (Stratagene). The sequence for the shRNA target sequence was changed to CGGCTTACGTTAAAAGAAGTA, leaving the amino acid sequence unchanged. For the inactive form of CNA␥, histidine 147 in the catalytic region of rat CNA␥ was mutated to alanine as described previously (24) using QuikChange site-directed mutagenesis (Stratagene). Rat CNA␣ and CNA␤ in the pcDNA3 vector were utilized for rescue experiments in CNA␥ shRNA-transfected neurons.
The kinetics of synaptic vesicle reacidification were measured, with minor modifications, as described previously (25). Neurons transfected with vGlut1-pHluorin with or without CNA␥ shRNA were stimulated at 50 Hz for 4 s. Surface vGlut1-pHluorin was acutely quenched by puffing MES-based Tyrode's buffer (pH 5.5), which permits the isolation of fluorescence from recently endocytosed synaptic vesicles. The decay of fluorescence was measured during this period, marking the reacidification of recently internalized vesicles.
Statistical Analyses-Data presented are mean Ϯ S.E. Mann-Whitney U test or Student's t test was used for comparisons performed in GraphPad Prism 4.03, with p Ͻ 0.05 considered statistically significant.

CNA␥ Protein Is Expressed in the Brain and Forms a Func-
tional Phosphatase-To examine the expression and localization of CNA␥ relative to ␣ and ␤, it was critical to develop antibodies specific to the individual isoforms. The C-terminal region of the proteins was chosen as the target for antibody generation because of its high level of amino acid sequence divergence (see "Experimental Procedures"). Custom rabbit polyclonal antibodies were developed against each isoform using peptides within this region as immunogens. The minimal sequence overlap between the corresponding peptides in the three calcineurin isoforms resulted in the absence of cross-reactivity of the antibodies (Fig. 1A).
Western blot analysis of mouse hippocampal protein extracts using these custom antisera demonstrated that CNA␥, as well as CNA␣ and CNA␤, are expressed in the brain (Fig. 1B). Tissue expression analysis demonstrated that, consistent with previous reports (3), CNA␣ and ␤ proteins are enriched in the mouse brain. In addition, we found that, although CNA␥ is expressed at the highest level in the testis, it is enriched in the brain relative to all other examined tissues (Fig. 1C). We confirmed the expression of CNA␥ in the human neocortex by performing immunoprecipitation followed by Western blot analysis using purified custom anti-CNA␥ antibodies (Fig. 1D).
A previous report on the basis of in situ hybridization analysis indicated that expression of Ppp3cc mRNA is regulated developmentally in specific rodent brain regions, with the highest expression during embryonic development, whereas expression of Ppp3ca and Ppp3cb peaks postnatally (26). To determine whether a similar regulation is observed at the protein level, we examined the developmental expression of the CNA␥ protein in the mouse neocortex. We found that the protein levels for all three CNA isoforms increased postnatally, reaching a peak around 2 weeks of age and remaining higher than embryonic levels well into adulthood (Fig. 1D).
Despite being divergent from CNA␣ and ␤ (2), CNA␥ forms a functional phosphatase (10). However, it is not known whether it dephosphorylates calcineurin substrates involved in neuronal function. To address this question, we examined whether CNA␥ can dephosphorylate the calcineurin substrate dynamin I, a protein critical for presynaptic function (27). We first generated expression constructs for constitutively active forms of the human CNA isoforms lacking the autoinhibitory domain and for dynamin I. We used these constructs to co-express each isoform with dynamin I in 293T cells and analyzed the level of phosphorylation of the dynamin I residue Ser-778, a known calcineurin substrate (28). Expression of all three isoforms of CNA, including CNA␥, resulted in a decreased level of phosphorylation of dynamin I Ser-778 (Fig. 1E), indicating that CNA␥ forms a functional phosphatase capable of modifying known synaptic calcineurin substrates.
CNA␥ Is Present in Presynaptic Terminals-Calcineurin is known to modulate neuronal processes occurring in multiple subcellular compartments (1). To investigate the neuronal functional role of CNA␥, we first analyzed whether it is localized to a specific neuronal subcellular compartment. Fluorescence immunocytochemistry analysis of CNA␥ performed on cultured rat hippocampal neurons revealed a punctate staining pattern colocalized with vGlut1, a marker of excitatory presynaptic terminals ( Fig. 2A). Densitometry measurements performed for CNA␥ and vGlut1 on 504 vGlut1 puncta showed that most excitatory synapses within hippocampal cultures contain measurable levels of CNA␥ and that the level of CNA␥ signal correlates with vGlut1 levels (Fig. 2B, R 2 ϭ 0.38, p Ͻ 0.0001). These data indicate that CNA␥ is localized to synaptic contacts.
We next performed immuno-EM analysis to further resolve the subcellular localization of CNA␥. Pre-embedding immuno-EM labeling for CNA␣ and CNA␤ in the mouse hippocampus showed a postsynaptic enrichment (Fig. 3, A and B), consistent with previous reports on the localization of calcineurin (29,30). In contrast, CNA␥ labeling was observed most prominently in the vicinity of synaptic vesicle clusters (Fig. 3,  C-E). This subcellular localization of CNA␥ was supported using post-embedding immunogold labeling in mouse hip-pocampal sections (Fig. 3, F-H). These results indicate that CNA␥ is present in presynaptic terminals.
CNA␥ Modulates Synaptic Vesicle Cycling-The localization of CNA␥ to presynaptic terminals suggests that it may have a role in modulating presynaptic function. Therefore, we examined the effects of shRNA-mediated CNA␥ knockdown on synaptic vesicle cycling in primary neurons using the vGlut1-pHluorin system (22). Rat hippocampal neurons were co-transfected with a Ppp3cc-specific shRNA and the vGlut1-pHluorin construct, and immunocytochemistry analysis for CNA␥ was performed to confirm knockdown of the CNA␥ protein (Fig. 4, A and B; Mann-Whitney U test, U ϭ 2289, p Ͻ 0.0001). FIGURE 1. CNA␥ is expressed in the brain and dephosphorylates synaptic calcineurin substrates. A, human (Hm) and mouse (Ms) CNA␣, CNA␤, and CNA␥ and rat (Rt) CNA␥ recombinant proteins were generated in 293T cells. Protein extracts were probed on a Western blot with anti-human CNA␣, CNA␤, and CNA␥ antisera; anti-mouse CNA␣, CNA␤, and CNA␥ antisera; and anti-rat CNA␥ antiserum to demonstrate antibody specificity. For the rat CNA␥ antiserum analysis, mouse CNA␣ and CNA␤ recombinant proteins were used because of complete amino acid sequence conservation. The higher molecular weight of the mouse relative to the rat CNA␥ protein is due to an N-terminal cycle 3 GFP tag present on the recombinant mouse form of the protein. B, hippocampus protein extract from 5-week-old mice was probed on a Western blot with anti-mouse CNA␣, CNA␤, and CNA␥ antisera. C, Western blotting was performed on mouse tissue protein extracts using anti-mouse CNA␣, CNA␤, and CNA␥ antisera and a GAPDH antibody. The lack of a Western blot signal in the pancreas tissue extracts is likely due to protein degradation. D, immunoprecipitation (IP) of CNA␥ from a human neocortex sample. Purified antibodies against different regions of the protein were used to immunoprecipitate (␥-1) and probe for human CNA␥ (␥-2). E, Western blot showing the expression of CNA␣, CNA␤, and CNA␥ (using custom antisera) and GAPDH in neocortex protein samples from mice at the indicated ages. E, embryonic day; P, postnatal day. F, 293T cells were co-transfected with GFP-tagged constitutively active versions of CNA␣, ␤, or ␥ (GFP-CNA CA ) and a dynamin I expression construct. Western blotting was performed on protein extracts for Ser(P)-778 and total dynamin I and for GFP.
We next analyzed the effects of CNA␥ knockdown on synaptic vesicle release and recycling. First, we measured vGlut1-pHluorin responses to a 10-Hz, 100-action potential stimulation from individual neurons transfected with Ppp3cc shRNA plus vGlut1-pHluorin or vGlut1-pHluorin alone. The fluorescence signals induced by the stimulation at each bouton were normalized to those obtained by rapid alkalization of the entire labeled vesicle pool using NH 4 Cl (23). These experiments showed that reduction of CNA␥ expression led to a modest reduction in vesicle exocytosis, but this difference did not achieve statistical significance (Fig. 4, C and D; Student's t test, t(25) ϭ 1.25, p ϭ 0.24). In contrast, when we examined the kinetics of vesicle endocytosis by fitting the fluorescence recovery with a single exponential curve (Fig. 4, C and E), we found that there was a significant slowing of the time constant of fluorescence recovery (Fig. 3E; Student's t test, t(26) ϭ 3.91, p Ͻ 0.001). Critically, restoration of the expression of CNA␥ by cotransfection with a shRNA-resistant expression vector resulted in a restoration of synaptic vesicle endocytosis (Fig. 4E;  Student's t test, t(18) ϭ 0.43, p ϭ 0.7 relative to the control).
These data indicate that CNA␥ modulates synaptic vesicle recovery. The recovery kinetics result from a combination of two processes: vesicle endocytosis and vesicle reacidification. Analysis of synaptic vesicle reacidification showed no effect of CNA␥ knockdown on this process ( Fig. 4F; Student's t test, t(9) ϭ 0.13, p ϭ 0.90), demonstrating that the loss of CNA␥ specifically affects synaptic vesicle endocytosis.
Because there are three isoforms of CNA, it is possible that this alteration in synaptic vesicle cycling following CNA␥ knockdown results from a reduction in total CNA levels and not from disruption of a specific role of CNA␥ in this process. Moreover, the observed alteration in synaptic vesicle endocytosis does not establish an enzymatic role for CNA␥ in this process because it is possible that a structural role could underlie its involvement in presynaptic function. To address these questions, we performed further synaptic functional analyses. As shown in Fig. 4G, we replicated the original observation that CNA␥ knockdown slows synaptic vesicle recovery ( Fig. 4G; Student's t test, t(17) ϭ 4.8, p Ͻ 0.001 relative to the control) and that this effect could be rescued by expression of an shRNA-resistant CNA␥ construct ( Fig. 4G; Student's t test, t(18) ϭ 0.95, p ϭ 0.35 relative to the control). In parallel, we coexpressed CNA␣ or CNA␤ with the CNA␥ shRNA, and, in both cases, synaptic vesicle recovery remained significantly slowed relative to control neurons ( Fig. 4G; Student's t test CNA␣: t(16) ϭ 3.9, p ϭ 0.001; Student's t test CNA␤: t(21) ϭ 4.4, p Ͻ 0.001 relative to the control), indicating that the effect of CNA␥ shRNA is specific for CNA␥. Lastly, we co-expressed an inactive version of CNA␥ that has a single amino acid point mutation in the catalytic domain shown previously to inactivate calcineurin (24). Co-expression of this construct with CNA␥ shRNA also failed to rescue the impairment in synaptic vesicle cycling ( Fig. 4G; Student's t test, t(14) ϭ 4.1, p ϭ 0.001 relative to the control). In sum, these data indicate that CNA␥ has a unique enzymatic role relative to the other CNA isoforms in synaptic vesicle cycling.

Discussion
PPP3CC encodes CNA␥, one of three isoforms of the catalytic subunit of calcineurin (2). Specific haplotypes of this gene have been associated with schizophrenia (9,(13)(14)(15)(16)(17), and the expression of its mRNA has been found to be reduced in the hippocampus of human schizophrenia patients (8). Because CNA␥ has long been thought to be a testis-specific protein (7), there has been no reported information on functions of CNA␥ relevant to its role in schizophrenia. In this study, we show that CNA␥ is present in presynaptic terminals, dephosphorylates known presynaptic calcineurin substrates, and modulates synaptic vesicle endocytosis. These findings raise the possibility that variations in PPP3CC contribute to psychiatric disease through altering presynaptic function.
Calcineurin and Presynaptic Function-Calcineurin is involved in multiple neuronal processes, including postsynaptic receptor trafficking, intracellular signaling, cytoskeletal regulation, synaptic vesicle release and recovery, and activity-dependent gene transcription (1). Presynaptically, calcineurin regulates multiple aspects of the synaptic vesicle cycle. Knockdown of the essential CNB1 regulatory subunit results in changes in recycling vesicle pool size and release kinetics (23). The change in release kinetics could be attributed to a modulation of calcium influx via N-type calcium channels by opposing activities of cyclin-dependent kinase 5 and CNA␣ (5). Furthermore, a set of phosphoproteins involved in vesicle endocytosis are substrates of calcineurin, and blockade of their dephosphorylation results in disruption of this process (6,28,31). Although calcineurin-mediated dephosphorylation of these proteins has been reported to play a role in bulk endocytosis (32), recently, dephosphorylation of dynamin has been pinpointed in controlling the acceleration of endocytosis during brief action potential trains (33).
Despite the importance of calcineurin in presynaptic function, relatively little is known about the specific roles of the calcineurin catalytic subunit isoforms in this process. CNA␣ has been implicated in controlling release probability via modulation of calcium influx (5), and a recent study has reported that knockout of the CNA␤ protein impairs synaptic vesicle endocytosis (6). The data presented in this report suggest that CNA␥ plays a key enzymatic role in the regulation by calcineurin of synaptic vesicle endocytosis. Because expression of either CNA␣ or CNA␤ did not overcome the effects of CNA␥ deficiency on synaptic vesicle endocytosis, it appears that the role of CNA␥ in this process can be separated from those of the other CNA isoforms. Precise definition of the specific roles of each isoform in presynaptic function will require further study. In addition, a modest decrease in the rate of exocytosis, which did not achieve significance, was observed following CNA␥ knockdown. It is possible that more pronounced effects of CNA␥ knockdown on exocytosis would occur in response to higher-frequency stimulation, as was observed for CNB1 knockdown (19). In this regard, further experiments to examine the effects of CNA␥ knockdown across a range of stimuli will be informative. Moreover, it will be of great interest, in future studies, to define the precise functional roles of the different calcineurin isoforms in synaptic vesicle cycling and to determine whether there is a distinction between structural and catalytic functions for the calcineurin subunits in this process. Last, our data do not preclude the localization of CNA␥ to additional subcellular compartments in which it may play additional roles in neuronal function.
A study of mRNA expression in the developing rat brain suggests a prenatal role of PPP3CC in schizophrenia pathogenesis (26). Here we show that CNA␥ protein levels in the cerebral cortex increase during postnatal development and remain relatively high into adulthood. This discrepancy may be due to species differences or developmental regulation of CNA␥ protein levels. Regardless, our data suggest that alterations in CNA␥ function could play a role in schizophrenia by altering cortical function during postnatal development or adulthood. In addition, given the high level of CNA␥ expression in adult testis, it is possible that it may affect schizophrenia pathogenesis through effects in the paternal germline (34).
Presynaptic Dysfunction and Psychiatric Disease-There is an expanding body of research linking alterations of presynaptic function with schizophrenia. A number of genes involved in this process have been reported to be associated with schizophrenia (35)(36)(37). The expression of the mRNA or protein encoded by presynaptic genes is altered in human schizophre- nia brain samples (38 -40), and a number of known schizophrenia risk genes modulate presynaptic function (41,42). Moreover, a recent study of mice with forebrain-specific deletion of calcineurin that have behavioral abnormalities relevant to schizophrenia provided evidence for a presynaptic mechanism for working memory impairments (19). Given these findings, it will be of considerable interest to study the behavioral function of mice following the specific deletion of CNA␥. In summary, the findings presented here suggest that alterations in PPP3CC may contribute to schizophrenia pathogenesis by modulating synaptic vesicle cycling and further implicate presynaptic dysfunction in psychiatric disease (19,20).  , and a CNA␥ shRNA-and shRNA-resistant CNA␥ co-transfected neuron (gamma-Rescue) in response to a 10-Hz, 10-s stimulation normalized to waveform peaks. Inset, representative non-normalized raw waveforms from neurons as described above. Red line, stimulus period. Scale bars ϭ 0.025 ⌬F/F (vertical) and 10 s (horizontal). D, mean Ϯ S.E. peak vGlut1-pHluorin fluorescence intensities in response to a 10-Hz, 10-s stimulation normalized to the peak NH 4 Cl fluorescence levels from control (n ϭ 8) neurons, CNA␥ shRNA-transfected (gamma-KD, n ϭ 6) neurons, and neurons co-transfected with CNA␥ shRNA and the shRNAresistant CNA␥ expression vector (gamma-Res, n ϭ 5). E, vGlut1-pHluorin fluorescence recovery time constants () were calculated as described under "Experimental Procedures." Shown are the mean Ϯ S.E. recovery values from waveforms as shown in C from control neurons (n ϭ 11), CNA␥ shRNAtransfected neurons (n ϭ 17), and neurons co-transfected with CNA␥ shRNA and the shRNA-resistant CNA␥ expression vector (gamma-Res, n ϭ 9). F, mean Ϯ S.E. synaptic vesicle reacidification kinetics measured in neurons transfected with either vGlut1-pHluorin alone (control, n ϭ 7) or vGlut1-pHluorin and CNA␥ shRNA (gamma-KD, n ϭ 6). G, mean Ϯ S.E. recovery values from control neurons (n ϭ 10), CNA␥ shRNA-transfected neurons (gamma-KD, n ϭ 9), and neurons co-transfected with CNA␥ shRNA and the shRNA-resistant CNA␥ expression vector (gamma-Res, n ϭ 10), a CNA␣ expression vector (alpha, n ϭ 8), a CNA␤ expression vector (beta, n ϭ 13), or an inactivated CNA␥ expression vector (gammaH147A, n ϭ 6). **, p Ͻ 0.01; ***, p Ͻ 0.001. Norm., normalized; A.U., arbitrary units.