Department of Neuroscience and Physiology, State University of New York Upstate Medical University, Syracuse, New York 13210
Submitted 15 December 2003 ; accepted in final form 27 May 2004
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ABSTRACT |
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calmodulin; exon selection
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MATERIALS AND METHODS |
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Cerebellar GC cultures.
Cerebellar cortices from postnatal day 8 (P8) Sprague-Dawley rats were minced, trypsinized, and triturated to dissociate cells as described previously by investigators at our laboratory (23). Cells were plated on poly-L-lysine-coated (10 µg/ml) 35-mm dishes at a density of 2.5 x 106 cells/2 ml of medium (for RNA and protein extraction studies) or 66.3 x 105/0.5 ml of medium in 24-well Corning dishes containing glass coverslips (for immunocytochemical studies) and then incubated at 37°C in a humidified atmosphere of 5% CO2-air. Cultures were supplemented with 10 µM cytosine -D-arabinofuranoside 24 h after plating to inhibit nonneuronal cell, i.e., glial, proliferation. The growth medium consisted of basal Eagle's medium with Earle's salts, supplemented with final concentration (f.c.) heat-inactivated fetal calf serum (10%), gentamicin sulfate (100 µg/ml), and L-glutamine (2 mM). Where indicated, standard growth medium (containing 5 mM KCl) was augmented with 20 mM KCl or 140 µM N-methyl-D-aspartate (NMDA) plus 5 mM KCl [Note: 10 mM KCl alone does not enhance long-term survival, but when used in conjunction with NMDA, it relieves the Mg2+-dependent voltage block of NMDA receptors (NR), thereby permitting activation of NR by NMDA].
Laser microdissection. Whole cell RNA was extracted from microdissected samples of the granular and medullary layers of slices from cerebellar cortex and compared for expression of mRNA encoding IP3R1 and CaMKIV. Actin was used as a loading control. Briefly, 10-µm sections were cut on a cryostat onto Leica polyethylene terephthalate foil slides at 18°C and returned to 80°C by placement on dry ice. Individual slides were then removed from dry ice and immediately placed upside down into the slideholder platform of the Leica AS LMD application solution laser microdissection instrument (Leica Microsystems, Bannockburn, IL). At x20 magnification, unstained sections provided adequate morphology to readily distinguish the cellular laminae of the cerebellar cortex. The specific cell layer of interest was outlined (by hand) using a PC mouse. Next, with the laser beam apertures set between 9 and 13 µm, the software was used to follow the predetermined path and dissect the layer of interest directly into the RNase-free microfuge tubes containing RLT lysis buffer (Qiagen RNeasy Mini kit) previously inserted into the carrier beneath the microscope platform. Approximately three sections per animal were used to isolate the cell layers within the cerebellum, followed by extraction of RNA and analysis by RT-PCR.
Cerebellar explant cultures. With the use of the method described by Connor et al. (5), cerebella from P3 or P4 Sprague-Dawley rats were isolated, pooled, and minced into 0.3- to 1-mm pieces while bathed in basal Eagle's medium with Earle's salts, supplemented with (f.c.) heat-inactivated fetal calf serum (10%), gentamicin sulfate (100 µg/ml), and L-glutamine (2 mM) (see Ref. 5). Pieces (n = 1020) were transferred to coverslips coated with 10 µg/ml poly-L-lysine and incubated at 37°C in a humidified atmosphere of 5% CO2-air.
Neuron survival. GC viability was examined using an assay in which 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) is converted to a blue formazan product by the mitochondria of viable cells as described previously (29). MTT was added directly to the culture medium at a final concentration of 0.5 mg/ml, and cultures were incubated at 37°C for 10 min. The solution was then aspirated and replaced with an equal volume of dimethyl sulfoxide. The absorbance of samples was quantified using a Hitachi U-2000 spectrophotometer at a wavelength of 540 nm. Our laboratory previously showed that the color yield was directly proportional to the number of viable cells, assessed by phase-contrast microscopy in conjunction with fluorescein diacetate/propidium iodide (FDA/PI; live/dead assay) (45).
Immunofluorescence/Western immunoblotting. GC or explants attached to coverslips were washed with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min. Afterward, cultures were washed three times for 5 min each with PBS, permeabilized with 0.3% Triton-X in 2% donkey serum, 1% bovine serum albumin (BSA) in PBS for 20 min at RT, rinsed three times for 5 min each with PBS, blocked with 2% donkey serum, 1% BSA in PBS for 30 min, and then incubated overnight with antibody specific for IP3R1 protein (1:10). Subsequently, the coverslips were washed three times for 5 min each in PBS, incubated with Texas red-labeled donkey anti-rabbit IgG (1:100 dilution, 1 h at RT), washed three times for 5 min each in PBS, and then mounted with Vectashield antifade on glass slides. Photomicrographs were produced using a Zeiss microscope.
For Western immunoblotting analysis, GC were rinsed with PBS, and then protein was collected in 100 µl of homogenization buffer (63 mM Tris·HCl, pH 7.5, 2 mM EDTA, 2 mM EGTA, 5 mM -mercaptoethanol, 100 µM PMSF). For IP3R1 protein detection, 15 µg/lane of whole cell homogenate was resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 5% gels. After electrophoresis, proteins were transferred to nitrocellulose and incubated with 1:100 dilution of polyclonal antibody specific for IP3R1 protein (49) and then peroxidase-conjugated secondary antibodies (1:500). Immunoreactivity was detected using enhanced chemiluminescence.
RT-PCR. Whole cell RNA from GC, whole cerebellum, and hippocampus were isolated using the guanidinium isothiocyanate method (4). After the final ethanol precipitation, the RNA pellet was resuspended in sterile water. The concentration and purity of RNA were assessed with a spectrophotometer using nucleotide absorption at 260 nm and ratio at 260/280 nm (nucleotide/protein), respectively. Where indicated, RNA was isolated using a Qiagen RNeasy Mini kit. Harvested RNA was used in a RT reaction to produce cDNA for use as a template in PCR. RNA (0.10.2 µg) in 4 µl of sterile water was heated to 94°C for 1 min and then cooled on ice. Six microliters of a mixture containing (f.c.) MMLV RT (100 U), RT buffer (in mM: 50 Tris·HCl, pH 8.3, 75 KCl, 3 MgCl2, 10 dithiothreitol), the four dNTPs (0.5 mM each), RNasin ribonuclease inhibitor (20 U), and hexamer random primers (5 µM) were added to each RNA sample (final volume 10 µl). The RT reaction was initiated by incubation at 37°C for 60 min to promote synthesis of cDNA and terminated by heating to 95°C for 5 min and then placing the tubes on ice and diluting to 0.15.0 ng/µl using sterile H2O. After RT, PCR was performed in a final volume of 100 µl containing Taq buffer (10 mM Tris·HCl, pH 8.8, 50 mM KCl), 2 mM MgCl2, 0.17 mg/ml BSA, 2.5 units of Taq polymerase (AmpliTaq Gold), 2 µCi [32P]dCTP, and 0.05 mM of each dNTP, as well as 25 pmol of each oligonucleotide primer as follows: S1 upstream (5'-GGTCAACTCCGTCAACTGTAA-3': sense) and S1 downstream (5'-AGCAGGAGAAACGGGGACTAT-3': antisense); S2 upstream (5'-GGTTCATCTGCAAGCTAATAAAAC-3': sense) and S2 downstream (5'-AATGCTTTCATGGAATACTCGGTC-3': antisense); and S3 upstream (5'-TGACAACCATCTTCCCCATTAG-3': sense) and S3 downstream (5'- TGGATAGATCTCATCACGTTGC-3': antisense). Before PCR, samples were incubated at 94°C (S1 and S2) or at 96°C (S3) for 7 min. PCR was performed for 35 cycles in a PerkinElmer Tempcycler model 480 as follows: S1, 94°C for 30 s, 58°C for 30 s, and 72°C for 45 s; S2, 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min; and S3, 96°C for 1 min, 55°C for 1 min, and 72°C for 1 min. After PCR, the samples were heated to 72°C for 5 min. The amplified products were resolved on 8% (S1 and S2) or 20% (S3) polyacrylamide gels, and radioactivity was exposed on a PhosphorImager. Where indicated, variants containing or lacking the S1 exon were distinguished by restriction analysis with BpmI.
PCR of the region flanking the mouse IP3R1 S1 splice site was amplified in a final volume of 100 µl containing Taq buffer (10 mM Tris·HCl, pH 8.8, 50 mM KCl), 2 mM MgCl2, 0.17 mg/ml BSA, 2.5 units of Taq polymerase (AmpliTaq Gold), and 0.05 mM of each dNTP, as well as 25 pmol of each oligonucleotide primer: upstream (5'-GGAAAAGAAGCAGAATGAGAC-3': sense) and downstream (5'-CAGGGGAAACAGGAACTATGG-3': antisense). Before PCR, the samples were incubated at 94°C for 9 min. PCR was performed for 40 cycles in a PerkinElmer model 480 Tempcycler as follows: 94°C for 30 s, 59°C for 30 s, and 72°C for 65 s. After PCR, the samples were heated to 72°C for 5 min. The amplified products were resolved on 1.5% agarose gels and photographed under UV illumination.
PCR for actin was performed using the primers specified by Kudo et al. (22). PCR for CaMKIV was performed in a final volume of 100 µl containing Taq buffer (10 mM Tris·HCl, pH 8.8, 50 mM KCl), 2 mM MgCl2, 0.17 mg/ml BSA, 2.5 units of Taq polymerase (AmpliTaq Gold), and 0.05 mM of each dNTP, as well as 25 pmol of each oligonucleotide primer: upstream (5'-TGCAAGGTAGAAGGGACTCG-3': sense) and downstream (5'-GTACTGGAGGTGACCGAGGA-3': antisense). Before PCR, the samples were incubated at 94°C for 7 min. PCR was performed for 40 cycles in a PerkinElmer model 480 Tempcycler as follows: 95°C for 30 s, 56°C for 30 s, and 72°C for 45 s. After PCR, the samples were heated to 72°C for 5 min. The amplified products were resolved on 8% polyacrylamide gels and photographed under UV illumination.
Statistical analysis. Values are means ± SE and were analyzed for significance using Student's t-test or ANOVA followed by Tukey's test as appropriate. P < 0.05 was considered significant.
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RESULTS |
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CaMK inhibitors attenuate Ca2+-dependent induction of IP3R1. To discern the possible role of CaMK in GC IP3R1 expression, cultures were grown in medium containing 5 or 25 mM KCl, and, where indicated, KN-62 (5 µM; IC50 in vitro 900 nM) was added at 4 DIV. The selection of agent for these studies was based on a rigorous analysis of the specificity of numerous kinase inhibitors in which the fidelity of KN-62 as a selective and potent inhibitor of CaMKII and CaMKIV was supported (8). Note that brief, overnight addition of KN-62 to the growth medium does not compromise GC viability, unlike longer-term exposure. GC were harvested at 5 DIV and processed for RT-PCR using the S1, S2, and S3 oligonucleotide primers (Fig. 2A). Consistent with the data in Fig. 2B, cultures grown in 25 mM KCl-containing media expressed amplicons corresponding to S1+, S2+, and S3+ IP3R1 mRNA. Importantly, overnight addition of KN-62 attenuated the KCl-mediated upregulation of all three variants (Fig. 3A) as well as attenuation of immunoreactive IP3R1 (Fig. 3B). Volumetric analysis of the S3 variants (sum total), readily measurable in the 5 mM samples, was performed to obtain estimates of the depolarization-induced increases in IP3R1 mRNA. These increases were statistically significant relative to 5 mM KCl or 25 mM KCl cultures receiving KN-62 (% of 5 mM KCl, set at 100; 25 mM + KN-62 = 273.3 ± 27.3; 25 mM KCl = 716.7 ± 112.0; n = 3). Note that attenuation of IP3R1 mRNA induction by 25 mM KCl was also observed after overnight treatment with KN-93, a less selective CaMK inhibitor that triggers some toxicity (not shown). These data indicate that activation of a target of KN-62, likely CaMKII or CaMKIV, is required for Ca2+-dependent induction of IP3R1 in GC.
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IP3R1 S1+ is the predominant variant in cerebella of mutant mice lacking Purkinje neurons. Investigators have largely ignored functional studies of IP3R1 in GC neurons in vivo because of the lack of detectable expression of mRNA and protein. As indicated, IP3R1 S1+ is prominent in the neonatal, but not adult, cerebellar cortex (30). After migration is complete (P21 in rat), there is a switch from prominent expression of IP3R1 S1+ to the adult form, S1. An intriguing possibility is that GC in vivo express IP3R S1+, and it contributes to the Ca2+-dependent process of migration and/or synaptic refinement. To explore this, oligonucleotide primers encoding the S1+ variant were used to compare the relative proportions of S1+ and S1 variants in the cerebella of mutant mice with specific Purkinje cell lesions (designated as pcd) vs. wild-type controls.
The two major neuronal types in cerebellar cortex are Purkinje and GC. In pcd mice, there is a rapid degeneration of nearly all Purkinje neurons beginning at approximately P15, without significant loss of GC (17). Thus, if GC in vivo predominately express IP3R1 S1+ mRNA (as observed in cultures under all conditions of assay), then an increase in the relative proportion of the S1+ variant should be observed in the cerebellar cortex of pcd/ mice, compared with wild-type (+/+) littermates. In other words, by eliminating the major source of IP3R1 in cerebellum, namely, IP3R1 S1 in Purkinje neurons, a detectable amount of IP3R1 S1+ mRNA may be revealed in the remaining GC. Initially, a RT-PCR assay specifying S1+ and S1 variants in mice was tested. Despite a 99.6% similarity in the amino acid sequences between rat and mouse, different oligonucleotide primers were designed because of potential mismatches between the rat primers and the murine sequences flanking the S1 site. The predicted sizes of the amplicons are 158 and 113 bp (Fig. 5A). On the basis of in situ hybridization and RNase protection analyses, the major variant in adult murine hippocampus is S1+, whereas S1 predominates in adult cerebellum (30, 31). To establish the validity of the RT-PCR assay, adult murine hippocampus and cerebellum were compared (Fig. 5B). In agreement with previously published reports (30, 31), the S1+ variant predominated in hippocampus, and the major variant in cerebellum was S1. The identity of this S1+ variant was verified by restriction site analysis (with BpmI; data not shown). Next, RNA from the cerebella of 30-day-old wild-type and pcd/ mice was compared (Fig. 5B). Again, the predominant variant in wild-type mice was S1. In contrast, a statistically significant increase in the relative proportion of amplicon corresponding to S1+ mRNA was observed in pcd mice (%S1 in wild-type vs. null mouse cerebellum: 82.5 ± 0.85 vs. 44.7 ± 2.02%; n = 4). In addition, the overall amount of IP3R1 amplicon was lower than that of wild type. These data verify that S1 is the major variant in Purkinje cells. Importantly, they suggest that GC predominately express IP3R1 S1+ in vivo.
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DISCUSSION |
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In the present study, evidence for activation of CaMK in IP3R1 induction in GC is based on the use of highly selective pharmacological agents. Accordingly, supplementation of the growth medium with elevated KCl or NMDA supported GC survival (11) and also induced IP3R1 protein and mRNA. Overnight addition of the L-type VSCC antagonists, nifedipine or nimodipine, effectively reversed 25 mM KCl-mediated induction of IP3R1 protein and mRNA (Figs. 1C and 2B). Likewise, the competitive and noncompetitive NR antagonists, AP5 and MK-801, respectively, effectively reversed NMDA-mediated induction of IP3R protein and mRNA (Fig. 2B). Thus, although the route of Ca2+ entry into neurons can differentially affect gene transcription, i.e., hippocampal neurons (28), both routes of Ca2+ entry induced IP3R1 in GC. The selective and potent cell-permeable CaMK inhibitor KN-62, which competes with calmodulin for binding to the regulatory domains of CaMKIV and CaMKII (6, 8, 44), also attenuates IP3R1 induction (Fig. 3). Similar effects were observed with the less selective agent, KN-93. On the basis of existing reports that these compounds attenuate transcriptional events mediated by CaMKIV in diverse cell types (e.g., Refs. 15, 38, 50), we suggest that nuclear CaMKIV is a component of this signaling cascade in GC. However, a role for CaMKII cannot formally be excluded.
Besides the present study and an earlier report (35), only one other group of investigators has examined depolarization-dependent induction of IP3R1 in GC (12). Those authors demonstrated that pharmacological blockade of calcineurin with FK506 or CsA attenuated depolarization-dependent expression of IP3R1 (independently verified herein, Fig. 4B). Depolarization- and calcineurin-dependent induction of IP3R1 was subsequently reported in hippocampal neurons, and a NF-ATc pathway was shown to be critical (13; see also Ref. 16). Taken together, these studies support roles for both calcineurin, acting through NF-ATc, and CaMK, probably CaMKIV, as regulators of IP3R1 expression in GC.
IP3R1 promoter activity appears to be regulated through several cis elements present in a short region within the 5'-flanking region, which contains a TATA box, and binding sites for the cAMP-responsive element, PU box motif, CCAAT-binding transcription factor/NF-1, AP2, basic helix-loop-helix factor, TPA-responsive element, AP3, B sequence, CCAGG-containing sequence, and POU/homeobox transcription factor binding sequence. These cis elements confer widespread and differential expression of the IP3R1 gene in brain and peripheral tissues (10). An interesting possibility in GC is that the basic helix-loop-helix promoter site in the IP3R1 gene is synergistically activated by calcineurin and CaMKIV. Along these lines, the myocyte enhancing factor-2 (MEF-2) can dimerize with basic helix-loop-helix proteins, and synergism between CaMKIV and calcineurin in transactivation through MEF-2 has been described in T cells, ventricular myocytes, and skeletal muscle (for review, see Refs. 18 and 39). Indeed, MEF-2 is expressed in newly generated GC and is activated by depolarization and Ca2+, where it contributes to survival (25). Moreover, MEF-2 activity in GC appears to be enhanced by calcineurin under depolarizing conditions because this process is sensitive to inhibition by CsA and FK506 (26). More recently, Groth and Mermelstein (16) reported that inhibition of NF-AT signaling only partially reduced expression of IP3R1 in response to brain-derived neurotropic factor (BDNF), indicating that multiple transcription factors can control the IP3R1 gene, some independently of NF-AT. In contrast, inhibition of NF-AT signaling completely abolished BDNF expression, indicating that although multiple transcription factors may regulate BDNF, NF-AT is required. Further studies are needed to determine whether a synergistic link between CaMKIV and calcineurin regulates Ca2+-dependent IP3R1 induction in GC.
Ca2+ and CaM kinases induce selection of a distinct IP3R1 variant in GC. In the present study, depolarizing agents that increase Ca2+ influx in GC produced splice variants that differed from those that predominate in Purkinje neurons or in GC grown in 5 mM KCl. GC grown in NMDA or in 25 mM KCl-containing medium exhibited significantly higher levels of amplicons that included all three exons. In contrast, Purkinje neurons predominantly express variants containing S2 but lacking the S1 and S3 exons (30, 34), while GC grown in 5 mM KCl predominantly express variants containing S1 with proportionately less S2+ and similar amounts of S3+/ (Figs. 2 and 3). These data suggest that Ca2+, calcineurin, and/or CaMK influence exon choice in IP3R1. Indeed, a handful of reports have indicated that Ca2+-dependent signaling cascades influence exon selection in excitable cells. For example, depolarization-dependent Ca2+ influx through VSCC or NR alters exon selection in pre-mRNA encoding Ca2+-ATPases (51), NR (46, 47), and the slo K+ channel (50). Using transfected cell lines, Xie and Black (50) specifically demonstrated that overexpression of CaMKIV regulates splicing of the STREX exon in slo K+, whereas overexpression of CaMKI, CaMKII, or dominant negative CaMKIV was ineffective. A cell-permeable CaMK inhibitor, KN-93, interfered with depolarization-dependent STREX exclusion, presumably by targeting CaMKIV, which is enriched in neuronal nuclei. Overexpression of CaMKIV produced similar effects on exon selection at three other sites: an alternative exon (SloII87) in the slo K+ channel and two exons (5 and 21) in NR1, hinting that CaMKIV may have a broad role in the regulation of alternative splicing. To identify specific features of the STREX exon needed for CaMKIV repression, deletions in the STREX exonic sequences that were similar to either purine-rich enhancer or pyrimidine-rich repressor elements, previously identified in other pre-mRNA, were mutated. The pyrimidine-rich repressor deletion increased exon inclusion with almost no repression by CaMKIV, leading to the conclusion that the pyrimidine-rich region is required for CaMKIV-mediated repression of the STREX exon (50). An additional repressor element in the upstream 3' splice site, termed the CaMKIV-responsive RNA element (CaRRE), that confers sensitivity to CaMKIV was also identified. However, a thorough sequence analysis of potential purine- and pyrimidine-rich sequences in and near the S2 and S3 exons in IP3R1 was unrevealing. Furthermore, we were unable to identify a CaRRE sequence in the 3' region of the S2 and S3 exons. In both cases, structurally distinct but functionally homologous sequences may underlie Ca2+-dependent exon selection, and GC should prove useful in studying such questions in the future.
Is IP3R1 physiologically relevant in cerebellar GC neurons in vivo? As indicated, previously available methods of assay did not reveal IP3R1 protein or mRNA in the cerebellar granular layer in vivo (24, 30, 32, 40). The inability to detect a low amount of immunoreactive IP3R1 may be further compounded by the fact that the adjacent Purkinje cell layer is by far the richest source known. Nevertheless, IP3R1-specific mRNA can be detected in GC grown in standard medium (5 mM KCl) when a highly sensitive RT-PCR assay is used (e.g., Figs. 24). Moreover, immunoreactive IP3R1 is detectable in migrating GC in explant cultures grown in culture medium containing standard concentrations of KCl (5 mM) (Fig. 6). In all conditions tested, we observed only the S1+ variant of IP3R1, the form that predominates in neonatal, but not adult, cerebellar cortex (30). These data suggest that IP3R1 S1+ may have a role in developing, but not mature, GC. Along these lines, Kirischuk et al. (19) compared agonist-induced Ca2+ mobilization in GC from acutely prepared slices from murine cerebella at two ages. IP3-mediated Ca2+ mobilization was observed in GC in slices from neonates (P6) but not adults (P30).
Herein we provide the first evidence that IP3R1 S1+ is expressed in GC in vivo. First, cerebella from pcd mice express proportionally more amplicon corresponding to the S1+ variant than do their wild-type littermates (Fig. 5). This result is consistent with the selective loss of the S1 variant due to degeneration of Purkinje cells relative to the S1+-expressing GC, which are spared. Second, microdissected tissue from the granular layer of cerebellar slices from neonatal rats specifically express amplicons corresponding to IP3R1 S1+ (Fig. 7). Although previous investigators failed to detect IP3R1 mRNA or immunoreactive protein in GC, there is evidence for IP3R1 promoter activity. Thus moderate expression of a transgene containing a strong IP3R1 promoter was detected in cerebellar GC neurons (10), and Ohkawa et al. (36) reported weak but significant AP2-dependent IP3R1 promoter activity in GC. Possibly, IP3R1 contributes to migration or synaptic refinement in developing GC, two Ca2+-dependent processes that depend critically on the concerted action of CaM kinases and phosphatases (for review, see Refs. 7, 20, 21). In this regard, emerging evidence suggests an important link between IP3R1 activity and neuronal morphogenesis (3, 9, 14, 16, 42). Notably, functional inactivation of IP3R1 in growth cones in dorsal root ganglia results in growth arrest and neurite retraction (42), and axonal outgrowth is compromised in NF-ATc null mice (14). Thus neurons may use IP3R1 in conjunction with other pathways known to integrate Ca2+ signals to strengthen maturing synapses. The data presented herein provide a strong rationale for pursuing functional studies of IP3R1 S1+ in developing GC.
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GRANTS |
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ACKNOWLEDGMENTS |
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Current address of J. Y. Choi: Dept. of Orthopedic Surgery, State Univ. of New York Upstate Medical Univ., Syracuse, NY 13210.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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