Activity-dependent Expression of Inositol 1,4,5-Trisphosphate Receptor Type 1 in Hippocampal Neurons*

There are several lines of evidence showing that synaptic activity regulates the level of expression of inositol 1,4,5-trisphosphate receptor type 1 (IP 3 R1) in neurons. In this study, we examined the effect of chronic activity blockade on the localization and level of IP 3 R1 expres- sion in cultured hippocampal neurons. We found that chronic blockade of NMDA receptors (NMDARs), one of the major Ca 2 (cid:1) -permeable ion channels, increased the number of neurons that express a high level of IP 3 R1 without any apparent changes in its intracellular localization. Interestingly, this up-regulation was time-de-pendent; there was no clear change in IP 3 R1 expression level up to day 5 of the NMDAR blockade, but expression increased at day 6, and the increased expression level persisted for at least a week. The up-regulation of IP 3 R1 depended on transcription and protein synthesis and required cAMP-dependent protein kinase activity. Moreover, although most of the control neurons did not respond to the metabotropic glutamate (mGluR) medium same except L

The inositol 1,4,5-trisphosphate receptor (IP 3 R) 1 is one of the important channels responsible for Ca 2ϩ release from intracel-lular Ca 2ϩ stores (1)(2)(3), and three types have been identified thus far: IP 3 R1, IP 3 R2, and IP 3 R3 (4,5). One of the members of the IP 3 R family, IP 3 R1, is thought to be the predominant isoform in the central nervous system (6), and various approaches, including gene knockout and functional blockade by specific antibodies, have shown that IP 3 R1 plays crucial roles in several neuronal functions, including synaptic plasticity (7)(8)(9)(10)(11), neurite extension (12), and nerve growth cone guidance (13). Thus, the precise regulation of Ca 2ϩ release through IP 3 R1 is one of the important factors in these physiological functions in the brain. IP 3 R channel activity largely depends on direct modulation by numerous intracellular signals, including Ca 2ϩ , ATP, calmodulin, FKBP12, calcineurin, BANK, IRAG, chromogranin, and Huntington-associated protein-1A, which acutely control the channel opening (3, 14 -18). In addition, several recent studies suggest that, by changing the sensitivity of Ca 2ϩ stores to IP 3 , the level of IP 3 R expression may also be an important factor affecting intracellular Ca 2ϩ mobilization (19). For example, retinoic acid up-regulates IP 3 R1 expression in HL-60 cells, a human leukocyte cell line, and HL-60 cells exposed to retinoic acid show a 2-3-fold increase in Ca 2ϩ mobilization in response to IP 3 stimulation (20). Overexpression of IP 3 R1 in Xenopus oocytes results in an increased velocity of Ca 2ϩ wave propagation (21). Mouse L fibroblasts stably expressing approximately an 8.5-fold amount of IP 3 R1 show 4-fold increase in the sensitivity to IP 3 -induced Ca 2ϩ release compared with control cells (22). These cells also show a greater frequency of agonistinduced Ca 2ϩ oscillation and have a lower threshold of agonist concentrations for the transition from oscillatory to peak-andplateau Ca 2ϩ patterns (23). Because the Ca 2ϩ release and Ca 2ϩ oscillation patterns profoundly affect the activity of downstream targets that play crucial roles in a myriad of cellular functions (24), it is important to determine how the IP 3 R expression level is controlled in various types of cells.
In neurons, control of the IP 3 R1 expression level is closely related to synaptic activity. In cerebellar granule cells, Ca 2ϩinflux through L-type Ca 2ϩ channels and N-methyl-D-aspartate receptors (NMDARs) increases the expression level of IP 3 R1 (25). Similarly, activation of L-type Ca 2ϩ channels and NMDA receptors increases IP 3 R1 expression through the calcineurin/NFATC4 (nuclear factor of activated T-cells) pathway in hippocampal neurons (26). In terms of more long term effects of synaptic activity, chronic activation of muscarinic or metabotropic glutamate receptors results in down-regulation of the IP 3 R1 expression level in neuroblastoma cells and cerebellar granule cells, respectively (27,28). However, although many studies have examined the effects of various stimuli on IP 3 R1 expression, little is known about the effect of chronic activity blockade on IP 3 R1 expression in neurons.
In this study, we examined the effect of chronic activity blockade on IP 3 R1 expression and its intracellular localization in cultured hippocampal neurons that have spontaneous synaptic activity (29). The results showed that chronic NMDAR blockade by 2-amino-5-phosphonopentanoic acid (APV) increases the IP 3 R1 expression level in hippocampal neurons. Transcription, protein synthesis, and cAMP-dependent protein kinase (PKA) activity were also found to be necessary for APVinduced IP 3 R1 expression. In addition, chronic NMDAR blockade increased the number of neurons that released Ca 2ϩ in response to the mGluR stimulation, and sensitivity to mGluR stimulation was correlated with the IP 3 R1 expression levels. Furthermore, we showed that neurons transiently overexpressing GFP-tagged IP 3 R1 become sensitive to mGluR and muscarinic acetylcholine receptor stimulation. Thus, these results indicate that chronic NMDAR blockade increases IP 3 R1 expression, which in turn enhances sensitivity of Ca 2ϩ stores to mGluR stimulation. Control of the IP 3 R1 expression level, which was regulated by the extracellular environment, may be one of the important mechanisms in maintaining intracellular Ca 2ϩ homeostasis.

MATERIALS AND METHODS
Cell Culture-Hippocampal neuronal cultures were prepared from 17-18-day embryonic ICR mice as described previously (30). Briefly, hippocampi were dissociated by trypsin treatment and trituration, and were plated on poly-L-lysine-coated glass coverslips in 15-mm culture dishes at a density of 5.0 -6.0 ϫ 10 4 cells/cm 2 in 1.0 ml of medium. After plating, cells were incubated at 37°C, 5% CO 2 /air, and 400 l of medium was replaced once a week. The composition of the medium is Neurobasal TM medium (Invitrogen) containing 2.0% B-27 supplement (Invitrogen), 0.5% insulin-transferrin-selenium A (Invitrogen), 1 mM L-glutamine (Nacalai Tesque, Kyoto, Japan), 100 units/ml penicillin, and 0.1 mg/ml streptomycin. When medium was replaced, medium of the same composition except L-glutamine was used.
Pharmacological Immunocytochemistry and Quantification-Neurons were fixed in 4% paraformaldehyde/PBS for 10 min and subsequently in 100% methanol (Ϫ20°C) for 10 min, and then permeabilized in 0.2% Triton X-100/ PBS for 10 min at room temperature. Coverslips were blocked with 5% skim milk/PBS and incubated with primary antibodies in 5% skim FIG. 1. Chronic NMDA receptor blockade increases the number of hippocampal neurons with intense IP 3 R1 immunoreactivity without any apparent change in IP 3 R1 intracellular localization. A, hippocampal neurons from E18 mice were cultured and exposed to (APV) or unexposed to (Control) 100 M APV for the 7 days from 14 to 21 DIV. At 21 DIV, cells were fixed and stained with anti-IP 3 R1 and anti-MAP2 antibodies. Arrowheads indicate neurons with intense IP 3 R1 immunostaining. Scale bar, 20 m. B, no difference was observed for IP 3 R1 subcellular distribution between control and the APV-exposed hippocampal neurons of intense IP 3 R1 immunoreactivity. Lower panels show the IP 3 R1 immunoreactivity in the proximal dendrites of hippocampal neurons. Scale bars, 10 m (upper panel) and 5 m (lower panel). C, a histogram of the intensity of the IP 3 R1 immunoreactivity in chronically APV-exposed (black bar) or unexposed (Control; white bar) hippocampal neurons. Fluorescence intensity of the IP 3 R1 immunoreactivity at soma of neurons (MAP2-positive cells) was measured, and a representative histogram from one experiment was shown here. The experiments were repeated six times, and similar results were obtained. D, Western blot analysis of the lysates from APV-exposed for 7 days (APV) or unexposed (Control) hippocampal neurons probed with anti-IP 3 R1 and anti-␤-actin antibodies. Experiments were performed three times, and representative data are shown here. milk/PBS overnight at 4°C. Double-label immunostaining was done with combinations of rat anti-IP 3 R1 monoclonal (18A10, 5.0 g/ml; see Ref. 31) and mouse anti-MAP2 monoclonal (1:200, Sigma) antibodies. Generally, five view fields were blindly and randomly chosen from each piece of coverslip, and fluorescence images were taken and analyzed with a cooled charge-coupled device (CCD) camera (Spot model 1.3.0, Diagnostic Instruments, Inc., Sterling Heights, MI) mounted on a Olympus BX50 using a custom-made software, TI Workbench. Neuronal somata were identified by MAP2 signal, fluorescence intensity of IP 3 R1 immunostaining of pixels corresponding to each soma was averaged, and background fluorescence was subtracted. Because absolute fluorescence intensity varied among different experiments, quantitative comparison of IP 3 R1 expression levels were always normalized by values of control cells treated in the same experiments if necessary.
Western Blot Analysis-The hippocampal cell cultures were rinsed with ice-cold PBS and lysed in 150 l of 2ϫ SDS-PAGE sample buffer. The cell lysates were boiled at 100°C for 3 min and centrifuged at 15,000 rpm at 4°C for 5 min. The homogenates were separated by 7.5% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk in PBS for 2 h at room temperature, the membrane was probed with anti-IP 3 R1 (18A10, 1.0 g/ml), mouse monoclonal anti-␤-actin antibodies (1:5000, Sigma), or rabbit polyclonal anti-calreticulin antibody (Affinity Bioreagents) at 4°C overnight. After extensive washing with PBS, the membrane was probed with the alkaline phosphatase-conjugated secondary antibodies. To detect signals, the polyvinylidene difluoride membrane was incubated with AttoPhos substrate (Amersham Biosciences) and scanned on FluorImager (Amersham Biosciences). Band intensities were analyzed using ImageQuaNT software (version 4.1, Amersham Biosciences).
Isolation of RNA and Reverse Transcription (RT)-PCR-Total RNA was prepared from hippocampal cell cultures by using TRIzol reagent according to instructions from the manufacturer (Invitrogen). First strand cDNA was prepared from the total RNA, using reverse transcriptase Superscript II (Invitrogen) and oligonucleotide (dT) primers. PCR was performed using specific primers for IP 3 R1 isoform (forward, 5Ј-CGTGGATGTTCTACACAGACCAG-3Ј; reverse, 5Ј-CCTTGAAGAA-CTTCTCTGATTTC-3Ј) (32). The primers used for ␤-actin were as follows: forward, 5Ј-GGAAATCGTGCGTGACATCAAAGAG-3Ј; reverse, 5Ј-ACCGATCCACACAGAGTACTTGCGC-3Ј. After an initial cycle of 2 min at 94°C, the reaction was cycled 30 times for 30 s at 94°C, 30 s at 50°C, and 70 s at 72°C. We confirmed that the PCR fragments were exponentially amplified at least from 25 to 35 cycles (data not shown). For analysis, the PCR products were separated on 3.5% polyacrylamide gel electrophoresis (PAGE) and stained with ethidium bromide. We performed densitometry by scanning the gel on FluorImager and analyzed the band intensities using ImageQuaNT software. The IP 3 R1 mRNA levels were normalized relative to those of ␤-actin. The PCR products were separated by electrophoresis in 2% agarose gel, extracted, and purified with the GeneClean kit (Bio 101, Vista, CA). Nucleotide sequencing was performed using a DNA sequencer (ABI Prism 377; PerkinElmer Applied Biosystems, Foster City, CA).
Intracellular Ca 2ϩ Imaging-The hippocampal neurons were grown on cover glass engraved with grids (CELLocate, Eppendorf, Hamburg, Germany) coated with 50 g/ml poly-L-lysine (Nacalai Tesque) and treated with or without 100 M APV for 7 days from 14 to 21 days in vitro (DIV). The neurons were loaded with 5 M fura-2-AM (Dojindo, Kumamoto, Japan) for 30 min at room temperature in recording solution containing (in mM): 115 NaCl, 5.4 KCl, 2 CaCl 2 , 1 MgCl 2 , 20 Hepes, 10 glucose, 1 M tetrodotoxin, pH 7.4, washed once, and then kept in the recording solution in the dark at room temperature for 30 min. Fura-2 fluorescence images were analyzed using an inverted microscope (IX50, Olympus, Tokyo, Japan) and a video image analysis system (Argus-50/ CA, Hamamatsu Photonics, Hamamatsu, Japan) with excitation filters at 340 Ϯ 10 and 380 Ϯ 10 nm, dichroic beam splitter at 400 nm, and a band pass emission filter at 520 and 550 nm. After taking basal fura-2 fluorescence images every 10 s for 2.5 min, the cells were stimulated with 50 mM KCl for 15 s to fill Ca 2ϩ stores. After another 4.5 min, the cells were stimulated with 100 M DHPG for 3 min. During these periods, fura-2 fluorescence images were taken every 5 s. After Ca 2ϩ imaging, the cells were fixed with 4% PFA for 10 min at room temperature, permeabilized with 0.2% Triton/PBS for 5 min, and subjected to immunostaining for IP 3 R1 and MAP-2 as described above. Scoring of IP 3 R1 staining intensity was done by two independent observers who were blind to the result of the DHPG sensitivity. The cells showing no IP 3 R1 staining intensity were counted as IP 3 R1-negative cells and indicated as (Ϫ); the cells showing saturated levels of staining (maximally stained cells) were considered as having strong staining intensity and indicated as (ϩϩ); the cells labeled with intermediate intensity between these two categories were considered to be moderately stained and expressed as (ϩ).
Transfection of Hippocampal Neurons-Hipppocampal neurons (1 ϫ 10 5 /well) were transfected with GFP-IP3R1 or GFP expression vectors with calcium phosphate method as described previously (33). After 36 h, the neurons were subjected to Ca 2ϩ imaging described above.
Statistical Analysis-Results are expressed as mean Ϯ S.E., and were statistically evaluated by Student's t test. p Ͻ 0.05 was considered to be statistically significant.
were few such neurons, and the IP 3 R1 expression level was relatively low in most of the neurons in the control cultures. To examine the effect of chronic activity blockade on expression of IP 3 R1 and its intracellular localization, we exposed hippocampal neurons to 100 M APV, an NMDAR antagonist, for the 7 days from 14 to 21 DIV. As shown in Fig. 1A, chronic exposure to APV increased the number of hippocampal neurons with intense IP 3 R1 immunoreactivity in their cell bodies (arrowheads in Fig. 1A, APV), but did not induce any apparent change in the intracellular localization of IP 3 R1; IP 3 R1 clusters remained diffused within the dendritic shafts and soma in a punctate pattern, and there was no apparent change in the size of IP 3 R1 clusters after APV exposure (Fig. 1B). Because IP 3 R1 immunoreactivity in the proximal dendritic shafts was similar in both the APV-treated neurons and control neurons, translocation of IP 3 R1 from dendrites to the cell body was unlikely to be the mechanism for the increase in IP 3 R1 intensity observed in the cell body (Fig. 1B, lower panels). Fig. 1C shows a histogram of the intensity of IP 3 R1 immunoreactivity in neuronal cell bodies (MAP2-positive cells), confirming the increased number of neurons with high IP 3 R1 immunoreactivity (arbitrary units, 9 -20) in the APV-exposed neurons (black bar) compared with the control neurons (white bar).
To further confirm the increase in IP 3 R1 expression level, we analyzed the amount of IP 3 R1 expression by Western blot analysis. As shown in Fig. 1D, the IP 3 R1 expression level in the chronically APV-exposed neurons was clearly increased compared with the control cells, but there was no difference in expression level of ␤-actin and calreticulin between the groups. Quantitative analysis revealed that the IP 3 R1 protein level in APV-exposed neurons was 1.64 Ϯ 0.34-fold higher than in the control cells (Control; n ϭ 65 wells, APV; n ϭ 24 wells, p Ͻ 0.01). Taken together, these results indicated that the chronic NMDAR blockade increased the IP 3 R1 expression level in hippocampal neurons.
Time Course of the APV-induced Increase in IP 3 R1 Expression Level in Hippocampal Neurons-To examine the time course of the APV-induced increase in IP 3 R1 expression, as shown in Fig. 2A, we exposed hippocampal neurons to 100 M APV for various times (1, 3, 5, 6, 7, and 14 days), and determined the IP 3 R1 expression level by Western blot analysis at 21 DIV. At days 1-5 of APV-treatment, there was no apparent change in IP 3 R1 expression level, except for slight decrease in IP 3 R1 expression at day 1 (Fig. 2B). Because NMDAR activation is known to increase the IP 3 R1 expression through L-type Ca 2ϩ channel activation (26), the slight decrease after 1 day of APV exposure was probably because of the suppression of basal NMDAR activity. After 6 days of APV exposure, however, the IP 3 R1 expression level clearly increased (Fig. 2, B and C), and the increase was observed until day 14. Thus, these results indicated that long term APV exposure, i.e. for more than 6 days, increases the IP 3 R1 expression level in hippocampal neurons.
Transcription and Protein Synthesis Were Required for the APV-induced Increase in IP 3 R1 Expression-To assess the contribution of protein synthesis to the APV-induced increase in IP 3 R1 expression level, a protein synthesis inhibitor, cycloheximide (CHX), was added to the APV-exposed cultures from 20 to 21 DIV, when the IP 3 R1 expression level had greatly increased (Fig. 3A). As shown in Fig. 3B, CHX significantly inhibited the APV-induced increase in IP 3 R1 expression and there was no significant difference in IP 3 R1 expression level between the APVϩCHX-exposed and CHX-exposed neurons, indicating that IP 3 R1 degradation occurred to a similar extent irrespective of the presence of APV (Fig. 3, B and C). Next we examined the contribution of transcription of IP 3 R1 to the APV-induced increase in IP 3 R1 expression by measuring IP 3 R1 mRNA by RT-PCR. Total RNA was prepared from cultured hippocampal cells exposed to and unexposed to APV for 7 days, and semiquantitative RT-PCR analysis was performed using specific primers for IP 3 R1. As shown in Fig. 3D, the amount of IP 3 R1 mRNA in the APV-exposed neurons increased compared with the controls, whereas the ␤-actin mRNA level and the SERCA 2b mRNA level (data not shown) did not change significantly. Normalization of the amount of IP 3 R1 mRNA to that of ␤-actin revealed that the amount of IP 3 R1 mRNA had increased by 177 Ϯ 34% in the APVexposed neurons (n ϭ 3, p Ͻ 0.02). These results demonstrated that long lasting NMDAR blockade increases the IP 3 R1 expression level through a mechanism involving transcription and protein synthesis rather than decreased IP 3 R1 degradation.

FIG. 3. Transcription and protein synthesis are involved in the APV-induced IP 3 R1 expression in cultured hippocampal neurons.
A, schematic illustration of the schedule of drug treatments. Hippocampal neurons were exposed to 100 M APV from 15 to 21 DIV and 5.0 M CHX from 20 to 21 DIV. B, Western blot analysis of cell lysates from APV-exposed, APVϩCHX-exposed, or CHX-exposed hippocampal neurons probed with anti-IP 3 R1 (upper panel) and ␤-actin (lower panel) antibodies. C, relative IP 3 R1 band intensity normalized to that of ␤-actin. Percentage and numbers of wells used for each analysis were as follows: control, 100 Ϯ 16% (n ϭ 16); APV, 126 Ϯ 25% (n ϭ 12); APVϩCHX, 70 Ϯ 23% (n ϭ 10); CHX, 61 Ϯ 3% (n ϭ 3). ** and * indicate significant difference from control cells by t test (p Ͻ 0.002 and p Ͻ 0.001, respectively). D, semiquantitative RT-PCR analysis with specific primers for IP 3 R1 and ␤-actin. Total RNA from hippocampal cells exposed to (APV) or unexposed to (Control) 100 M APV was used as a template. Experiments were performed three times, and representative data are shown here.

Involvement of PKA Activity in the APV-induced IP 3 R1 Ex-
pression-Chronic NMDAR blockade has been shown to activate cAMP-dependent protein kinase (36). To assess the involvement of PKA activity in the APV-induced IP 3 R1 expression, we added a specific PKA inhibitor, KT5720, to the APV-exposed cells for the 24 h from 20 to 21 DIV (Fig. 4A). The result was complete abolition of the increase in IP 3 R1 expression by APV (Fig. 4, C and D). Moreover, activation of PKA by a mixture of IBMX (an inhibitor of phosphodiesterase), and 8-bromo-cAMP, a membrane-permeable analogue of cAMP, for the 48 h from 19 to 21 DIV was sufficient for the increase in IP 3 R1 expression level (Fig. 4, C and D). The histograms of IP 3 R1 immunoreactivity in neuronal soma (MAP2-positive; Fig. 4B) clearly show a decreased population of cells with relatively high IP 3 R1 immunoreactivity (arbitrary units, 9 -12) and an increased population of cells with relatively low IP 3 R1 immunoreactivity (arbitrary units, 1-3) among the APVϩKT5720-exposed neurons compared with the APV-exposed neurons. These results demonstrated that the APV-induced increase in IP 3 R1 expression required PKA activity. APV-exposed Hippocampal Neurons with High IP 3 R1 Expression Are More Sensitive to mGluR Stimulation-To explore the functional effect of the increased IP 3 R1 expression on IP 3induced calcium release, we imaged Ca 2ϩ signals evoked by application of 100 M DHPG, an agonist of group I metabotropic glutamate receptors, in chronically APV-exposed and unexposed hippocampal neurons. To distinguish neurons from other types of cells and quantify the IP 3 R1 expression level, we immunostained neurons with anti-IP 3 R1 and anti-MAP2 antibodies after Ca 2ϩ imaging and analyzed only the Ca 2ϩ signals from MAP2-positive cells. Glial cells were also distinguished from neurons by the sustained shape of Ca 2ϩ transients (data not shown; see Ref. 37). In the control cultures (Control), application of 100 M DHPG did not evoke Ca 2ϩ release in any of the hippocampal neurons except for transient Ca 2ϩ release by two neurons (control: 0.6%, 2 of 353 MAP2-positive cells; Fig.  5), a finding that is consistent with the previous observations (37). In contrast to the control cells, however, some of the APV-exposed neurons responded to DHPG application and transiently released Ca 2ϩ from Ca 2ϩ stores (Fig. 5, lower pan-els). The number of DHPG-responding cells among the chronically APV-exposed neurons significantly increased by approximately 10 times compared with the unexposed control cells (APV: 6.9%, 31 of 448 MAP2-positive cells; Fig. 5). Although many strongly IP 3 R1-positive (ϩϩ) neurons did not respond to the mGluR stimulation (e.g. neurons indicated by asterisks in Fig. 5, lower panel), there was a correlation between IP 3 R1expression level and sensitivity of Ca 2ϩ stores to mGluR stimulation in the chronically APV-exposed neurons; only 3.6% (13 of 366) of the IP 3 R1-negative or weakly positive neurons (e.g. neurons with red and yellow-green arrows in Fig. 5, lower panel; Ϫ or ϩ) responded to DHPG stimulation, whereas 22% (18 of 82) of strongly IP 3 R1-positive neurons (e.g. neurons with pink, orange, light blue, and blue arrowheads in Fig. 5, lower panel; ϩϩ) transiently released Ca 2ϩ in response to mGluR stimulation. Thus, although there maybe other factors regulating the mGluR-induced Ca 2ϩ release in the APV-treated neurons, these findings suggested that the increased IP 3 R1 expression level in APV-exposed neurons is an important factor in the increased number of DHPG-responsive neurons among the APV-exposed neurons.
Hippocampal Neurons Expressing GFP-tagged IP 3 R1 Released Ca 2ϩ in Response to G-protein-coupled Receptor Stimulation-To further confirm the relationship between the IP 3 R1 expression level and the sensitivity of Ca 2ϩ store to mGluR stimulation in hippocampal neurons, we transiently expressed GFP-tagged IP 3 R1 (38) and examined Ca 2ϩ release in response to mGluR stimulation. As shown in Fig. 6, the cells exogenously expressing GFP-tagged IP 3 R1 showed transient Ca 2ϩ release upon DHPG application (7 of 8 neurons), whereas no cells expressing GFP alone responded to the agonist stimulation (n ϭ 4 neurons). The transient Ca 2ϩ release in GFP-tagged IP 3 R1expressing cells was also observed by stimulation of muscarinic acetylcholine receptor, another type of G-protein-coupled receptor (Fig. 6), suggesting that this increased sensitivity of Ca 2ϩ store is not a specific up-regulation of mGluRs but rather a general phenomenon for G-protein-coupled receptor signaling. Taken together, these results indicated that the expression level of IP 3 R1 is a crucial determinant for the Ca 2ϩ release upon G-protein-coupled receptor stimulation in hippocampal neurons. FIG. 4. Involvement of PKA activity in the activity-dependent expression of IP 3 R1 in hippocampal neurons. A, schedule of the drug treatments. 2.0 M KT5720 (a specific inhibitor of PKA) was added to the cultured cells for the 24 h from 20 to 21 DIV, and 25 M IBMX (an inhibitor of cAMP phosphodiesterase) and 10 M 8-bromo-cAMP (Br-cAMP, an agonist of PKA) were added for the 48 h from 19 to 21 DIV. Then, the cells were fixed and stained with antibodies for IP 3 R1 and MAP2. B, representative histograms of the IP 3 R1 immunoreactivity in hippocampal neurons after exposure to various drugs. Intensity of the IP 3 R1 immunoreactivity in the soma of neurons (MAP2-positive cells) was quantified. C, Western blot analysis of the cell lysates from IBMXϩ8-bromo-cAMP-, APVϩKT5720-, or KT5720-exposed hippocampal neurons probed with anti-IP 3 R1 (upper panel) and anti-␤-actin (lower panel) antibodies. D, relative IP 3 R1 band intensity normalized to that of ␤-actin. Experiments were performed twice, and each experiment contained at least six wells per each drug treatment. Control, 100 Ϯ 29%; APV, 216 Ϯ 39%; APVϩKT5720, 57 Ϯ 22%; KT5720, 55 Ϯ 26; IBMXϩ8-bromo-cAMP, 171 Ϯ 37%. Asterisks indicate significant difference compared with nontreated cells (p Ͻ 0.02).

DISCUSSION
The results of this study showed that chronic blockade of NMDAR activity by APV for more than 6 days leads to an increase in IP 3 R1 expression level, and that this up-regulation of IP 3 R1 requires RNA transcription, protein synthesis, and PKA activity. They also showed that APV-exposed neurons acquire the ability to mobilize intracellular Ca 2ϩ in response to mGluR stimulation and that this Ca 2ϩ -mobilizing property is correlated with the IP 3 R1 expression level in the APV-exposed neurons. Thus, our findings indicated that long lasting NMDAR blockade increases IP 3 R1 expression and enhances the sensitivity of hippocampal neurons to mGluR stimulation. Consistently, we demonstrated that overexpression of IP 3 R1 in hippocampal neurons was sufficient for Ca 2ϩ release upon Gprotein-coupled receptor stimulation. As far as we know, this is the first direct evidence that the expression level of IP 3 R1 in hippocampal neurons determines the sensitivity of Ca 2ϩ stores upon G-protein-coupled receptor stimulation.
Numerous factors, including Ca 2ϩ , calmodulin, ATP, and phosphorylation, have been reported to directly modulate IP 3 R channel activity (3,19), and, in addition to these direct modifications, regulation of the expression level and subunit combination of IP 3 Rs can also affect Ca 2ϩ mobilization from Ca 2ϩ stores (19). The former can immediately and directly change channel activity, although the effects on intracellular Ca 2ϩ mobilization may be transient, whereas the latter takes a longer time to influence intracellular Ca 2ϩ mobilization, but can affect it for a longer time. Such prolonged regulation of IP 3 R-mediated signaling would be important for cells to respond to protracted events, including development and lengthy changes in inhibitory or excitatory environment in various pathological states. The results of the present study revealed a new form of long term regulation of IP 3 R1 expression levels. Up-regulation of IP 3 R1 expression might respond to a demand for a Ca 2ϩ source to maintain the intracellular Ca 2ϩ level after blockade of another Ca 2ϩ source through the NMDAR channel. It should be mentioned that there are multiple lines of evidence indicating that chronic stimulation of plasma membrane receptors linked to IP 3 production leads to a decrease in IP 3 R protein level via calpain, caspase, and ubiquitin-proteasome pathways, which would attenuate excessive Ca 2ϩ release from Ca 2ϩ stores (19). Control of the IP 3 R1 expression level on the endoplasmic reticulum in response to changes in the extracellular environment would be an important mechanism in maintaining intracellular Ca 2ϩ homeostasis.
DHPG-insensitive cells were still observed among the strongly IP 3 R1-positive neurons, suggesting the existence of other determinants of mGluR-mediated Ca 2ϩ release in those neurons. Differences in Ca 2ϩ signaling upon mGluR stimulation were also observed in acute hippocampal slices; mGluRmediated Ca 2ϩ release was observed in CA1 (39,40) and CA3 (41,42), but not in the granule cells of the dentate gyrus 2 or 2 T. Nakamura, personal observation. interneurons (see discussion in Ref. 43). Because IP 3 R1 is predominantly expressed in CA1, with substantially less expression in CA3 and only moderate levels in the granule cells of the dentate gyrus (34,35), other factors, including the expression level and the cell surface expression level of mGluRs (44,45), and mGluR-IP 3 R coupling status through an anchoring protein, Homer (46), may also be responsible for these differences in mGluR-IP 3 R signaling in different types of neurons.
NMDAR activation has been reported to increase the IP 3 R1 expression level in hippocampal neurons (26). This apparent contradiction of our results is probably caused by the difference in the duration of NMDAR blockade, because we observed a slight decrease in the IP 3 R1 expression level after 1 day of APV exposure (Fig. 2). Thus, NMDAR activation up-regulating IP 3 R1 expression in the short term seems consistent with their findings (26). After longer NMDAR blockade, however, the decreased IP 3 R1 expression level recovered to the basal level by day 3, and then increased after 6 days of APV exposure (Fig. 2). Thus, our results indicated that, although the IP 3 R1 expression level tends to decrease in response to NMDAR blockade for a short time, long lasting NMDAR blockade increases the IP 3 R1 expression level. This, together with the report that NMDAR activation increases IP 3 R1 expression though the calcineurin-NFAT pathway (26) and the finding that PKA activity is necessary for APV-induced IP 3 R1 expression in this study, suggests that various signaling pathways downstream of NMDAR regulate IP 3 R1 expression. Further study is necessary to understand the mechanism of the APV-induced IP 3 R1 expression.
PKA is one of the key factors in long term plasticity, namely, in late phase long term potentiation through a transcription factor, cAMP response element-binding protein (47). Our finding that the IP 3 R1 up-regulation by prolonged NMDAR blockade involved PKA activation suggests a new signal cross-talk between PKA and phosphatidylinositol pathways over a span of days. Because IP 3 R1 plays an important role in synaptic plasticity (7)(8)(9)(10)(11), PKA probably regulates long term synaptic plasticity by phosphorylating cAMP response element-binding protein, as well as by controlling IP 3 R1 expression at the transcription level. One of the candidates for the link between PKA and IP 3 R1 expression is activating protein-2 (AP-2), a transcription factor. AP-2 is highly expressed in the hippocampus and cerebellar Purkinje cells, where IP 3 R1 is predominantly expressed (48). In addition, AP-2 has been shown to directly regulate the IP 3 R1 promoter (49), and it is directly phosphorylated and activated by PKA (50). Further studies on the correlation between AP-2 phosphorylation levels and IP 3 R1 expression levels in APV-exposed hippocampal neurons would help to understand the mechanism of the up-regulation of IP 3 R1 by prolonged NMDAR inhibition. In summary, we have shown that chronic NMDAR blockade increases IP 3 R1 expression and that the neurons highly expressing IP 3 R1 protein are more sensitive to mGluR stimulation. Moreover, we have shown that the neurons overexpressing GFP-tagged IP 3 R1 became to show Ca 2ϩ release upon G-protein-coupled receptor stimulation. Our findings, thus, suggest that the IP 3 R1 expression level is an important determinant of the sensitivity of Ca 2ϩ stores to mGluR activation in hippocampal neurons. Further studies, including in vivo exper- iments, may elucidate the physiological significance of the APV-induced IP 3 R1 expression in hippocampal neurons.