Involvement of NMDAR2A tyrosine phosphorylation in depression‐related behaviour

The NR2A subunit of the NMDA receptor contains 25 tyrosine residues in the intracellular C‐terminal region. To determine the Src family kinase‐mediated NR2A phosphorylation sites, we constructed GST fusion proteins containing truncated segments of the NR2A C‐terminal region (termed GST–C1, GST–C2, and GST–C3) (Figure 1A). These fusion proteins were phosphorylated by baculovirally expressed Fyn in the presence of [γ‐³²P]ATP. The phosphorylated proteins were subjected to tryptic phosphopeptide mapping. As shown in Figure 1B, highly phosphorylated peptides P1–P7 were generated from the phosphorylated GST–fusion proteins. To identify the tyrosine residue in most prominently phosphorylated P7 peptide, we constructed Y1325F (conversion of Tyr 1325 to Phe 1325)–GST–C2 protein by site‐directed mutagenesis, and performed the in vitro phosphorylation assay as above. Conversion of Tyr 1325 to Phe 1325 resulted in generation of a tryptic phosphopeptide map that lacked phosphopeptide P7, suggesting that Tyr 1325 was most prominently phosphorylated by Fyn in vitro (Figure 1B). Similarly, phosphorylated tyrosines in other phosphopeptides were examined by introducing YF mutations in the GST–C1 and GST–C2 proteins. We found that Tyr 943, Tyr 1105, Tyr 1118, Tyr 1187, Tyr 1246, and Tyr 1267 were also phosphorylated in vitro (data not shown). As Tyr 1325 was most strongly phosphorylated in vitro, we examined whether Tyr 1325 was phosphorylated in cells (Figure 1C). HEK 293T cells were transfected with expression plasmids encoding wild‐type or Y1325F mutant forms of the NR2A subunit along with a plasmid encoding Src YF, a constitutively active form of Src. Immunoblotting of NR2A immunoprecipitates with the 4G10 anti‐phosphotyrosine antibody revealed that Y1325F mutation significantly decreased the tyrosine phosphorylation level of the NR2A subunits (n=4; ^*P<0.001, Student's t test; Figure 1C). The data suggest that Tyr 1325 was one of the principal Src‐mediated phosphorylation sites in HEK 293T cells as well as in vitro.

We next addressed the biological significance of Tyr 1325 phosphorylation. As NR2A phosphorylation by Src is suggested to potentiate NMDA receptor currents (Wang and Salter, 1994; Kohr and Seeburg, 1996; Yu et al, 1997), we examined the effect of null phosphorylation at Tyr 1325 by introducing Y1325F mutation in NR2A. Heteromeric NMDA receptor channels consisting of NR1 and wild‐type NR2A or Y1325F mutant (Y1325F–NR2A) were transiently expressed in HEK 293T cells and NMDA‐evoked whole cell currents were recorded. The current mediated by the NMDA receptor channel with wild‐type NR2A subunits was potentiated by the application of the Src protein through patch pipette (Figure 2A). In contrast, the current mediated by the NMDA receptor channel with Y1325F–NR2A subunits was minimally potentiated by the Src protein (Figure 2A). To confirm the lack of Src‐dependent NMDA receptor potentiation by Y1325F mutation, we examined the effect on Ca²⁺ influx. HEK293T cells were transfected with expression plasmids encoding NR1 and wild‐type (WT) or Y1325F mutant forms of NR2A together with or without active Src. After that the cells were loaded with fura2/AM and stimulated using NMDA. As shown in Figure 2B, a marked increase of Ca²⁺ concentration was observed in cells expressing NR1/WT–NR2A and active Src after NMDA stimulation, as compared with that in cells expressing NR1/WT–NR2A alone. In contrast, the level of increase in Ca²⁺ in cells expressing NR1/Y1325F–NR2A and active Src was virtually identical to that in cells expressing NR1/Y1325F–NR2A alone (Figure 2B). These data suggest that Tyr 1325 phosphorylation is required for Src‐mediated potentiation of NMDA receptor currents to modulate Ca²⁺ signalling downstream of NMDA receptors.

To examine the physiological role of NR2A Tyr 1325 phosphorylation, we generated mutant mice in which Tyr 1325 was substituted with Phe 1325 by a knock‐in technique (Figure 3A). The success of these procedures was confirmed by Southern blot and PCR analysis (data not shown). We confirmed the absence of Tyr 1325 phosphorylation in homozygous knock‐in mice (YF/YF mice) by immunoblot analysis using the anti‐pY1325 antibody that specifically recognizes NR2A phosphorylated at Tyr 1325 (Figure 3B and Supplementary Figure 1). The expression level of NR2A in YF/YF mice was virtually identical to that in wild‐type littermates (WT/WT mice) (Figure 3B). In YF/YF mice, the level of NR2A tyrosine phosphorylation was significantly reduced as compared with that in WT/WT mice (n=3; ^*P<0.001, Student's t test; Figure 3C), indicating that Tyr 1325 was one the major phosphorylation sites in vivo as well as in vitro (Figure 1). YF/YF mice were born according to Mendelian genetics and appeared healthy (data not shown). Histological analysis with Nissl‐stained coronal sections of central nervous system structures from YF/YF mice did not show any gross abnormalities in cytoarchitecture (Figure 3D). These findings suggest that Tyr 1325 phosphorylation may be relevant to the NMDAR function in mature brain.

To examine the effect of Tyr 1325 phosphorylation on NMDA receptor‐mediated excitatory postsynaptic currents (EPSCs) in vivo, we performed electrophysiological experiments on medium spiny neurons. Two glass micropipettes were placed in the cerebral cortex or in the underlying white matter to stimulate the corticostriatal axons. Stimulus pulses (duration: 0.1 ms; intensity: 0–80 V) were then applied between the pipettes to evoke EPSCs in medium spiny neurons. Application of the Src protein through a patch pipette potentiated the NMDA receptor‐mediated EPSCs in WT/WT slices (Figure 4). In contrast, NMDA receptor‐mediated EPSCs were not significantly changed by the Src protein in YF/YF slices (Figure 4). These results suggest that Tyr 1325 phosphorylation is required for Src‐induced potentiation of the NMDA receptor‐mediated EPSCs in medium spiny neurons, which is consistent with findings from recombinantly expressed NMDA receptor in HEK 293T cells (Figure 2).

We performed a battery of behavioural tests to examine sensory and motor functions as well as cognition and anxiety of YF/YF mice. The tests revealed that YF/YF mice were less immobile than WT/WT mice in the tail suspension test (Figure 5A and B), one of the most widely used models for assessing antidepressant‐like activity in mice (Cryan et al, 2005). YF/YF mice also showed significantly less immobility than WT/WT mice in the forced swim test (Figure 5C and D), another test for assessing antidepressant‐like activity (Petit‐Demouliere et al, 2005). Given that, the higher the mobility, mice are less depressive (Cryan et al, 2005; Petit‐Demouliere et al, 2005), we postulated that YF/YF mice were less susceptible to depression than WT/WT mice. Alternatively, the reduced immobility of YF/YF mice in these tests might have been caused by the increase in spontaneous activity (Crawley, 2007). To check this possibility, we performed the open field test to measure spontaneous locomotor activity of YF/YF mice (Figure 5E). The results showed that there were no significant differences between WT/WT and YF/YF mice in the number of rearing (measure the number of beam breaks) (WT/WT, 47.9±4.10, n=13; YF/YF, 51.5±4.49, n=13; F_(1,24)=0.35, P=0.56, one‐way ANOVA) and the total distance travelled during the test (WT/WT, 3435±191 cm, n=13; YF/YF, 3400±182 cm, n=13; F_(1,24)=0.018, P=0.89, one‐way ANOVA), suggesting that spontaneous activity was virtually normal in YF/YF mice. As alteration in fearfulness can also affect the performance in the tests for depression (Crawley, 2007), we also analysed the anxiety‐related behaviour. We did not detect any significant abnormality in YF/YF mice in the open field test (the time spent in the centre, WT/WT, 13.0±1.78%, n=13; YF/YF, 10.2±1.10%, n=13; F_(1,24)=1.83, P=0.19, one‐way ANOVA; Figure 5E), the light–dark emergence test (the time spent in the lighted area: WT/WT, 40.9±3.15%, n=13; YF/YF, 42.6±4.14%, n=13; F_(1,24)=0.11, P=0.74, one‐way ANOVA; the number of transitions: WT/WT, 24.9±2.10, n=13; YF/YF, 21.7±2.96, n=13; F_(1,24)=0.79, P=0.38, one‐way ANOVA; Figure 5F), the social interaction test (the number of social interactions: WT/WT, 14.5±1.7, n=11; YF/YF, 14.5±0.8, n=11; F_(1,20)=0.002, P=0.96, one‐way ANOVA; the social interaction time, WT/WT, 12.7±2.06%, n=11; YF/YF, 16.4±2.66, n=11; F_(1,20)=1.18, P=0.28, one‐way ANOVA; Figure 5G), and the elevated plus maze test in YF/YF mice (the time on open arms: WT/WT, 15.68±1.48%, n=13; YF/YF, 16.45±2.85%, n=13; F_(1,24)=0.06, P=0.81, one‐way ANOVA; the number of entries into open arms: WT/WT, 28.2±2.02, n=13; YF/YF, 30.7±2.76, n=13; F_(1,24)=0.55, P=0.47, one‐way ANOVA; the number of head dips: WT/WT, 42.5±7.69, n=10; YF/YF, 43.5±9.89, n=10; F_(1,18)=0.006, P=0.94, one‐way ANOVA; Figure 5H). In addition, the performance of YF/YF mice in the Morris water maze test, contextual fear conditioning test, auditory fear conditioning test, acoustic startle response test, pre‐pulse inhibition test, tail flick test, and hot plate test appeared normal (Supplementary Figure 2‐4). These results suggest that YF/YF mice showed reduced susceptibility to depression, but other general behaviours, such as locomotor activity, cognitive function, anxiety‐related behaviour, startle response, and pain behaviour are normal.

We next investigated whether the level of Tyr 1325 phosphorylation in the striatum, one of the brain regions involved in depression (Petit‐Demouliere et al, 2005), was affected by the forced swim test. At 5 min after the test, the mice were sacrificed, the striata were dissected out, and the lysates were prepared. The NR2A immunoprecipitates from these lysates were then subjected to immunoblotting with the anti‐pY1325 antibody followed by re‐blot with the anti‐NR2A antibody. As shown in Figure 6A, the level of Tyr 1325 phosphorylation was increased significantly in the striatum in mice exposed to the forced swim test compared with control (n=3; ^*P<0.05, Student's t test). Likewise, the level of Tyr 1325 phosphorylation was also increased in the striatum in mice exposed to the tail suspension test (n=5; ^*P<0.05, Student's t test; Figure 6B). We also found that Src was activated by the forced swim test using anti‐Src (pY418) antibody specific to the active form of Src (Figure 6A; n=3; ^*P<0.05, Student's t test). Neither Ser 896 phosphorylation on NR1 (Figure 6A) nor Tyr 1472 phosphorylation on NR2B (Figure 6A and B) was affected by the same stimulation. In addition, the level of Tyr 1325 phosphorylation in the striatum was not at all affected by the elevated plus maze and the open field test (Supplementary Figure 5). These data suggest that Tyr 1325 phosphorylation of NR2A is relevant to the depression‐related behaviour.

Given that depression‐related behaviour is affected by monoamine systems (Cryan and Mombereau, 2004), we measured the amounts of monoamines and their metabolites in various brain regions, including the hippocampus, hypothalamus, prefrontal cortex, and striatum of WT/WT and YF/YF mice (Figure 7A–F and data not shown). We did not detect any significant abnormalities in the levels of dopamine and serotonin and their metabolites in any of the brain region we examined, including the striatum of YF/YF mice (Figure 7A–F and data not shown), suggesting that monoamine systems are normal in YF/YF mice. In addition, the levels of glutamate and GABA were also unaltered in the striatum of YF/YF mice compared with those of WT/WT mice (Figure 7G and H).

DARPP‐32, a dopamine‐ and cAMP‐regulated phosphoprotein of 32 kDa, is a signal transduction molecule that is highly enriched in medium spiny neurons of the neostriatum (Svenningsson et al, 2004). There is a report that DARPP‐32 phosphorylation at Thr 34 is involved in the regulation of depression‐related behaviours (Svenningsson et al, 2002). This led us to examine whether Thr 34 phosphorylation of DARPP‐32 is altered in YF/YF mice. As shown in Figure 8A, Thr 34 phosphorylation of DARPP‐32 was significantly increased in the striatum of YF/YF mice as compared with that of WT/WT mice (^*P<0.05, Student's t test; Figure 8A). We then examined the ERK activity because Thr 34 phosphorylation of DARPP‐32 occurs upstream of ERK activation (Valjent et al, 2005). We observed that the activity of ERK, evaluated by the anti‐phospho‐ERK specific antibody, was significantly higher in YF/YF mice compared with that in WT/WT mice (^*P<0.05, Student's t test; Figure 8B). Therefore, we conclude that Thr 34 phosphorylation on DARPP‐32 and the subsequent ERK activity are elevated in the absence of Tyr 1325 phosphorylation on the NR2A subunit. Given that Thr 34 phosphorylation of DARPP‐32 is important for depression‐related behaviour (Svenningsson et al, 2002, 2004), antidepressant‐like phenotype of YF/YF mice may be attributable to increased Thr 34 phosphorylation of DARPP‐32.

We next addressed the mechanism by which DARPP‐32 phosphorylation at Thr 34 was increased in YF/YF mice. We focused on the Ca²⁺/calmodulin‐dependent protein phosphatase calcineurin, as it dephosphorylates Thr 34 of DARPP‐32 downstream of NMDA receptor activation (Nishi et al, 2005). We analysed calcineurin activity in the striatum by measuring the free phosphate released from the synthetic RII phosphopeptide substrate, the most efficient and well‐known peptide substrate of calcineurin (Enz et al, 1994). As expected, calcineurin activity was significantly decreased in YF/YF mice as compared with that in WT/WT mice (^*P<0.05, Student's t test; Figure 8C). These results suggest that loss of Tyr 1325 phosphorylation downregulates calcineurin activity, and the impaired calcineurin activity is responsible for the increased DARPP‐32 phosphorylation at Thr 34 (Figure 9). Intriguingly, however, we did not detect any significant reduction in CaMKII activity in YF/YF mice (Figure 8D), despite the fact that CaMKII activity is also regulated by Ca²⁺ (see Discussion).

Fusion proteins, GST–C1, GST–C2, GST–C3, and GST–C2–Y1325F were expressed in Escherichia coli BL21. Purification of GST fusion proteins was performed as described previously by Nakazawa et al (2001).

Tryptic peptide mapping of GST fusion proteins was performed as described previously by Nakazawa et al (2001).

The culture of HEK 293T cells and their transient transfection were performed as described previously by Nakazawa et al (2001).

Preparation of lysate from the mouse brain, immunoprecipitation, and immunoblotting were performed as described previously by Nakazawa et al (2006). For quantification, the immunoreacted protein bands were analysed using the NIH image software.

Rabbit polyclonal antibodies against phospho‐Tyr 1325 were raised using a keyhole limpet hemocyanin‐conjugated synthetic phosphopeptide around Tyr1325 as immunogen. The antibody was purified from sera of the immunized rabbits by successive affinity chromatography. The anti‐NR2A antibody and anti‐pY1472/NR2B antibodies have been described in previous studies (Tezuka et al, 1999; Nakazawa et al, 2001). Commercially available antibodies used in this study are: anti‐phosphotyrosine (4G10; Upstate Biotechnology, Waltham, MA, USA); anti‐phospho Thr 34 DARPP‐32 (PhosphoSolutions, Aurora, CO, USA); anti‐DARPP‐32 (PhosphoSolutions); anti‐phospho ERK (Cell Signaling Technology, Beverly, MA, USA); anti‐ERK2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti‐phospho‐Src (Cell Signaling Technology); anti‐Src family (Santa Cruz Biotechnology); anti‐phospho‐CaMKII (pT286; Promega, Madison, WI, USA); and anti‐CaMKII (Chemicon International, Temecula, CA, USA) antibodies.

Cloned cDNAs for the rat NMDA receptor subunits NR1E and NR2A, inserted into the eukaryotic expression vector pME, were used to transfect HEK 293T cells as described by Nakazawa et al (2001). Whole‐cell recordings were performed as described previously by Miwa et al (2008). Cells on coverslips were kept in a chamber continuously perfused with Ringer's solution (135 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 5 mM HEPES (pH 7.2), 10 mM glucose), which was saturated with 95% O₂ and 5% CO₂. During giga‐seal formation, single captured cells were lifted from the bottom of the dishes. After establishing the whole‐cell configuration, short pulses of NMDA stimulus solution (100 μM NMDA and 10 μM glycine in Ringer's solution: 0.5 s) were applied to the recorded cell every 30 s with the local perfusion system (SF‐77B Perfusion Fast‐Step, Harvard Apparatus, Hamden, CT, USA). The patch pipette solution contained: 140 mM CsCl, 1.0 mM MgCl₂, 0.1 mM EGTA, 10 mM HEPES (pH 7.2), 4 mM ATP with or without recombinant Src (30 U/ml) (Upstate Biotechnology). The NMDA receptor‐mediated currents were recorded at a holding potential of −60 mV. MultiClamp 700A amplifier and Digidata 1322A (Molecular Devices, Sunnyvale, CA, USA) were used to record and store data using the pClamp software (Molecular Devices), respectively. All experiments were performed at 25–28°C.

Measurements of cytosolic free calcium concentrations using an acetoxymethyl ester of fura‐2 (fura‐2/AM; Invitrogen, Carlsbad, CA, USA) were performed as described previously by Yokoyama et al (2002). The ratio of increase in Ca²⁺ concentration, during the 30‐s interval after NMDA stimulation (500 μM) relative to that during the 30‐s interval before the NMDA stimulation in cells expressing NR1/WT–NR2A (without Src), was assigned the arbitrary value 1.

To construct the targeting vector, a genomic DNA fragment carrying the carboxy‐terminal coding region of the NR2A gene was isolated from a C57BL/6 genomic library (Stratagene, La Jolla, CA, USA). The 3′ fragment of NR2A contained the Tyr 1325 mutated to phenylalanine. Embryonic stem cell line E14.1 (129/Ola‐derived) was electroporated with the linearized targeting vector, and positive clones were identified by PCR and confirmed by Southern blotting using the outer probe. Positive clones were used to generate chimeric mice through the aggregation method. The chimeric mice were crossed to the transgenic mice carrying the CAG–cre transgene with C57BL/6 background (Sakai and Miyazaki, 1997) to remove the neo cassette through Cre/lox‐P‐mediated excision. After confirmation of the loss of the neo gene by PCR and Southern blotting, the mice were crossed to C57BL/6J mice to yield heteromeric F2 mice with a 75% pure C57BL/6J genetic background. Heterozygous mice were backcrossed successively to C57BL/6J mice to yield subsequent generations with more pure C57BL/6J genetic backgrounds. F5–7 heterozygous mice were crossed to each other to yield homozygous mice and wild‐type littermates. Experiments using animals were carried out in accordance with the guidelines for animal use issued by the Committee of Animal Experiments, Institute of Medical Science, University of Tokyo.

Histological analyses were performed as described previously by Nakazawa et al (2006).

Coronal brain slices containing the cortex and the striatum (300 μm thick) were prepared from WT/WT or YF/YF mice at postnatal day 16–28 as described previously by Narushima et al (2006). In brief, mice were decapitated under anaesthesia with 100% CO₂, and the brains were cooled in ice‐cold, modified external solution(120 mM Choline–Cl, 2 mM KCl, 8 mM MgCl₂, 28 mM NaHCO₃, 1.25 mM NaHPO₄, and 20 mM glucose (bubbling with 95% O₂ and 5% CO₂)). Slices were cut using a Leica VT1200 slicer (Wetzlar, Germany). For recovery, slices were incubated for at least 1 h in the normal bathing solution(125 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 1.25 mM NaH₂PO₄, 26 mM NaHCO₃, and 20 mM glucose (pH 7.4) (bubbling with 95% O₂ and 5% CO₂)).

Whole‐cell recordings were made from medium spiny neurons in the dorsolateral region of the striatum using an upright microscope (BX50WI; Olympus Optical, Tokyo, Japan) equipped with an infra‐red CCD camera system (Hamamatsu Photonics, Hamamatsu, Japan). Medium spiny neurons were identified visually through their medium‐sized, spherical somata as well as their electrophysiological properties (Kita et al, 1984). Resistance of the patch pipette was 2–3 MΩ when filled with the intracellular solution 60 mM CsCl, 10 mM Cs‐gluconate, 20 mM TEA–Cl, 30 mM HEPES, 20 mM BAPTA‐K₄, 4 mM MgCl₂, 4 mM Na₂‐ATP, 0.4 mM Na₂‐GTP (pH 7.3, adjusted with CsOH). Membrane currents were recorded with an EPC9/2 amplifier (HEKA Electronik, Lambrecht/Pfalz, Germany) and the pipette access resistance was compensated by 80%. The PULSE software (HEKA Electronik) was used for stimulation and data acquisition. The signals were filtered at 3 kHz and digitized at 20 kHz. To stimulate corticostriatal axons, two glass micropipettes filled with the normal saline were placed in the cerebral cortex or in the underlying white matter. Stimulus pulses (duration: 0.1 ms; intensity: 0–80 V) were applied between the pipettes to evoke EPSCs in medium spiny neurons.

For assessing the effect of Src on NMDA receptor‐mediated EPSCs, Src (60 U/ml) was applied intracellularly from the recording pipette by diffusion. At the holding potential of +50 mV, NMDA receptor‐mediated EPSCs were monitored after every 20 s starting from 2 min after the establishment of whole‐cell recording. The bath solution was supplemented with picrotoxin (100 μM) and NBQX (10 μM) to block GABA_A receptor‐mediated inhibitory post‐synaptic currents and AMPA receptor‐mediated EPSCs, respectively.

Mice (8–10‐week‐old) were suspended above the floor by fixing the end of their tail to wire netting. The mice were manually observed for the presence or absence of immobility during the 6‐min test session. All behavioural experiments were performed blindly.

Mice (8–10‐week‐old) were placed in a cylinder filled with water temperature of which was between 23 and 25°C. The mice were manually observed for the presence or absence of immobility during the 10‐min test session.

The apparatus was a square arena (50 × 50 × 33.3 cm (W × D × H)) made of polyvinyl chloride. A mouse was placed in the perimeter and allowed to explore the apparatus for 15 min. The total distance travelled in the arena, the time spent in the centre (30 × 30 cm), and rearing activity were analysed with a Macintosh computer using Image OFCR 1.00 × and Image OF circle 1.01 × (O'Hara & Co. Ltd., Tokyo, Japan), a modified software based on the public domain of NIH Image programme.

The light–dark box consisted of two compartments: a transparent polyvinyl chloride box (100 lux) and a black polyvinylchloride box (both 19 × 19 × 19 cm). Two boxes were separated by a vertical sliding door that remained open (5 × 5 cm). The amount of time spent in the transparent and black boxes was measured for 10 min.

A male subject mouse was placed in a neutral cage for 30 min, after which a juvenile (5 weeks of age) conspecific male subject was introduced. The interaction between the male subject and the juvenile conspecific male subject was recorded using a video camera for 3 min. Social interaction was determined as the total amount of time that the male subject spent on interaction with the juvenile conspecific male subject.

The elevated plus maze (EP‐3002; O′ Hara & Co. Ltd.) consisted of two open arms (25 × 5 cm) and two enclosed arms of the same size extending from a central area (5 × 5 cm) and elevated 50 cm from the ground. Mice were placed in the central square of the maze facing one of the open arms. Mouse behaviour was recorded during a 10‐min test period by means of a Macintosh computer using Image OFCR 1.00 × and Image OF circle 1.01 × (O'Hara & Co. Ltd.), a modified software based on the public domain of NIH Image programme. The following conventional parameters were recorded: the number of entries into open or closed arms and the time spent in open or closed arms. The number of head dips (a behaviour in which the mouse dips its head into open space and observes the environment while the body is in an open arm) was also recorded.

Circular tissue punches (1 mm diameter) were taken from 150‐μm thick frozen coronal brain sections obtained from 8‐week‐old mice (n=6 per group) and stored at −80°C until assayed. Monoamines, amino acids, and metabolites were extracted by sonication in 0.1 M perchloric acid containing a mixture of internal standards (200 nM isoproterenol, 200 μM l‐norleucine, and 5 μM ethylhomocholine). Protein contents were measured using a DC Protein Assay Kit (Bio‐Rad, Hercules, CA, USA). The remainder of the sample was centrifuged for 15 min at 15 000 r.p.m. and the supernatant filtered through 0.22‐mm micropore, polyvinylidene fluoride filters (Millipore Corp. Billerica, MA, USA) by centrifugation (14 000 r.p.m. for 5 min). The filtrate was analysed by high‐pressure liquid chromatography coupled to electrochemical detection (Eicom, Kyoto, Japan). Briefly, an Eicompak SC‐5ODS 3.0 × 150 mm column was used for separations, perfused with a mobile phase consisting of citrate (41.4 mM), sodium acetate (39.2 mM), methanol (17%), sodium 1‐octanesulfonate (190 mg/l), and EDTA (5 mg/l), adjusted to pH 3.7 using glacial acetic acid and pumped at a rate of 0.5 ml/min. The working electrode (WE‐3G) potential was set at +0.75 V. Amino acid content was analysed by HPLC using fluorometric detection. Amino acids were derivatized with o‐phthalaldehyde (OPA) before automated injection into the HPLC column (methods essentially similar to Murphy and Maidment (1999)) and resolved using a gradient on a reverse‐phase column with sodium acetate (35 mM, pH 5.9 adjusted using glacial acetic acid), 1% tetrahydrofuran, as aqueous solvent. The organic mobile phase consisted of 70% acetonitrile, 15% methanol, and 35 mM sodium acetate (pH 7.65, adjusted using glacial acetic acid). The flow rate was 0.6 ml/min. Data were collected and analysed by Ezchrom Elite software (Scientific Software, Lincolnwood, IL, USA). All analysis information, including the retention times, peak heights, concentrations, and recovery rate of internal standards, were calculated by comparison to standard curves generated for known concentrations of external standards that were run daily. Turnover rates were calculated as follows: dopamine (DA) turnover rate=[3,4‐dihydroxyphenylacetic acid (DOPAC)]+[homovanillic acid (HVA)]/[DA]; serotonin (5‐HT) turnover rate=[5‐hydroxyindoleacetic acid (5‐HIAA)]/[5‐HT].

Calcineurin activity was measured by using the synthetic phosphorylated RII peptide using a calcineurin assay kit (Biomol, Plymouth Meeting, PA, USA) following the manufacturer's instructions.

All data are expressed as mean±s.e.m values. Statistical analysis was done using Student's t‐test, paired t‐test, and one‐way ANOVA. Differences with P<0.05 were considered as significant.

Supplementary data are available at The EMBO Journal Online (http://www.embojournal.org).