J. Neurochem. (2011) 118, 773–783.
Regulatory mechanisms of ryanodine receptor (RyR) expression are not well known, although methamphetamine (METH) has been reported to up-regulate RyRs in mouse brain. This study investigate regulatory mechanisms of RyR expression by dopaminergic system using the midbrain and cerebral cortical neurons in primary culture intermittently exposed to METH and dopamine receptor (DR) agonists (1 h/day, for 3 days). Intermittent METH (10 μM) exposure enhanced RyR-1 and -2 proteins and their mRNA, but not RyR-3 expression in the both types of the neurons. These METH-induced increases of RyR proteins and their mRNA were dose-dependently blocked by SCH23390 (a selective D1DR antagonist), but not a D2DR antagonist sulpiride, suggesting a regulatory role of D1DRs in RyR expression by METH in these neurons. In cerebral cortical neurons, intermittent SKF82958 (a selective D1DR agonist) exposure increased RyR-1 and -2 proteins and their mRNA, whereas quinpirole (a selective D2DR agonist) showed no effects. KT5720, a protein kinase A inhibitor, dose-dependently attenuated the METH-stimulated RyR-1 and -2 expressions in cerebral cortical neurons. METH significantly increased phosphorylation of cAMP-response element-binding protein, which was completely suppressed by SCH23390. These results indicate that RyR-1 and -2 expressions are regulated by D1DRs via the signal transduction linked to D1DRs.
l-amino-aromatic acid decarboxylase
cAMP-response element-binding protein
- D1DRs and D2DRs
dopamine D1 and D2 receptors
Dulbecco’s modified Eagle’s medium
Krebs-Ringer bicarbonate buffe
protein kinase A
Neuronal calcium signals produce diverse physiological responses in different neuronal functions including neurotransmitter release, synaptic plasticity, gene expression, and axonal growth. Calcium signals can arise in many types of cells via calcium entry through calcium channels present on plasma membrane, and this calcium entry is activated by voltage-dependent membrane depolarization and neurotransmitter receptor stimulation (Berridge 1998; Berridge et al. 2003; Dolmetsch 2003). In addition to such extracellular calcium entry into cells, calcium release from intracellular calcium store into cytosol also plays a biochemical role as a member of calcium signaling system (Hidalgo et al. 2004, 2005; Hidalgo 2005). Many investigations have revealed that ryanodine receptors (RyRs) are one of channel proteins to release calcium from intracellular calcium store into cytosol (Meissner et al. 1988; McPherson et al. 1991). RyRs are classified to multigene family of channel proteins that mediate calcium release from intracellular calcium stores such as the endoplasmic reticulum into cytosol. Three different genes coding for isoforms of RyRs have been identified and cloned (RyR-1, RyR-2 and RyR-3). RyR-1 is expressed preferentially in skeletal muscle, whereas RyR-2 is mainly cardiac (Otsu et al. 1990). All of three isoforms of RyRs and their mRNA were detected in brain with RyR-2 mRNA showing the most abundant expression (Furuichi et al. 1994; Giannini et al. 1995). In addition, enhancement of calcium release from intracellular calcium stores by stimulation of ryanodine receptors (RyRs) as well as of inositol-1, 4, 5-tisphosphate receptors also gives rise to calcium signals recognized as calcium-induced calcium release that is considered to propagate small and localized calcium signals to mitochondria and nucleus with signal amplification (Meldolesi 2001). Although several studies have demonstrated modification of expression of RyRs under several pathophysiological conditions such as cerebral ischemia and in mouse model of Alzheimer’s disease (Bull et al. 2008; Supnet et al. 2009), there are few available data to define regulatory mechanisms of RyRs in the CNS under physiological and pathophysiological conditions (Edwards and Rickard 2007; Forrest et al. 2010).
Among the factors involving in the development of psychostimulant dependence, the intracellular increase in calcium concentration via RyRs (Kurokawa et al. 2010a) as well as l-type voltage-gated calcium channels (Pierce et al. 1998; Licata et al. 2000) has been reported to be important for psychostimulant-induced behavioral and neurochemical changes. In the case of the former event, increased expression of RyRs in the mouse frontal cortex and limbic forebrain, the brain regions participating in the development of psychological dependence, occurs in mice showing methamphetamine (METH)-induced place preference, which is considered to be one of suggestive behavioral makers defined as the development of psychostimulant-induced psychological dependence, and this METH-induced place preference was significantly suppressed by dantrolene, a RyR antagonist (Kurokawa et al. 2010a). It is therefore considered that RyRs may one of important players to be involved in the development of METH-induced place preference via modification of intracellular Ca2+ dynamics.
METH-induced elevation of extracellular dopamine concentration results in complex neurochemical changes and profound psychiatric effects (Segal and Kuczenski 1997; Rothman and Baumann 2003). Dopamine signals are mediated by two major subfamilies of dopamine receptors, termed as dopamine D1 and D2 receptors (D1DRs and D2DRs), for producing rewarding effects of drugs of abuse (Hanson et al. 1992; Mizoguchi et al. 2004; Kurokawa et al. 2010b). We also demonstrated that increased expression of RyRs in the brain of mouse showing METH-induced place preference was significantly suppressed by blockade of D1DRs, but not of D2DRs (Kurokawa et al. 2010b). These results suggest that RyRs play a significant role in METH-induced psychological dependence and that DRs may participate, in part, in up-regulation of RyR expression in the brain of mice showing METH-induced place preference.
The present study therefore attempts to confirm mechanisms to regulate RyR expression, especially how D1DRs regulate it, using mouse midbrain and cerebral cortical neurons in primary culture exposed intermittently to METH and drugs directly acting on DRs.
Materials and methods
Primary culture of midbrain and cerebral cortical neurons
Pregnant female ddY mice with gestation of 15 days (12 weeks old; Japan SLC, Inc., Hamamatsu, Japan) were used to isolate neurons.
Isolation and primary culture of midbrain and cerebral cortical neurons were carried out according to the method described previously (Ohkuma et al. 1986; Kurokawa et al. 2011) with a minor modification. In brief, the midbrain and neopallium free of meninges were dissected from 15-day-old fetuses of ddY strain mice anesthetized, minced, treated with trypsin, and centrifuged. The pellet obtained was suspended with Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 15% bovine calf serum, and an aliquot of cell suspension (cell number 3 × 106 cells/mL) was innoculated in a poly-l-lysine-pre-coated culture dish and cultured in humidified 95% air–5% CO2 at 37°C for 3 days. The cells were further cultured in DMEM containing 10% horse serum under the same conditions described above after exposing the cells to 10 μM cytosine arabinoside dissolved in DMEM with 10% horse serum for 24 h to inhibit the proliferation of non-neuronal cells. The neurons were used for the following experiments on 13th day of the culture. More than 95% of cultured cells were identified as the neurons by immunohistochemical analysis (Ohkuma et al. 1986).
All experiments presented in this study were approved by the Animal Research Committee of Kawasaki Medical School and conducted according to the ‘Guide for Care and Use of Laboratory Animals’ of Kawasaki Medical School that is based on the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996.
Exposure of the neurons to agents
In this study, the neurons were intermittently exposed to METH. That is, the neurons were exposed to METH in DMEM supplemented with 10% horse serum for 1 h and were then cultured for following 23 h in the same medium without METH after two times washings of the neurons with Hank’s solution. This exposure schedule of the neurons to METH was carried out for 3 days and then protein for analyzing RyRs expression was extracted.
SKF82958 (a full D1DR agonist) and quinpirole (a selective D2DR agonist) were exposed to the neurons with the protocol similar to that of METH exposure. SCH23390 (a D1DR antagonist), sulpiride (a D2DR antagonist), and KT570 (an inhibitor of protein kinase A) were dissolved in Hank’s solution and directly added in the culture medium 10 min before the addition of METH or DR agonists and then the neurons were exposed to METH or agonists for 1 h. Such exposure schedule of the neurons to agents was carried out for 3 days and then protein for analyzing RyRs expression was extracted. Accordingly, the duration of exposure of the neurons to DR antagonists used here were 70 min every day.
Even after the exposure to METH and agents acting on DRs, the activity of the neurons to exclude trypan blue did not change (data not shown), indicating that the present experimental conditions to expose the neurons to these drugs induce no cellular toxicity.
The neurons were scraped off from culture dishes and homogenized in 10 volumes of ice-cold buffer containing 10 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 0.5 mM EDTA, 10 mM NaF, 0.5% Triton X-100, and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) using a Potter-Elvehjem tissue grinder with a Teflon pestle. The homogenate thus obtained was centrifuged at 1000 g for 10 min at 4°C. The resultant supernatant was thereafter centrifuged at 100 000 g for 60 min at 4°C to obtain the pellets that were retained as the sample for western blot analysis.
The electrophoresis (applied protein: 20 μg/lane for RyR-1 and -3, 5 μg/lane for RyR-2) was carried out using 3–8% Tris-acetate gel (Invitrogen, Carlsbad, CA, USA) with size of 8 × 8 cm and thickness of 1.0 mm (150 V, 60 min) (Kurokawa et al. 2011). The proteins separated on the gel were transferred to a nitrocellulose filter with a wet type transblotter (90 V, 60 min), and the nitrocellulose filter was incubated over night at 4°C with primary antibodies against RyR-1 (mouse monoclonal anti-ryanodine receptor type-1), RyR-2 (mouse monoclonal anti-ryanodine receptor type-2), RyR-3 (rabbit polyclonal anti-ryanodine receptor type-3), cAMP-response element binding protein (CREB) (rabbit polyclonal anti-CREB), and phospho (ser133)-CREB (mouse monoclonal anti-phospho (ser133)-CREB: pCREB) diluted in phosphate-buffered saline containing 5% non-fat dried milk and then further incubated for 2 h at 25°C with horseradish peroxidase-conjugated goat anti-mouse IgG or horseradish peroxidase-conjugated goat anti-rabbit IgG diluted 1 : 5000 in phosphate-buffered saline containing 5% non-fat dried milk. Finally, separated proteins were detected with chemiluminescence (Thermo Fisher Scientific, Rockford, IL, USA).
Monoclonal antibodies for RyR-1 and -2 obtained from Sigma-Aldrich (St. Louis, MO, USA) show that each of RyR-1 and -2 is identified as one band with the expected molecular weight of approximately 550–560 kDa as reported previously (Ouardouz et al. 2003). However, we analyzed RyR-1 and -2 expressions using polyclonal antibodies. As a results, these antibodies detected several bands including one band with the expected molecular weight of RyR-1 or -2. In addition, polyclonal antibody for RyR-3 used here show only one band with the expected molecular weight and other several bands with molecular weight smaller than that of RyR-3 on western blotting. Based on these experimental data, we concluded that the polyclonal antibody for RyR-3 used here would be satisfactory to detect the expression of RyR-3 (Appendix S1, Figure S6).
Using a Nonodrop spectrophotometer (Wilmington, NC, USA), the amount of total RNA, which was prepared from the neurons using TRIzol reagents (Invitrogen Co., Tokyo, Japan) according to the standard protocol for RNA extraction, was quantified to measure OD260. Total RNA (200 ng) was reacted with Prime Script reverse transcriptase (Takara, Kyoto, Japan). Quantitative PCR was performed with One SYBR® PrimeScript ®RT-PCR kit II (Takara) using 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the protocol supplied by the manufacturer. The data were analyzed by 7500 system SDS Software 1.3.1 (Applied Biosystems) using the standard curve method. The sequences of the primers for RyR-1 and RyR-2 were as followed: RyR-1 forward primer, 5′-AAGTCCCACAACTTTAAGCG-3′ and reverse primer: 5′-TCTTCTTGGTGCGTTCCTG-3′ (NM_009109.2). RyR-2 forward primer: 5′-AGCTTGAAAGACACCGAGGA-3′ and reverse primer: 5′-TAGAGAGCCATCTGCCACCT-3′ (NM_023868.2) (Kurokawa et al. 2011).
Methamphetamine hydrochloride was obtained from Dainippon Pharmaceutical Co. (Tokyo, Japan). SCH23390, sulpiride, SKF82958, quinpirole, KT570, and actinomycin D were the products of Sigma-Aldrich. Antibodies against RyR-1 (mouse monoclonal anti-ryanodine receptor type-1), RyR-2 (mouse monoclonal anti-ryanodine receptor type-2), and phospho (ser133)-CREB (mouse monoclonal anti-phospho (ser133)-CREB) were also purchased from Sigma-Aldrich. Antibodies against RyR-3 (rabbit polyclonal anti-ryanodine receptor type-3) and CREB (rabbit polyclonal anti-CREB) were obtained from Millipore Bioscience Research Reagents (Temecula, CA, USA). Horseradish peroxidase-conjugated goat anti-rabbit IgG and horseradish peroxidase-conjugated goat anti-mouse IgG were the products of Southern Biotechnology Associates Inc., (Birmingham, AL, USA). Other agents used here were locally available and of analytical grade.
Protein concentration in samples was assayed by the method of Lowry et al. (1951) using bovine serum albumin as standard.
Each of data is presented as the mean ± SEM. Statistical analysis was performed using Prism 5 (GraphPad, Inc., San Diego, CA, USA). Independent group t-test were used for comparisons between two experimental groups. For multiple groups, the statistical significance of differences was assessed by the methods described in each figure legend after the application of one-way anova followed by Bonferroni multiple comparisons test with the significance level set at p < 0.05 or Dunnett’s post hoc test where appropriate.
Methamphetamine increased RyR expression in mouse midbrain and cerebral cortical neurons
To study whether there was the difference in RyR expression after intermittent METH exposure in the presence or absence of DR antagonists in the midbrain and cerebral cortical neurons of mice, we first determined how METH affected RyR protein levels in these two types of neurons. In the midbrain neurons, the intermittent exposure to METH (10 μM) increased the expression of RyR-1 and -2 proteins in a dose-dependent manner, whereas such manipulation showed no effects on RyR-3 expression (Fig. 1a) [anova analysis: RyR-1 F(3,8) = 11.42, p = 0.0029; RyR-2 F(3,12) = 32.48, p < 0.0001; RyR-3 F(3,12) = 0.49, p = 06928, not significant]. Dunnett’s multiple comparison post hoc tests confirmed that one or three tested doses of METH significantly increased RyR-1 and -2 expressions, respectively, when compared with control: [RyR-1; 10 μM (q = 5.51, p < 0.05), RyR-2; 1 μM (q = 3.18, p < 0.05), 3 μM (q = 7.65, p < 0.001), 10 μM (q = 8.67, p < 0.001) vs. control group by post hoc test]. In the cerebral cortical neurons the intermittent exposure to METH (10 μM) increased the expression of RyR-1 and -2 proteins in a dose-dependent manner, but not the expression of RyR-3 (Fig. 1b) [anova analysis: RyR-1 F(3,12) = 4.81, p = 0.02; RyR-2 F(3,12) = 6.81, p = 0.0062; RyR-3 F(3,12) = 0.09539, p = 0.9611, not significant]. Dunnett’s multiple comparison post hoc tests confirmed that one tested dose of METH significantly enhanced RyR-1 and -2 expressions when compared with control: [RyR-1; 10 μM (q = 3.5, p < 0.05), RyR-2; 10 μM (q = 4.09, p < 0.01) vs. control group by post hoc test].
Effects of DR blockade on METH-induced increase of RyR expression in mouse midbrain and cerebral cortical neurons
As METH produces its pharmacological action via increased extracellular dopamine concentration, the possibility that the increase of RyR proteins by METH may be regulated via dopamine receptor-mediated signal transduction system is considered. In addition, we have reported that D1 and D2 subtypes of DRs are predominantly enriched in the cerebral cortical neurons (Kurokawa et al. 2010b). We therefore checked effects of DR blockade on METH-induced increase of RyR-1 and -2 expressions using antagonists selective to D1DRs or D2DRs, SCH23390 and sulpiride, respectively, in the mouse midbrain and cerebral cortical neurons. As shown in Fig. 2, SCH23390 significantly suppressed the METH-induced enhancement of both RyR-1 and -2 proteins in a dose-dependent manner in the midbrain [anova analysis: RyR-1; F(5,18) = 6.11, p = 0.0018, RyR-2; F(5,18) = 6.33, p = 0.0015] and cerebral cortical neurons [anova analysis: RyR-1; F(5,18) = 3.89, p = 0.015, RyR-2; F(5,18) = 27.38, p < 0.0001]. Post hoc comparisons showed that the reduction in METH-induced increase of RyR expression was statistically significant at one or two doses of SCH23390: (Fig. 2a) in the midbrain neurons; RyR-1, 1 μM (q = 4.05), 10 μM (q = 4.37), p < 0.01; RyR-2, 10 μM (q = 3.93), p < 0.01 versus control group by post hoc test, (Fig. 2b) in the cerebral cortical neurons; RyR-1, 1 μM (q = 2.83), 10 μM (q = 2.76), p < 0.05; RyR-2, 1 μM (q = 3.74), 10 μM (q = 6.34), p < 0.01 versus control group by post hoc test.
Figure 3 shows that a D2DR antagonist, sulpiride, did not suppress the METH-induced enhancement of both RyR-1 and -2 proteins. Using real-time PCR, we examined the level of RyR-1 and -2 mRNA in the midbrain and cerebral cortical neurons intermittently exposed to METH (10 μM). The intermittent exposure to METH significantly increased mRNA of these receptor proteins [(Fig. 4), in the midbrain neurons; RyR-1 (p < 0.05), RyR-2 (p < 0.01): in the cerebral cortical neurons; RyR-1 (p < 0.05), RyR-2 (p < 0.001)]. These data indicate that the response of RyR and their mRNA expression to METH and the behaviors of METH-induced enhancement of RyR expression by DR blockade are considered to be almost same in both types of the neurons used here.
Effect of actinomycin D on METH-induced increase of RyR expression in cerebral cortical neurons
Actinomycin D completely abolished the significant increase of RyR-1 [anova analysis: F(3,12) = 34.47, p < 0.0001, t = 7.71, p < 0.001 vs. METH group by post hoc test] and -2 [anova analysis: F(3,12) = 13.13, p = 0.0004, t = 5.0, p < 0.01 vs. METH group by post hoc test) proteins produced by the intermittent exposure to METH in the cerebral cortical neurons (Fig. 5a).
Similarly, SCH23390 (10 μM) significantly suppressed the METH-induced enhancement of RyR-1 [anova analysis: F(3,12) = 15.71, p = 0.0002, t = 3.88, p < 0.05 vs. METH group by post hoc test] and -2 [anova analysis: F(3,12) = 8.37, p = 0.0028, t = 3.91, p < 0.05 vs. METH group by post hoc test] mRNA in the cerebral cortical neurons (Fig. 5b).
Based on these data, it is reasonable to conclude that the METH-induced increased expression of RyR-1 and -2 proteins is mediated via the increased synthesis of these receptor proteins following increased synthesis of their mRNA after the activation of D1DRs.
Effects of D1 and D2DR activation on RyR expression in cerebral cortical neurons
We further examined whether selective D1DR activation with SKF82958, a full D1DR agonist, potentiated RyR-1 and -2 expressions. Figure 6a shows that the intermittent exposure (1 h exposure per day, three times) to SKF82958 dose-dependently increases the levels of RyR-1 [anova analysis: F(3,12) = 41.37, p = 0.0001] and -2 [anova analysis: F(3,12) = 17.44, p = 0.0001] proteins. Dunnett’s multiple comparison post hoc tests showed that two and three tested doses of SKF82958 significantly enhanced the expression of RyR-1 and -2 proteins, respectively, when compared with control: [RyR-1; 3 μM (q = 7.91, p < 0.001), 10 μM (q = 9.34, p < 0.001) vs. control group by post hoc test: RyR-2; 1 μM (q = 2.93, p < 0.05), 3 μM (q = 3.45, p < 0.05), 10 μM (q = 7.19, p < 0.001) vs. control group by post hoc test].
SKF82958 (10 μM)-stimulated increase of RyR-1 [anova analysis: F(3,12) = 24.86, t = 4.99, p < 0.01 vs. SKF82958 group by post hoc test] and -2 [anova analysis: F(3,12) = 9.92, t = 4.04, p < 0.01 vs. SKF82958 group by post hoc test) proteins were significantly blocked by 10 μM SCH23390 (Fig. 6b). SKF82958 (10 μM) also shows significant stimulatory effect on RyR-1 and -2 mRNA expression and this enhancement was also completely abolished by the concomitant exposure to SKF82958 and SCH23390 (10 μM) (Fig. 6c) [anova analysis: RyR-1; F(3,12) = 18.62, p < 0.0001, t = 5.97, p < 0.01 vs. SKF82958 group by post hoc test, RyR-2; F(3,12) = 9.48, p = 0.0017, t = 4.29, p < 0.01 vs.SKF82958 group by post hoc test]. These results clearly indicate that D1DRs have potential to regulate RyR-1 and -2 expressions via increased transcription of their genes.
However, a D2DR agonist, quinpirole, showed no effects on the expression of both RyR-1 and -2 proteins (Fig. 6d). Taken together with these data, it is concluded that D2DRs do not participate in the regulation of RyR-1 and -2 expressions.
Involvement of protein kinase A in METH-induced increase of RyR-1 and -2 expressions in cerebral cortical neurons
D1DRs coupling with Gαs-protein activate adenylate cyclase to increase cAMP formation and subsequent activation of protein kinase (PKA), which suggests that D1DRs may participate in the METH-induced up-regulation of RyRs via PKA activation. We therefore attempted to examine how PKA played a role in the increase of synthesis of RyR 1 and 2 proteins induced by D1DR stimulation. Figure 7 demonstrates that a PKA inhibitor, KT5720, attenuates the METH-induced increase of both RyR-1 and -2 proteins in a dose-dependent manner [anova analysis: RyR-1; F(5,18) = 4.54, p = 0.0075, q = 3.60, p < 0.01 vs. METH group by post hoc test, RyR-2; F(5,18) = 11.28, p < 0.0001, q = 4.23, p < 0.001]. Thus, PKA activation is required in the up-regulation of RyR-1 and -2 protein expression produced by D1DR stimulation.
Effect of D1DR blockade on METH-induced CREB activation
It is well known that the transcription factor CREB is abundantly expressed in the nucleus of neurons, is activated through phosphorylation at serine 133 by cAMP and Ca2+ signals, and is involved in transferring transcription signals mediated by PKA to the nucleus (Lu and Tsai 2007). Furthermore, stimulation of D1DRs by dopamine has been well documented to induce a rapid and transient increase in CREB phosphorylation (Arnauld et al. 1998). In this study, therefore, we attempted to examine the role of active CREB in synthesizing RyR-1 and -2 proteins by METH exposure. As shown in Fig. 8, the short-term exposure of the cerebral cortical neurons to METH (10 μM for 1 h) elevated pCREB, but not CREB, and enhancement of pCREB level by transient exposure to METH was completely abolished by SCH23390, a selective D1DR antagonist [anova analysis: F(3,12) = 6.6, p = 0.007, t = 3.49, p < 0.05 vs. METH group by post hoc test].
Our previous investigation clearly demonstrated that intermittent administration of METH-induced significant enhancement of RyR-1 and -2 proteins in the cerebral cortex of mice as well as produced possible psychological dependence with increased METH-induced place preference (Kurokawa et al. 2010a). This study was therefore carried out for the purpose of define mechanisms of increase in RyRs-1 and -2 proteins by METH using the primary cultures of mouse midbrain and cerebral cortical neurons.
The present study used the intermittent exposure of METH to the neurons as a protocol of METH exposure, because mice were usually administered METH or other psychostimulants every 2 days (3–5 times for 6–10 days) for conditioning place preference. Therefore, the duration and the pattern of the cultured neurons to be exposed to METH in vitro is supposed to resemble that of neurons in brain to be exposed to METH with administration protocol of METH for conditioning place preference test, because the duration of neurons in brain to be exposed to METH is supposed for approximately 1 h after each administration of METH when considering the data based on pharmocokinetic profile of METH in rodents (Zombeck et al. 2009). The protocol with intermittent exposure to amphetamine, that is similar to that used here, was also employed in the previous investigation (Park et al. 2002), in which they reported that intermittent exposure to amphetamine (1 μM, for 5 min/day, for 5 days) showed a neurotrophic effect in PC12 cells. Under the intermittent METH exposure to the neurons, METH increased RyR-1 and -2 proteins with no changes of RyR-3, which is considered to be similar to the previous data that intermittent administration of METH up-regulated expression of RyR-1 and-2 proteins, but not RyR-3, in association with the development of conditioned place preference (Kurokawa et al. 2010a). Furthermore, it was noted that the single exposure to METH for 1 h did not up-regulated RyR-1 and -2 proteins when measured them up to 12 h after the exposure (Appendix S1, Figure S5). These results indicate that the increase in RyR expression requires repeated stimulation by METH as demonstrated in this study.
In the present study, we checked whether dopaminergic neurons were contained in the neurons in primary culture derived from the midbrain and cerebral cortex of mice and whether the cerebral cortical neurons had potential to release dopamine by its membrane depolarization with stimulation by high KCl (30 mM) or METH, as METH exhibited its pharmacological action by increasing dopamine concentration in extracellular space via both stimulating dopamine release from neurons to extracellular space and suppressing dopamine re-uptake by dopamine transporter (DAT) inhibition. The data presented as supplementary data show the presence of dopaminergic neurons in neurons in primary cultures prepared from the mouse cerebral cortex (Appendix S1, Figure S2) and the ability of these neurons to release dopamine in response to 30 mM KCl and METH (Appendix S1, Figure S4). Dopamine content in Krebs-Ringer bicarbonate buffer (KRB) after the stimulation by METH was 118 pmol/mL of KRB and, when evaluating as concentration, it is equivalent to about 120 nM. This concentration in extracellular space is supposed to be lower than that in synaptic space immediately after its release, because this concentration in extracellular space is considered to reflect that after dilution of dopamine by its diffusion from synaptic space to extracellular space. Therefore, it is likely that dopamine concentration could be higher in synaptic space immediately after its release and could be sufficient to stimulate both D1 and D2DRs. In addition, immunohistochemical studies showing the presence of the neurons having positive immunoreactivities to tyrosine hydroxylase (TH), l-amino-aromatic acid decarboxylase (AADC), a well-known neurochemical marker for dopaminergic neurons, and DAT, indicate that the neurons were considered to be appropriate for the purpose of this study. Several reports suggested that TH and AADC are markers for dopaminergic neurons (Gao and Wolf 2007; Inoue et al. 2007; Stephenson et al. 2007). DAT is also considered to be a possible marker for dopaminergic neurons (Stephenson et al. 2007). Therefore, the neurons with the expression of TH, AADC and DAT are considered to be able to use as a dopaminergic neuron model.
The present results show that the response of RyR expression to METH exposure in the presence or absence of D1 and D2DR antagonist in the neurons prepared from the midbrain and cerebral cortex are almost similar. In the cerebral cortical neurons, the presence of both TH and AAAD (Appendix S1, Figure S2) and the potential to release dopamine in response to stimulation with high KCl and METH (Appendix S1, Figure S4) were found. In addition, the previous reports demonstrate the localization of D1 and D2DRs in the neurons both positive and negative to immunoreactivity of TH (Kihara et al. 2002; Lezcano and Bergson 2002; Song et al. 2002; Kurokawa et al. 2010b). Similarly, our previous data also demonstrate that the expression of both D1 and D2DRs has been found in both TH-positive and -negative neurons prepared from the cerebral cortex of mice (Kurokawa et al. 2010b). It is considered to be reasonable that intracellular signal transduction system after DRr activation in the neurons from these two brain regions is almost same. Taken together with these, we consider that the use of the cerebral cortical neurons has no problems to investigate effects of modification of DR function on RyR expression in the following experiment. Moreover, immunohistochemical study shows the co-localization of immunoreactivity for three types of proteins, TH, AAAD, and DAT, in both types of the neurons prepared from the midbrain and cerebral cortex of mice (Appendix S1, Figures S1 and S2), although the percentage of TH-positive neurons in the cultured of the cerebral cortex is one-third of that of the midbrain (Appendix S1, Figure S3).
The intermittent METH exposure to the midbrain neurons and cerebral cortical neurons increased RyR-1 and -2 proteins. DR blockade with its selective antagonist SCH23390 dose-dependently suppressed METH-induced the up-regulation of RyR-1 and -2 proteins. It is noteworthy that the same manipulation also completely inhibited the increased expression of RyR-1 and -2 mRNA. On the other hand, sulpiride, a selective antagonist for dopamine D2 receptors, showed no effects on METH-induced increase in proteins of RyR-1 and -2. As demonstrated here the METH-induced increase of RyR proteins was accompanied with increase of their mRNA in the midbrain neurons and cerebral cortical neurons. Furthermore, the METH-induced increases of RyR proteins were completely abolished by actinomycin D in the cerebral cortical neurons, indicating that the up-regulation of these receptor proteins is mediated via increased transcription of their genes. These results also indicate that METH up-regulates RyR-1 and -2 proteins via D1DR activation in association with increased induction of their gene transcription.
Previous investigations have reported that the activation of D2DRs in the nucleus accumbens can produce place conditioning effect (White et al. 1991) and that the development of conditioned place preference induced by amphetamine is suppressed by D2DR antagonists (Liao 2008). The latter data are in good agreement with our previous report obtained from the mice with METH-induced conditioned place preference (Kurokawa et al. 2010a), in which Kurokawa et al. reported that, in mice showing METH-induced place preference, dopamine D2 receptor antagonism had potential to suppress METH-induced place preference although it showed no effects on METH-induced increase in RyR-1 and -2 proteins. These data are considered to be certain evidence that D2DRs participate in the development of conditioned place preference induced by drugs of abuse. Taken together with these data and the results presented here, it is likely that D2DRs may be involved in the development of METH-induced place preference via neurochemical processes not related to regulatory pathway of RyR synthesis, although it is not clear which pathways among D2DR-coupling neurochemical pathways except processes associated with the changes in RyR expression are involved in the development of conditioned place preference induced by drugs of abuse. We further investigated regulatory role of dopamine D1 and D2DRs in RyR expression by direct exposure of the cerebral cortical neurons to their selective agonists. As shown in this study, a selective D1DR agonist SKF82958 enhanced RyR-1 and -2 proteins, which is considered to mimic the stimulatory effect of METH on RyR-1 and -2 protein expression, whereas a D2DR agonist, quinpirole, did not show any effects on RyR expression.
This study investigated the changes of expression of RyRs by D1DR activation with a full D1DR agonist (Taymans et al. 2005; Mannoury la Cour et al. 2007), SKF82958, and a partial D1DR agonist, SKF38393. Although the data obtained by using SKF38393 are not shown here, the pattern of responses of RyR-1 and -2 expressions to these agonists were similar, but the minimal dose of SKF82958 to stimulate the expression was about one-third of that of SKF38393.
As D1 and D2DRs are coupled with Gs and Gi/o proteins, respectively, and thus they have opposite effects on intracellular signaling via cAMP/PKA pathway (Zanassi et al. 2001), we examined the involvement of PKA and of CREB in RyR expression. Our data demonstrate the inhibitory effect of a PKA inhibitor, KT5720, on the METH-induced increase of RyR expression and the abolishment of METH-induced enhancement of phospho-CREB by SCH23390, a selective D1DR antagonist. The latter event is consequent to decreased cAMP production by D1DR blockade, because PKA, one of cytosolic second messengers including Ca2+ and calmodulin kinases, phosphorylates CREB to facilitate target gene transcription in heterogeneous systems and cultured neurons (Lonze and Ginty 2002; Carlezon et al. 2005). Previous investigations revealed that D1DRs activated immediate early gene expression and locomotion in dopamine-depleted rats (Paul et al. 1992; Keefe and Gerfen 1995). Moreover, the role of D1DRs in the neurochemical and behavioral adaptation induced by amphetamine (Pierce et al. 1996; White and Kalivas 1998) were reported. Repeated amphetamine administration has also been reported to stimulate D1DR-mediated signal transduction pathways including activation of several protein kinases (Pierce et al. 1996; Hu et al. 2002; Licata and Pierce 2003), to enhance neuroadaptation in association with alterations in phosphorylation of transcription factors such as CREB (Turgeon et al. 1997), and to change expression of neurotrophins including fibroblast growth factors (Forster and Blaha 2000) and brain-derived neurotrophic factor (Meredith et al. 2002). Based on the experimental results demonstrated here and the previously reported data described above, it is reasonable to conclude that METH-induced up-regulation of RyR proteins is mediated by increased transcription of their genes via stimulated D1DR-related signal transduction system.
This work was supported in part by Grants-in-Aid for Ministry of health, Labour and Welfare, and by a Research Project Grant from Kawasaki Medical School (No. 21-611, No.22-A25). All authors declare that no conflict of interest exists in the present work.