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Dopamine-D1 and -D2 receptors differentially regulate synapsin II expression in the rat brain
Neuroscience 138 (2006) 587–599
DOPAMINE-D1 AND -D2 RECEPTORS DIFFERENTIALLY REGULATE SYNAPSIN II EXPRESSION IN THE RAT BRAIN V. Z. CHONG, K. SKOBLENICK, F. MORIN, Y. XU AND R. K. MISHRA*
and their primary function of regulating synaptic vesicle localization is mediated by phosphorylation (Greengard et al., 1993). Three synapsin types have been discovered: synapsin I, synapsin II and synapsin III (Sudhof et al., 1989; Kao et al., 1998). Among these subtypes, synapsin II was demonstrated to be significantly upregulated in the striatum at both mRNA and protein levels following chronic treatment with the typical antipsychotic drug, haloperidol (Chong et al., 2002). This observation is significant because synapsin II is essential in the primary stages of synapsin-regulated synapse formation (Ferreira et al., 1994, 1995), while brain concentrations of this protein have been shown to be reduced in schizophrenia (Mirnics et al., 2000) whose etiology has been linked to allelic variations in the synapsin II gene (Chen et al., 2004). In addition, this finding may be important to our understanding of haloperidol-induced synaptic changes in the brain, which have been associated with the therapeutic and side effects of this antipsychotic agent (Zhang et al., 1989; Kerns et al., 1992; Chong et al., 2002). Unfortunately, the mechanism by which haloperidol regulates synapsin II expression remains elusive. Dopamine (DA) receptors are likely participants in the regulation of synapsin II expression by haloperidol because the antipsychotic agent is a DA-D2 receptor antagonist (Seeman, 1987; Bennett, 1998), and DA receptors have been suggested to be involved in the haloperidolregulated expression of various genes (Robinet et al., 2001; Nakahara et al., 2001; Stork et al., 1994). One way DA receptors can control gene expression is by mediating the activity of the cyclic AMP (cAMP)-synthesizing enzyme, adenylyl cyclase, which is positively and negatively linked to DA-D1 and -D2 receptors, respectively. Both DA-D1 and -D2 receptors have been shown to be colocalized on medium spiny neurons in the striatum (LaHoste et al., 1993; Lester et al., 1993; Shetreat et al., 1996; Xu et al., 2005). Since cAMP can upregulate genes via cAMP-dependent transcription modulators (Vaccarino et al., 1993; Shaywitz and Greenberg, 1999), haloperidol treatment has been suggested to increase gene expression through such factors by allowing DA to exert more of its stimulatory effects on adenylyl cyclase through DA-D1 receptors (Morris et al., 1988; Taymans et al., 2003). This mechanism may also function in the observed synapsin II expression increases by chronic haloperidol treatment because the synapsin II gene promoters, early growth response factor-1 (EGR-1) and activating protein-2␣ (AP-2␣) (Petersohn et al., 1995), have been shown to be cAMP inducible (Vaccarino et al., 1993; Philipp et al., 1994; Garcia et al., 1999; Meaney et al., 2000). This pos-
Department of Psychiatry and Behavioural Neurosciences, McMaster University, 1200 Main Street West, Hamilton, HSC 4N78 Ontario, Canada L8N 3Z5
Abstract—We previously demonstrated that chronic treatment with the dopamine-D2 receptor antagonist, haloperidol, increases mRNA and protein content of the phosphoprotein, synapsin II, in the rat striatum. Since dopamine-D2 receptor antagonism and dopamine-D1 receptor blockade can have opposing effects on gene expression, the present investigation compared the effects of haloperidol with those of the dopamine-D1 receptor antagonist, R-[ⴙ]-7-chloro-8-hydroxy3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390), on the expression of synapsin II protein. Haloperidol and SCH23390 respectively elevated and reduced concentrations of the molecule in mouse primary midbrain cell cultures. Additional experiments revealed that the dopamine-D1 receptor agonist, R-[ⴙ]-1-phenyl-2,3,4,5-tetrahydro(1H)-3-benzapezine-7,8-diol (SKF38393), upregulated the phosphoprotein in these cells. Furthermore, in vivo rat studies demonstrated that chronic haloperidol treatment increases synapsin II protein expression in the medial prefrontal cortex and nucleus accumbens, as was observed in the striatum. In contrast, chronic SCH23390 administration reduced concentrations of this protein in all of these regions, although the reductions seen in the medial prefrontal cortex were insignificant. Neither haloperidol nor the dopamine-D1 receptor antagonist affected synapsin I protein expression in any of the studied brain areas. Based on these findings, we propose dopamine receptors may specifically regulate synapsin II expression through a cyclic AMP-dependent pathway. Since synapsin II is involved in neurotransmitter release and synaptogenesis, and changes in synaptic efficacy and structure are suggested in schizophrenia as well as in haloperidol treatment, our findings offer insight into the mechanistic actions of the antipsychotic agent at the synaptic level. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: haloperidol, SCH23390, schizophrenia, synaptic plasticity, transcription factor.
Synapsins are a family of phosphoproteins involved in both neurotransmitter release and synaptogenesis (Hilfiker et al., 1999; Ferreira et al., 1994; Gitler et al., 2004a). They are highly concentrated in neurons (Valtorta et al., 1992), *Corresponding author. Tel: ⫹1-905-525-9140x22396; fax: ⫹1-905522-8804. E-mail address: [email protected] (R. K. Mishra). Abbreviations: AP-2␣, activating protein-2␣; cAMP, cyclic AMP; DA, dopamine; EGR-1, early growth response factor-1; PBS, phosphatebuffered saline; PKA, protein kinase A; SCH23390, R-[⫹]-7-chloro8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SKF38393, R-[⫹]-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzapezine-7, 8-diol; TBS-T, Tris-buffered saline-Tween 20.
0306-4522/06$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.11.037
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sible mode of regulation is supported by the fact that chronic haloperidol treatment can elevate cAMP concentrations in the rat brain (Marshall and Mishra, 1980). If this model is correct, DA-D1 receptor inhibition should reduce the expression of synapsin II protein. Therefore, we compared the effects of haloperidol with those of the DA-D1 receptor antagonist, R-[⫹]-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3, 4,5-tetrahydro-1H-3-benzazepine (SCH23390), on synapsin II expression in primary cell cultures derived from mouse midbrain. These cultures were selected for study because they contain high concentrations of dopaminergic neurons as well as DA-D1 and -D2 receptors, and because they have been shown to be useful in the immunocytochemical investigation of DA receptor-regulated protein expression changes (Perrone-Capano and Di Porzio, 2000; Eells et al., 2001; Yasumoto et al., 2004). In addition, we evaluated the chronic effects of haloperidol and SCH23390 treatments on synapsin II expression in DA-regulated regions of the rat brain. These experiments were performed not only to verify our in vitro results in an in vivo setting, but also to determine whether haloperidol-mediated synapsin II upregulation occurs in DAregulated regions (i.e. medial prefrontal cortex and nucleus accumbens) other than the striatum. The effects of these interventions on synapsin I protein concentrations were also examined in the striatum, medial prefrontal cortex and nucleus accumbens to evaluate the specificity of these treatments in modulating synapsin II expression. Finally, we investigated the consequences of long-term administration with haloperidol or SCH23390 on EGR-1 and AP-2␣ protein expression in these brain regions to examine a potential role of these transcription factors in DA receptormediated synapsin II protein expression.
EXPERIMENTAL PROCEDURES Primary culture of mouse midbrain neurons Midbrain sections were removed from 15 day old mouse embryos (Charles River Canada, St. Constant, QC, Canada; n⫽10) and mechanically dissociated using a Pasteur pipette tip against a 0.05 mm2 mesh made of stainless steel screen. Cells passing through the wire screen were collected into calcium- and magnesium-free phosphate-buffered saline (PBS) solution (Invitrogen, Burlington, ON, Canada). The cellular concentration in the suspension was quantified and subsequently plated onto 10 mm round coverslips at 104 cells per coverslip. Neurobasal media (500 L) supplemented with B27 and Glutamax 2 mM (Invitrogen) was added to each well. Plated cells were cultured at 37 °C and 5% CO2.
Primary midbrain cell culture treatment Cell cultures were used 5 days post-plating and received haloperidol (1 M), SCH23390 (1 M) or R-[⫹]-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzapezine-7,8-diol (SKF38393, 10 M) diluted in 1⫻ PBS. The drug concentrations used were selected for their abilities to regulate adenylyl cyclase activity based on dose-response studies performed in these cultures in our laboratory (data not shown). Control slides received an equivalent volume of 1⫻ PBS. Time course images were obtained by fixing the slides with 4% formaldehyde (diluted in 1⫻ PBS) at 30 min, 1 h, 3 h and 6 h following each treatment to examine the time-dependent effects of the studied treatments on synapsin II protein expression.
Immunocytochemistry Following each fixation period, coverslips were washed twice with 1⫻ PBS and incubated in 5% donkey serum (SigmaAldrich, Oakville, Ontario, Canada) for 1 h. After blocking, the serum solution was replaced with primary synapsin II antibody (Stressgen, Victoria, BC, Canada) at a concentration of 1:150 diluted in 1⫻ PBS containing 0.6% Triton X-100 (BioRad, Mississauga, ON, Canada). Cells were incubated overnight in the primary antibody solution, then washed three times in PBS for 5 min each wash. FITC-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) was applied to the cells at a concentration of 1:100 for 4 h, also diluted in 1⫻ PBS containing 0.6% Triton X-100. Upon completion of secondary antibody incubation, coverslips were washed six times in 1⫻ PBS (5 min/wash) and mounted to slides using VectaShield mounting media (Vector Laboratories, Burlingame, CA, USA). Slides were visualized on a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss, North York, ON, Canada) connected to a computer running the LSM 510 Image Examiner software. Scale bars were measured using the LSM 510 Image Browser software.
Animal housing and drug treatment Male Sprague–Dawley rats (Charles River Canada) weighing 200 –250 g were used in this investigation and housed at McMaster University Health Sciences Centre’s Central Animal Facility where they were handled according to the Canadian Council for Animal Care guidelines. All experiments conformed to international guidelines on the ethical use of animals. Procedures causing suffering were not used and the numbers of animals used were minimized. The rats were divided equally into four groups (n⫽8 per group): two drug-treated groups (haloperidol and SCH23390) and their respective control groups. The haloperidol group received haloperidol (SigmaAldrich) i.p. at a concentration of 2 mg/kg, and the SCH23390 group received SCH23390 hydrochloride (Sigma-Aldrich) i.p. at a concentration of 0.5 mg/kg. The control groups were administered vehicle i.p. at volume per weight amounts comparable to those of their respective drug-treated groups. Injections were made daily between 10:00 a.m. and 11:00 a.m. for 28 consecutive days, and rats were killed for brain tissue dissection immediately after final injections on the 28th day of drug treatments. The selected haloperidol dose has been extensively implemented and shown to regulate DA-D2 receptor function (Okada et al., 1996), while the selected SCH23390 dose has been demonstrated to be sufficient for antagonizing DA-D1 receptors and inhibiting DA-D1 receptor-regulated events (Hyttel, 1983; Kita et al., 1999; Bourne, 2001).
Animal dissection and tissue handling/storage Animals were anesthetized with methoxyflurane and killed by decapitation at the end of drug treatments. Following the killing, rat brains were quickly removed, and the striatum (caudateputamen), medial prefrontal cortex and nucleus accumbens were dissected from each brain on ice. Tissues were stored at ⫺80 °C until further use.
Immunoblotting Protein preparation and quantification, SDS-PAGE procedures and antibody incubation times were performed as previously described (Chong et al., 2002). The following proteins were examined in this study: synapsin II (reacts with both a- and b-type isoforms), synapsin I, EGR-1 and AP-2␣. The amounts loaded for these proteins were 5 g, 2.5 g, 5 g and 10 g, respectively, per lane. These quantities were within the linear
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Fig. 1. Standard curve immunoblots for (a) synapsin I (Syn I; 80 kDa), (b) synapsin IIa (Syn IIa; 74 kDa), (c) synapsin IIb (Syn IIb; 54 kDa), (d) EGR-1 (80 kDa) and (e) AP-2␣ (50 kDa) antibodies. Representative immunoblots of the curves are shown above graphs. The standard curve immunoblots were established by plotting incremental rat brain protein quantities against their respective optical densities resulting from immunoreactions.
range of the standard immunoblots made for the antibodies implemented in studying the expression of these proteins (Fig. 1). The antibody standard immunoblots were established by
plotting incremental rat brain protein quantities against their respective optical densities resulting from the immunoreactions. The primary antibodies for synapsins I and II were ob-
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tained from Stressgen (rabbit polyclonal), while the primary antibodies for EGR-1 and AP-2␣ were purchased from Santa Cruz Biotechnology (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The following antibody concentrations were implemented: 1:35,000 for anti-synapsin II, 1:280,000 for anti-synapsin I, 1:3000 for anti-EGR-1, and 1:850 for anti-AP-2␣. The secondary antibody (horseradish-peroxidaseconjugated donkey anti-rabbit; AmershamPharmacia Biotech, Baie d’Urfe, PQ, Canada) used for these immunoreactions was diluted 1:5000, and the primary and secondary antibodies were prepared in TBS-T (50 mM Tris, 150 mM NaCl, 0.2% Tween-20, pH 8.5), as done previously.
Following the antibody incubation steps, the immunoblots were developed using enhanced chemiluminescence reagent procedures (AmershamPharmacia Biotech). The signals resulting from the enhanced chemiluminescence reactions were quantified using Northern Eclipse 6.0 software (Empix Imaging, Mississauga, ON, Canada). The densities of these signals reflect the expression of the studied protein in each sample. To further ensure protein loading accuracy, all the blots were stripped with Reblot Plus antibody stripping solution (Chemicon International, Temecula, CA, USA) and incubated with -actin antibody (1:10,000 –1:20:000 in TBS-T; mouse monoclonal; Novus Biologicals, Littleton, CO, USA). The stripping procedure
Fig. 2. Representative immunocytochemical pictures showing relative differences in synapsin II protein concentrations among control, haloperidoltreated, SCH23390-treated and SKF38393-treated cell cultures at 30 min, 1 h, 3 h and 6 h following each treatment.
V. Z. Chong et al. / Neuroscience 138 (2006) 587–599 was performed according to the instructions provided by the manufacturer of the solution. The secondary antibody used in the -actin analyses was horseradish-peroxidase-conjugated donkey anti-mouse, which was also purchased from Amersham Pharmacia Biotech.
Quantification of immunofluorescence in immunocytochemical studies Quantification was performed using ImageJ software (NIH, Bethesda, MD, USA). Multiple visual fields (n⫽4) of each experimental group were subjected to a threshold in order to eliminate all background and non-relevant pixels. The images were then quantified using the total pixel count above threshold, and the resulting data were imported into GraphPad Prism software (GraphPad Software, San Diego, CA, USA) for statistical analysis.
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Statistical analysis of immunofluorescence levels in immunocytochemistry studies Using GraphPad Prism software, a one-way analysis of variance was performed to compare mean immunofluorescence levels among experimental groups. A Tukey’s multiple comparison post hoc test was employed to determine where significant differences existed among these groups. Immunofluorescence level differences were considered significant at P⬍0.05. Data are presented as a percent (⫾standard error of the mean) of the mean immunofluorescence level observed for the control groups.
Statistical analysis of protein expression in immunoblot studies Analysis was performed using GraphPad Prism software as well. Unpaired two-tailed Student t-tests were employed to
Fig. 3. (a) Graphs depicting relative immunofluorescence levels (i.e. synapsin II protein concentrations) among control, haloperidol-treated, SCH23390-treated, and SKF38393-treated midbrain primary cultures. Also included are graphs depicting relative immunofluorescence levels across time within (b) haloperidol-treated, (c) SCH23390-treated, and (d) SKF38393-treated midbrain primary cultures (* P⬍0.05 relative to control; ** P⬍0.01 relative to control; *** P⬍0.001 relative to control).
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compare -actin-normalized protein expression between the control groups and their respective drug-treated groups. An expression change was considered significant at P⬍0.05. Data are presented as a percent (⫾standard error of the mean) of the mean optical density of the immunoreaction signals observed for the control groups.
RESULTS Haloperidol and SCH23390 respectively increase and decrease synapsin II protein concentrations in mouse primary midbrain cell cultures Synapsin II expression was examined in mouse primary midbrain cell cultures using immunocytochemical procedures in which the intensity of fluorescence observed reflected synapsin II protein concentrations (Fig. 2). Cul-
tures treated with haloperidol exhibited greater fluorescence intensity than those treated with vehicle, while SCH23390-treated cells showed less fluorescence intensity relative to control cultures. Interestingly, cultures exposed to SKF38393 also displayed greater fluorescence intensity than those treated with haloperidol or with vehicle. For all treatments investigated, the fluorescence intensity differences between drug-treated and control cultures were observed 30 min after the treatments and maintained 1 h, 3 h and 6 h post-treatment. The punctuated synapsin II protein expression patterns observed in the images were consistent with those reported in previous studies (Gitler et al., 2004b). Fig. 3 depicts the relative immunofluorescence levels (i.e. synapsin II protein concentrations) among the experimental groups.
Fig. 4. Graphs depicting relative synapsin IIa (Syn IIa; 74 kDa) and IIb (Syn IIb; 54 kDa) protein concentrations between control (C) and haloperidol-treated (H) groups in the (a) medial prefrontal cortex (mPFX) and (b) nucleus accumbens (NA). Representative immunoblots of synapsin IIa and IIb protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown to the left of graphs (* P⬍0.05; ** P⬍0.02). (c) Relative haloperidol-induced increases in synapsin II subtypes among the striatum (STR), mPFX and NA (* P⬍0.05 relative to control; ** P⬍0.02 relative to control).
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Fig. 5. Graphs depicting relative synapsin I (Syn I; 80 kDa) protein concentrations between control and haloperidol-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of synapsin I protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below graphs.
Chronic haloperidol treatment significantly increases synapsin II protein concentrations in the medial prefrontal cortex and nucleus accumbens Immunoblotting was used to determine synapsin I and II protein concentrations in the investigated brain regions following chronic treatment with haloperidol. The antipsychotic drug caused about a 25% increase in synapsin IIa protein expression (* P⬍0.05) and 16% increase in synapsin IIb protein expression (** P⬍0.02) in the medial prefrontal cortex (Fig. 4a). On the other hand, chronic haloperidol treatment elevated the concentrations of both isoforms by approximately 10% (** P⬍0.02) in the nucleus accumbens (Fig. 4b). However, the haloperidol-induced synapsin II protein expression increases observed in these
brain regions were less than those seen in the striatum (i.e. approximately 108% and 67% increase in synapsin IIa and synapsin IIb, respectively) in earlier studies (Chong et al., 2002). Fig. 4c shows the relative haloperidol-induced increases in the synapsin II subtypes among the studied brain regions. Haloperidol had no effect on synapsin I protein expression in any of the investigated brain areas (Fig. 5). Chronic SCH23390 treatment significantly decreases synapsin II protein concentrations in the striatum and nucleus accumbens Immunoblotting was also used to examine synapsin I and II protein concentrations in the investigated brain
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Fig. 6. Graphs depicting relative synapsin IIa (Syn IIa; 74 kDa) and IIb (Syn IIb; 54 kDa) protein concentrations between control (C) and SCH23390-treated (S) groups in the (a) striatum (STR), (b) medial prefrontal cortex (mPFX) and (c) nucleus accumbens (NA). Representative immunoblots of synapsin IIa and IIb protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown to the left of graphs (* P⬍0.05; ** P⬍0.02). (d) Relative SCH23390-induced decreases in synapsin II subtypes among the STR, mPFX and NA (* P⬍0.05 relative to control; ** P⬍0.02 relative to control).
regions following chronic SCH23390 administration. SCH23390 reduced striatal synapsin IIa protein content by approximately 30% (* P⬍0.05) and decreased striatal synapsin IIb protein concentrations by about 22% (* P⬍0.05)(Fig. 6a). Smaller decreases in the expression of both isoforms were observed in the nucleus accumbens where the treatment yielded about a 10% reduction in synapsin IIa protein content (** P⬍0.02) and about a 20% decrease in synapsin IIb protein concentrations (* P⬍0.05) (Fig. 6c). In addition, SCH23390 had a tendency to reduce synapsin II concentrations in medial prefrontal cortex, although these changes were not significant (Fig. 6b). Fig. 6d shows the relative SCH23390-induced decreases in the synapsin II subtypes among the studied brain regions. The DA-D1 re-
ceptor antagonist caused no changes in synapsin I protein content in any of the investigated brain areas (Fig. 7). Chronic treatments with haloperidol or SCH23390 do not affect EGR-1 protein concentrations in the striatum, medial prefrontal cortex or nucleus accumbens This experiment examined the possible role of EGR-1 in DA receptor-regulated synapsin II expression. DA-D1 receptor antagonism and DA-D2 receptor blockade have been shown to alter EGR-1 mRNA content in the same way they were demonstrated to modulate synapsin II protein expression in this study (Mailleux et al., 1992; Nguyen
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Fig. 7. Graphs depicting relative synapsin I (Syn I; 80 kDa) protein concentrations between control and SCH23390-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of synapsin I protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below graphs.
et al., 1992; Chong et al., 2002). However, the concentrations of EGR-1 protein were not changed by chronic treatment with haloperidol or SCH23390 in any of the brain regions investigated (data not shown). Chronic treatments with haloperidol and SCH23390 respectively increase and decrease AP-2␣ protein concentrations significantly in the striatum Chronic haloperidol treatment increased AP-2␣ protein expression in the striatum by approximately 30% (* P⬍0.05) (Fig. 8a), while chronic SCH23390 administration reduced striatal protein concentrations of this transcription factor by about 20% (* P⬍0.05) (Fig. 9a). Although AP-2␣ protein concentration changes in the medial prefrontal cortex and nucleus accumbens were insignificant following either of these interventions, both treatments modulated AP-2␣ pro-
tein expression in these regions with trends similar to their effects on AP-2␣ content in the striatum (Figs. 8b, 8c, 9b and 9c).
DISCUSSION The in vitro studies demonstrated that DA-D2 receptor antagonism increases synapsin II protein expression, while DA-D1 receptor blockade reduces concentrations of this phosphoprotein. Since synapsin II expression is suggested to be promoted by the cAMP-inducible transcription factors, EGR-1 and AP-2␣ (Chin et al., 1994; Petersohn et al., 1995; Vaccarino et al., 1993; Philipp et al., 1994; Garcia et al., 1999; Meaney et al., 2000), these findings support the proposal that haloperidol may increase synapsin II protein expression by elevating cAMP content, while
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Fig. 8. Graphs depicting relative AP-2␣ (50 kDa) protein concentrations between control and haloperidol-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of AP-2␣ protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below the graphs (* P⬍0.05).
SCH23390 may reduce concentrations of the phosphoprotein by decreasing cAMP levels. This notion is further supported by the finding that the cell cultures treated with the DA-D1 receptor agonist, SKF38393, exhibited greater synapsin II protein expression than those treated with haloperidol or with vehicle. This suggestion is also strengthened by the in vivo experiments in which haloperidol and SCH23390 modulated synapsin II protein concentrations in various regions of the rat brain in the same manner they regulated the expression of the phosphoprotein in the cell culture experiments. Since both agents failed to alter synapsin I protein content in striatum, medial prefrontal cortex or nucleus accumbens, our observations suggest that the regulatory effects of DA receptors on synapsin II expression may be specific to subtype II of the synapsin family.
From a clinical perspective, the observed haloperidolmediated upregulation of synapsin II may be relevant to the motor side effects of the drug since this antipsychotic agent caused the greatest increase in concentrations of the phosphoprotein in the striatum (Hallett, 1993). In particular, this upregulation may contribute to the ability of haloperidol to increase neurotransmitter release and synapse formation (Zhang et al., 1989; Klinzova et al., 1990), and enhancement of such plastic events in the striatum is implicated in the debilitating extrapyramidal consequences of the drug (Staton and Brumback, 1980; Kerns et al., 1992). Furthermore, the observation that haloperidol increased synapsin II protein concentrations in the medial prefrontal cortex to a lesser degree may explain why this drug is relatively ineffective for treating the negative symp-
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Fig. 9. Graphs depicting relative AP-2␣ (50 kDa) protein concentrations between control and SCH23390-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of AP-2␣ protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below the graphs (* P⬍0.05).
toms of schizophrenia, which have been related to decreased expression of the phosphoprotein in this brain region (Mirnics et al., 2000). However, since haloperidol was able to elevate synapsin II protein expression in this area to some extent, the possibility that synapsin II upregulation functions in the therapeutic actions of this antipsychotic agent cannot be discounted. To gain further insight into DA receptor-regulated synapsin II expression, the effects of haloperidol and SCH23390 on the expression of the transcriptional modulators of synapsin II were examined. Three possible transcription factors have been suggested to mediate synapsin II expression: EGR-1, AP-2␣ and polyoma enhancer activator-3 (Chin et al., 1994; Petersohn et al., 1995). However, this investigation focused only on EGR-1 and AP-2␣ as possible gene regulators participating in DA receptor-controlled synapsin
II concentration changes. The reason for these selections is that the haloperidol and SCH23390 studies suggest synapsin II upregulation involves elevations in cAMP content. In particular, such increases have only been demonstrated to enhance the expression of EGR-1 and AP-2␣ among the potential transcription factors of the synapsin II gene (Vaccarino et al., 1993; Philipp et al., 1994; Meaney et al., 2000). Unexpectedly, EGR-1 protein expression was found to be unchanged by either of the treatments implemented in this investigation, disputing the idea that EGR-1 is a significant contributor to DA receptor-mediated synapsin II expression. This result was unforeseen because studies have shown that the expression of this transcription factor can be regulated by DA receptors similar to the way in which these receptors modulated synapsin II concentra-
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tions in the present study (Mailleux et al., 1992; Nguyen et al., 1992; Chong et al., 2002). However, these investigations examined DA receptor actions on brain EGR-1 concentrations acutely rather than chronically as was analyzed in the current study. Thus, the finding is significant because it reveals that brain EGR-1 expression resulting from acute DA receptor interventions may not be maintained after long-term modulation of DA receptor activity. In addition, transient changes in EGR-1 expression resulting from acute dopaminergic modulation may also contribute to chronic DA receptor-mediated synapsin II protein expression. Hence, the potential involvement of this transcription factor in this regulation cannot be ruled out. Nevertheless, the lack of effects of the investigated agents on synapsin I protein expression reduces the likelihood of this possibility because the promoter region of the synapsin I gene also contains binding sites for EGR-1 (Thiel et al., 1994). These details open the prospect that AP-2␣ may be more significantly contributing to the observed DA receptor-mediated synapsin II expression changes, as AP-2␣ is a suggested promoter of synapsin II, but not synapsin I, expression (Thiel et al., 1994; Petersohn et al., 1995). Interestingly, chronic treatments with haloperidol and with SCH23390 respectively increased and decreased concentrations of this transcription factor in the striatum. Tendencies for a similar effect in the medial prefrontal cortex and nucleus accumbens were also observed, but were statistically insignificant. These findings strengthen the suggestion that AP-2␣ may participate in the observed DA receptor-mediated synapsin II expression changes, as the investigated dopaminergic modulators altered concentrations of the transcription factor in the same manner they changed synapsin II protein content in the rat brain. One may argue that the lack of significant effects observed for the haloperidoland SCH23390-induced AP-2␣ protein concentration changes in the medial prefrontal cortex and nucleus accumbens contradicts the proposal that this transcription factor is a significant contributor to DA-receptor-regulated synapsin II expression. However, these regions exhibited the least amount of synapsin II protein content changes following these treatments and therefore most likely experienced minute alterations in the expression of transcriptional regulators of the phosphoprotein. Unfortunately, the mechanism by which DA receptors modulate AP-2␣ concentrations via cAMP remains unclear, and comprehension of this control is needed to support the hypothesized model of DA receptor-mediated synapsin II expression. One way cAMP could be mediating AP-2␣ concentrations is through a positive autoregulatory pathway involving the cAMP-activated enzyme, protein kinase A (PKA) (Chin et al., 2002). This mechanism is plausible because AP-2␣ activity is stimulated by PKAmediated phosphorylation (Garcia et al., 1999), while the AP-2␣ gene can upregulate itself through the AP-2␣-binding sites in its regulatory upstream region (Bauer et al., 1994). This idea could explain how haloperidol-induced increases in cAMP concentrations and SCH23390-induced decreases in cAMP content could respectively elevate and reduce AP-2␣ protein concentrations in the brain. In fact,
preliminary studies from our laboratory demonstrate that haloperidol loses its ability to increase synapsin II protein concentrations when PKA activity or AP-2␣ expression is inhibited in cell cultures (K. Skoblenick, unpublished observations). These findings support our proposed PKA/AP2␣-dependent mechanism of DA receptor-regulated synapsin II expression. Alternatively, the observed synapsin II concentration changes could be occurring through DA receptor-mediated modulation of glutamate receptor activity, which can direct gene expression through calcium-dependent processes that may or may not involve AP-2␣ (McGinty, 1999). However, this possibility has yet to be investigated.
CONCLUSION In conclusion, our study may provide insight into the mode by which DA-D1 and -D2 receptors regulate synapsin II expression. Our investigation may therefore also contribute to our understanding of the mechanistic actions of haloperidol at the synaptic level. Acknowledgments—This work was supported by the Ontario Mental Health Foundation and National Institutes of Health (USA). R.K.M. is a recipient of the senior fellowship of the Ontario Mental Health Foundation. V.Z.C. is a recipient of the NSERC Canada Graduate Scholarship.
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(Accepted 19 November 2005) (Available online 18 January 2006)
DOPAMINE-D1 AND -D2 RECEPTORS DIFFERENTIALLY REGULATE SYNAPSIN II EXPRESSION IN THE RAT BRAIN V. Z. CHONG, K. SKOBLENICK, F. MORIN, Y. XU AND R. K. MISHRA*
and their primary function of regulating synaptic vesicle localization is mediated by phosphorylation (Greengard et al., 1993). Three synapsin types have been discovered: synapsin I, synapsin II and synapsin III (Sudhof et al., 1989; Kao et al., 1998). Among these subtypes, synapsin II was demonstrated to be significantly upregulated in the striatum at both mRNA and protein levels following chronic treatment with the typical antipsychotic drug, haloperidol (Chong et al., 2002). This observation is significant because synapsin II is essential in the primary stages of synapsin-regulated synapse formation (Ferreira et al., 1994, 1995), while brain concentrations of this protein have been shown to be reduced in schizophrenia (Mirnics et al., 2000) whose etiology has been linked to allelic variations in the synapsin II gene (Chen et al., 2004). In addition, this finding may be important to our understanding of haloperidol-induced synaptic changes in the brain, which have been associated with the therapeutic and side effects of this antipsychotic agent (Zhang et al., 1989; Kerns et al., 1992; Chong et al., 2002). Unfortunately, the mechanism by which haloperidol regulates synapsin II expression remains elusive. Dopamine (DA) receptors are likely participants in the regulation of synapsin II expression by haloperidol because the antipsychotic agent is a DA-D2 receptor antagonist (Seeman, 1987; Bennett, 1998), and DA receptors have been suggested to be involved in the haloperidolregulated expression of various genes (Robinet et al., 2001; Nakahara et al., 2001; Stork et al., 1994). One way DA receptors can control gene expression is by mediating the activity of the cyclic AMP (cAMP)-synthesizing enzyme, adenylyl cyclase, which is positively and negatively linked to DA-D1 and -D2 receptors, respectively. Both DA-D1 and -D2 receptors have been shown to be colocalized on medium spiny neurons in the striatum (LaHoste et al., 1993; Lester et al., 1993; Shetreat et al., 1996; Xu et al., 2005). Since cAMP can upregulate genes via cAMP-dependent transcription modulators (Vaccarino et al., 1993; Shaywitz and Greenberg, 1999), haloperidol treatment has been suggested to increase gene expression through such factors by allowing DA to exert more of its stimulatory effects on adenylyl cyclase through DA-D1 receptors (Morris et al., 1988; Taymans et al., 2003). This mechanism may also function in the observed synapsin II expression increases by chronic haloperidol treatment because the synapsin II gene promoters, early growth response factor-1 (EGR-1) and activating protein-2␣ (AP-2␣) (Petersohn et al., 1995), have been shown to be cAMP inducible (Vaccarino et al., 1993; Philipp et al., 1994; Garcia et al., 1999; Meaney et al., 2000). This pos-
Department of Psychiatry and Behavioural Neurosciences, McMaster University, 1200 Main Street West, Hamilton, HSC 4N78 Ontario, Canada L8N 3Z5
Abstract—We previously demonstrated that chronic treatment with the dopamine-D2 receptor antagonist, haloperidol, increases mRNA and protein content of the phosphoprotein, synapsin II, in the rat striatum. Since dopamine-D2 receptor antagonism and dopamine-D1 receptor blockade can have opposing effects on gene expression, the present investigation compared the effects of haloperidol with those of the dopamine-D1 receptor antagonist, R-[ⴙ]-7-chloro-8-hydroxy3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390), on the expression of synapsin II protein. Haloperidol and SCH23390 respectively elevated and reduced concentrations of the molecule in mouse primary midbrain cell cultures. Additional experiments revealed that the dopamine-D1 receptor agonist, R-[ⴙ]-1-phenyl-2,3,4,5-tetrahydro(1H)-3-benzapezine-7,8-diol (SKF38393), upregulated the phosphoprotein in these cells. Furthermore, in vivo rat studies demonstrated that chronic haloperidol treatment increases synapsin II protein expression in the medial prefrontal cortex and nucleus accumbens, as was observed in the striatum. In contrast, chronic SCH23390 administration reduced concentrations of this protein in all of these regions, although the reductions seen in the medial prefrontal cortex were insignificant. Neither haloperidol nor the dopamine-D1 receptor antagonist affected synapsin I protein expression in any of the studied brain areas. Based on these findings, we propose dopamine receptors may specifically regulate synapsin II expression through a cyclic AMP-dependent pathway. Since synapsin II is involved in neurotransmitter release and synaptogenesis, and changes in synaptic efficacy and structure are suggested in schizophrenia as well as in haloperidol treatment, our findings offer insight into the mechanistic actions of the antipsychotic agent at the synaptic level. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: haloperidol, SCH23390, schizophrenia, synaptic plasticity, transcription factor.
Synapsins are a family of phosphoproteins involved in both neurotransmitter release and synaptogenesis (Hilfiker et al., 1999; Ferreira et al., 1994; Gitler et al., 2004a). They are highly concentrated in neurons (Valtorta et al., 1992), *Corresponding author. Tel: ⫹1-905-525-9140x22396; fax: ⫹1-905522-8804. E-mail address: [email protected] (R. K. Mishra). Abbreviations: AP-2␣, activating protein-2␣; cAMP, cyclic AMP; DA, dopamine; EGR-1, early growth response factor-1; PBS, phosphatebuffered saline; PKA, protein kinase A; SCH23390, R-[⫹]-7-chloro8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; SKF38393, R-[⫹]-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzapezine-7, 8-diol; TBS-T, Tris-buffered saline-Tween 20.
0306-4522/06$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.11.037
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sible mode of regulation is supported by the fact that chronic haloperidol treatment can elevate cAMP concentrations in the rat brain (Marshall and Mishra, 1980). If this model is correct, DA-D1 receptor inhibition should reduce the expression of synapsin II protein. Therefore, we compared the effects of haloperidol with those of the DA-D1 receptor antagonist, R-[⫹]-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3, 4,5-tetrahydro-1H-3-benzazepine (SCH23390), on synapsin II expression in primary cell cultures derived from mouse midbrain. These cultures were selected for study because they contain high concentrations of dopaminergic neurons as well as DA-D1 and -D2 receptors, and because they have been shown to be useful in the immunocytochemical investigation of DA receptor-regulated protein expression changes (Perrone-Capano and Di Porzio, 2000; Eells et al., 2001; Yasumoto et al., 2004). In addition, we evaluated the chronic effects of haloperidol and SCH23390 treatments on synapsin II expression in DA-regulated regions of the rat brain. These experiments were performed not only to verify our in vitro results in an in vivo setting, but also to determine whether haloperidol-mediated synapsin II upregulation occurs in DAregulated regions (i.e. medial prefrontal cortex and nucleus accumbens) other than the striatum. The effects of these interventions on synapsin I protein concentrations were also examined in the striatum, medial prefrontal cortex and nucleus accumbens to evaluate the specificity of these treatments in modulating synapsin II expression. Finally, we investigated the consequences of long-term administration with haloperidol or SCH23390 on EGR-1 and AP-2␣ protein expression in these brain regions to examine a potential role of these transcription factors in DA receptormediated synapsin II protein expression.
EXPERIMENTAL PROCEDURES Primary culture of mouse midbrain neurons Midbrain sections were removed from 15 day old mouse embryos (Charles River Canada, St. Constant, QC, Canada; n⫽10) and mechanically dissociated using a Pasteur pipette tip against a 0.05 mm2 mesh made of stainless steel screen. Cells passing through the wire screen were collected into calcium- and magnesium-free phosphate-buffered saline (PBS) solution (Invitrogen, Burlington, ON, Canada). The cellular concentration in the suspension was quantified and subsequently plated onto 10 mm round coverslips at 104 cells per coverslip. Neurobasal media (500 L) supplemented with B27 and Glutamax 2 mM (Invitrogen) was added to each well. Plated cells were cultured at 37 °C and 5% CO2.
Primary midbrain cell culture treatment Cell cultures were used 5 days post-plating and received haloperidol (1 M), SCH23390 (1 M) or R-[⫹]-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzapezine-7,8-diol (SKF38393, 10 M) diluted in 1⫻ PBS. The drug concentrations used were selected for their abilities to regulate adenylyl cyclase activity based on dose-response studies performed in these cultures in our laboratory (data not shown). Control slides received an equivalent volume of 1⫻ PBS. Time course images were obtained by fixing the slides with 4% formaldehyde (diluted in 1⫻ PBS) at 30 min, 1 h, 3 h and 6 h following each treatment to examine the time-dependent effects of the studied treatments on synapsin II protein expression.
Immunocytochemistry Following each fixation period, coverslips were washed twice with 1⫻ PBS and incubated in 5% donkey serum (SigmaAldrich, Oakville, Ontario, Canada) for 1 h. After blocking, the serum solution was replaced with primary synapsin II antibody (Stressgen, Victoria, BC, Canada) at a concentration of 1:150 diluted in 1⫻ PBS containing 0.6% Triton X-100 (BioRad, Mississauga, ON, Canada). Cells were incubated overnight in the primary antibody solution, then washed three times in PBS for 5 min each wash. FITC-conjugated anti-rabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) was applied to the cells at a concentration of 1:100 for 4 h, also diluted in 1⫻ PBS containing 0.6% Triton X-100. Upon completion of secondary antibody incubation, coverslips were washed six times in 1⫻ PBS (5 min/wash) and mounted to slides using VectaShield mounting media (Vector Laboratories, Burlingame, CA, USA). Slides were visualized on a Carl Zeiss LSM 510 confocal microscope (Carl Zeiss, North York, ON, Canada) connected to a computer running the LSM 510 Image Examiner software. Scale bars were measured using the LSM 510 Image Browser software.
Animal housing and drug treatment Male Sprague–Dawley rats (Charles River Canada) weighing 200 –250 g were used in this investigation and housed at McMaster University Health Sciences Centre’s Central Animal Facility where they were handled according to the Canadian Council for Animal Care guidelines. All experiments conformed to international guidelines on the ethical use of animals. Procedures causing suffering were not used and the numbers of animals used were minimized. The rats were divided equally into four groups (n⫽8 per group): two drug-treated groups (haloperidol and SCH23390) and their respective control groups. The haloperidol group received haloperidol (SigmaAldrich) i.p. at a concentration of 2 mg/kg, and the SCH23390 group received SCH23390 hydrochloride (Sigma-Aldrich) i.p. at a concentration of 0.5 mg/kg. The control groups were administered vehicle i.p. at volume per weight amounts comparable to those of their respective drug-treated groups. Injections were made daily between 10:00 a.m. and 11:00 a.m. for 28 consecutive days, and rats were killed for brain tissue dissection immediately after final injections on the 28th day of drug treatments. The selected haloperidol dose has been extensively implemented and shown to regulate DA-D2 receptor function (Okada et al., 1996), while the selected SCH23390 dose has been demonstrated to be sufficient for antagonizing DA-D1 receptors and inhibiting DA-D1 receptor-regulated events (Hyttel, 1983; Kita et al., 1999; Bourne, 2001).
Animal dissection and tissue handling/storage Animals were anesthetized with methoxyflurane and killed by decapitation at the end of drug treatments. Following the killing, rat brains were quickly removed, and the striatum (caudateputamen), medial prefrontal cortex and nucleus accumbens were dissected from each brain on ice. Tissues were stored at ⫺80 °C until further use.
Immunoblotting Protein preparation and quantification, SDS-PAGE procedures and antibody incubation times were performed as previously described (Chong et al., 2002). The following proteins were examined in this study: synapsin II (reacts with both a- and b-type isoforms), synapsin I, EGR-1 and AP-2␣. The amounts loaded for these proteins were 5 g, 2.5 g, 5 g and 10 g, respectively, per lane. These quantities were within the linear
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Fig. 1. Standard curve immunoblots for (a) synapsin I (Syn I; 80 kDa), (b) synapsin IIa (Syn IIa; 74 kDa), (c) synapsin IIb (Syn IIb; 54 kDa), (d) EGR-1 (80 kDa) and (e) AP-2␣ (50 kDa) antibodies. Representative immunoblots of the curves are shown above graphs. The standard curve immunoblots were established by plotting incremental rat brain protein quantities against their respective optical densities resulting from immunoreactions.
range of the standard immunoblots made for the antibodies implemented in studying the expression of these proteins (Fig. 1). The antibody standard immunoblots were established by
plotting incremental rat brain protein quantities against their respective optical densities resulting from the immunoreactions. The primary antibodies for synapsins I and II were ob-
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tained from Stressgen (rabbit polyclonal), while the primary antibodies for EGR-1 and AP-2␣ were purchased from Santa Cruz Biotechnology (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The following antibody concentrations were implemented: 1:35,000 for anti-synapsin II, 1:280,000 for anti-synapsin I, 1:3000 for anti-EGR-1, and 1:850 for anti-AP-2␣. The secondary antibody (horseradish-peroxidaseconjugated donkey anti-rabbit; AmershamPharmacia Biotech, Baie d’Urfe, PQ, Canada) used for these immunoreactions was diluted 1:5000, and the primary and secondary antibodies were prepared in TBS-T (50 mM Tris, 150 mM NaCl, 0.2% Tween-20, pH 8.5), as done previously.
Following the antibody incubation steps, the immunoblots were developed using enhanced chemiluminescence reagent procedures (AmershamPharmacia Biotech). The signals resulting from the enhanced chemiluminescence reactions were quantified using Northern Eclipse 6.0 software (Empix Imaging, Mississauga, ON, Canada). The densities of these signals reflect the expression of the studied protein in each sample. To further ensure protein loading accuracy, all the blots were stripped with Reblot Plus antibody stripping solution (Chemicon International, Temecula, CA, USA) and incubated with -actin antibody (1:10,000 –1:20:000 in TBS-T; mouse monoclonal; Novus Biologicals, Littleton, CO, USA). The stripping procedure
Fig. 2. Representative immunocytochemical pictures showing relative differences in synapsin II protein concentrations among control, haloperidoltreated, SCH23390-treated and SKF38393-treated cell cultures at 30 min, 1 h, 3 h and 6 h following each treatment.
V. Z. Chong et al. / Neuroscience 138 (2006) 587–599 was performed according to the instructions provided by the manufacturer of the solution. The secondary antibody used in the -actin analyses was horseradish-peroxidase-conjugated donkey anti-mouse, which was also purchased from Amersham Pharmacia Biotech.
Quantification of immunofluorescence in immunocytochemical studies Quantification was performed using ImageJ software (NIH, Bethesda, MD, USA). Multiple visual fields (n⫽4) of each experimental group were subjected to a threshold in order to eliminate all background and non-relevant pixels. The images were then quantified using the total pixel count above threshold, and the resulting data were imported into GraphPad Prism software (GraphPad Software, San Diego, CA, USA) for statistical analysis.
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Statistical analysis of immunofluorescence levels in immunocytochemistry studies Using GraphPad Prism software, a one-way analysis of variance was performed to compare mean immunofluorescence levels among experimental groups. A Tukey’s multiple comparison post hoc test was employed to determine where significant differences existed among these groups. Immunofluorescence level differences were considered significant at P⬍0.05. Data are presented as a percent (⫾standard error of the mean) of the mean immunofluorescence level observed for the control groups.
Statistical analysis of protein expression in immunoblot studies Analysis was performed using GraphPad Prism software as well. Unpaired two-tailed Student t-tests were employed to
Fig. 3. (a) Graphs depicting relative immunofluorescence levels (i.e. synapsin II protein concentrations) among control, haloperidol-treated, SCH23390-treated, and SKF38393-treated midbrain primary cultures. Also included are graphs depicting relative immunofluorescence levels across time within (b) haloperidol-treated, (c) SCH23390-treated, and (d) SKF38393-treated midbrain primary cultures (* P⬍0.05 relative to control; ** P⬍0.01 relative to control; *** P⬍0.001 relative to control).
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compare -actin-normalized protein expression between the control groups and their respective drug-treated groups. An expression change was considered significant at P⬍0.05. Data are presented as a percent (⫾standard error of the mean) of the mean optical density of the immunoreaction signals observed for the control groups.
RESULTS Haloperidol and SCH23390 respectively increase and decrease synapsin II protein concentrations in mouse primary midbrain cell cultures Synapsin II expression was examined in mouse primary midbrain cell cultures using immunocytochemical procedures in which the intensity of fluorescence observed reflected synapsin II protein concentrations (Fig. 2). Cul-
tures treated with haloperidol exhibited greater fluorescence intensity than those treated with vehicle, while SCH23390-treated cells showed less fluorescence intensity relative to control cultures. Interestingly, cultures exposed to SKF38393 also displayed greater fluorescence intensity than those treated with haloperidol or with vehicle. For all treatments investigated, the fluorescence intensity differences between drug-treated and control cultures were observed 30 min after the treatments and maintained 1 h, 3 h and 6 h post-treatment. The punctuated synapsin II protein expression patterns observed in the images were consistent with those reported in previous studies (Gitler et al., 2004b). Fig. 3 depicts the relative immunofluorescence levels (i.e. synapsin II protein concentrations) among the experimental groups.
Fig. 4. Graphs depicting relative synapsin IIa (Syn IIa; 74 kDa) and IIb (Syn IIb; 54 kDa) protein concentrations between control (C) and haloperidol-treated (H) groups in the (a) medial prefrontal cortex (mPFX) and (b) nucleus accumbens (NA). Representative immunoblots of synapsin IIa and IIb protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown to the left of graphs (* P⬍0.05; ** P⬍0.02). (c) Relative haloperidol-induced increases in synapsin II subtypes among the striatum (STR), mPFX and NA (* P⬍0.05 relative to control; ** P⬍0.02 relative to control).
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Fig. 5. Graphs depicting relative synapsin I (Syn I; 80 kDa) protein concentrations between control and haloperidol-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of synapsin I protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below graphs.
Chronic haloperidol treatment significantly increases synapsin II protein concentrations in the medial prefrontal cortex and nucleus accumbens Immunoblotting was used to determine synapsin I and II protein concentrations in the investigated brain regions following chronic treatment with haloperidol. The antipsychotic drug caused about a 25% increase in synapsin IIa protein expression (* P⬍0.05) and 16% increase in synapsin IIb protein expression (** P⬍0.02) in the medial prefrontal cortex (Fig. 4a). On the other hand, chronic haloperidol treatment elevated the concentrations of both isoforms by approximately 10% (** P⬍0.02) in the nucleus accumbens (Fig. 4b). However, the haloperidol-induced synapsin II protein expression increases observed in these
brain regions were less than those seen in the striatum (i.e. approximately 108% and 67% increase in synapsin IIa and synapsin IIb, respectively) in earlier studies (Chong et al., 2002). Fig. 4c shows the relative haloperidol-induced increases in the synapsin II subtypes among the studied brain regions. Haloperidol had no effect on synapsin I protein expression in any of the investigated brain areas (Fig. 5). Chronic SCH23390 treatment significantly decreases synapsin II protein concentrations in the striatum and nucleus accumbens Immunoblotting was also used to examine synapsin I and II protein concentrations in the investigated brain
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Fig. 6. Graphs depicting relative synapsin IIa (Syn IIa; 74 kDa) and IIb (Syn IIb; 54 kDa) protein concentrations between control (C) and SCH23390-treated (S) groups in the (a) striatum (STR), (b) medial prefrontal cortex (mPFX) and (c) nucleus accumbens (NA). Representative immunoblots of synapsin IIa and IIb protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown to the left of graphs (* P⬍0.05; ** P⬍0.02). (d) Relative SCH23390-induced decreases in synapsin II subtypes among the STR, mPFX and NA (* P⬍0.05 relative to control; ** P⬍0.02 relative to control).
regions following chronic SCH23390 administration. SCH23390 reduced striatal synapsin IIa protein content by approximately 30% (* P⬍0.05) and decreased striatal synapsin IIb protein concentrations by about 22% (* P⬍0.05)(Fig. 6a). Smaller decreases in the expression of both isoforms were observed in the nucleus accumbens where the treatment yielded about a 10% reduction in synapsin IIa protein content (** P⬍0.02) and about a 20% decrease in synapsin IIb protein concentrations (* P⬍0.05) (Fig. 6c). In addition, SCH23390 had a tendency to reduce synapsin II concentrations in medial prefrontal cortex, although these changes were not significant (Fig. 6b). Fig. 6d shows the relative SCH23390-induced decreases in the synapsin II subtypes among the studied brain regions. The DA-D1 re-
ceptor antagonist caused no changes in synapsin I protein content in any of the investigated brain areas (Fig. 7). Chronic treatments with haloperidol or SCH23390 do not affect EGR-1 protein concentrations in the striatum, medial prefrontal cortex or nucleus accumbens This experiment examined the possible role of EGR-1 in DA receptor-regulated synapsin II expression. DA-D1 receptor antagonism and DA-D2 receptor blockade have been shown to alter EGR-1 mRNA content in the same way they were demonstrated to modulate synapsin II protein expression in this study (Mailleux et al., 1992; Nguyen
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Fig. 7. Graphs depicting relative synapsin I (Syn I; 80 kDa) protein concentrations between control and SCH23390-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of synapsin I protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below graphs.
et al., 1992; Chong et al., 2002). However, the concentrations of EGR-1 protein were not changed by chronic treatment with haloperidol or SCH23390 in any of the brain regions investigated (data not shown). Chronic treatments with haloperidol and SCH23390 respectively increase and decrease AP-2␣ protein concentrations significantly in the striatum Chronic haloperidol treatment increased AP-2␣ protein expression in the striatum by approximately 30% (* P⬍0.05) (Fig. 8a), while chronic SCH23390 administration reduced striatal protein concentrations of this transcription factor by about 20% (* P⬍0.05) (Fig. 9a). Although AP-2␣ protein concentration changes in the medial prefrontal cortex and nucleus accumbens were insignificant following either of these interventions, both treatments modulated AP-2␣ pro-
tein expression in these regions with trends similar to their effects on AP-2␣ content in the striatum (Figs. 8b, 8c, 9b and 9c).
DISCUSSION The in vitro studies demonstrated that DA-D2 receptor antagonism increases synapsin II protein expression, while DA-D1 receptor blockade reduces concentrations of this phosphoprotein. Since synapsin II expression is suggested to be promoted by the cAMP-inducible transcription factors, EGR-1 and AP-2␣ (Chin et al., 1994; Petersohn et al., 1995; Vaccarino et al., 1993; Philipp et al., 1994; Garcia et al., 1999; Meaney et al., 2000), these findings support the proposal that haloperidol may increase synapsin II protein expression by elevating cAMP content, while
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Fig. 8. Graphs depicting relative AP-2␣ (50 kDa) protein concentrations between control and haloperidol-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of AP-2␣ protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below the graphs (* P⬍0.05).
SCH23390 may reduce concentrations of the phosphoprotein by decreasing cAMP levels. This notion is further supported by the finding that the cell cultures treated with the DA-D1 receptor agonist, SKF38393, exhibited greater synapsin II protein expression than those treated with haloperidol or with vehicle. This suggestion is also strengthened by the in vivo experiments in which haloperidol and SCH23390 modulated synapsin II protein concentrations in various regions of the rat brain in the same manner they regulated the expression of the phosphoprotein in the cell culture experiments. Since both agents failed to alter synapsin I protein content in striatum, medial prefrontal cortex or nucleus accumbens, our observations suggest that the regulatory effects of DA receptors on synapsin II expression may be specific to subtype II of the synapsin family.
From a clinical perspective, the observed haloperidolmediated upregulation of synapsin II may be relevant to the motor side effects of the drug since this antipsychotic agent caused the greatest increase in concentrations of the phosphoprotein in the striatum (Hallett, 1993). In particular, this upregulation may contribute to the ability of haloperidol to increase neurotransmitter release and synapse formation (Zhang et al., 1989; Klinzova et al., 1990), and enhancement of such plastic events in the striatum is implicated in the debilitating extrapyramidal consequences of the drug (Staton and Brumback, 1980; Kerns et al., 1992). Furthermore, the observation that haloperidol increased synapsin II protein concentrations in the medial prefrontal cortex to a lesser degree may explain why this drug is relatively ineffective for treating the negative symp-
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Fig. 9. Graphs depicting relative AP-2␣ (50 kDa) protein concentrations between control and SCH23390-treated groups in the (a) striatum, (b) medial prefrontal cortex and (c) nucleus accumbens. Representative immunoblots of AP-2␣ protein concentrations and corresponding control -actin (42 kDa) protein concentrations are shown below the graphs (* P⬍0.05).
toms of schizophrenia, which have been related to decreased expression of the phosphoprotein in this brain region (Mirnics et al., 2000). However, since haloperidol was able to elevate synapsin II protein expression in this area to some extent, the possibility that synapsin II upregulation functions in the therapeutic actions of this antipsychotic agent cannot be discounted. To gain further insight into DA receptor-regulated synapsin II expression, the effects of haloperidol and SCH23390 on the expression of the transcriptional modulators of synapsin II were examined. Three possible transcription factors have been suggested to mediate synapsin II expression: EGR-1, AP-2␣ and polyoma enhancer activator-3 (Chin et al., 1994; Petersohn et al., 1995). However, this investigation focused only on EGR-1 and AP-2␣ as possible gene regulators participating in DA receptor-controlled synapsin
II concentration changes. The reason for these selections is that the haloperidol and SCH23390 studies suggest synapsin II upregulation involves elevations in cAMP content. In particular, such increases have only been demonstrated to enhance the expression of EGR-1 and AP-2␣ among the potential transcription factors of the synapsin II gene (Vaccarino et al., 1993; Philipp et al., 1994; Meaney et al., 2000). Unexpectedly, EGR-1 protein expression was found to be unchanged by either of the treatments implemented in this investigation, disputing the idea that EGR-1 is a significant contributor to DA receptor-mediated synapsin II expression. This result was unforeseen because studies have shown that the expression of this transcription factor can be regulated by DA receptors similar to the way in which these receptors modulated synapsin II concentra-
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tions in the present study (Mailleux et al., 1992; Nguyen et al., 1992; Chong et al., 2002). However, these investigations examined DA receptor actions on brain EGR-1 concentrations acutely rather than chronically as was analyzed in the current study. Thus, the finding is significant because it reveals that brain EGR-1 expression resulting from acute DA receptor interventions may not be maintained after long-term modulation of DA receptor activity. In addition, transient changes in EGR-1 expression resulting from acute dopaminergic modulation may also contribute to chronic DA receptor-mediated synapsin II protein expression. Hence, the potential involvement of this transcription factor in this regulation cannot be ruled out. Nevertheless, the lack of effects of the investigated agents on synapsin I protein expression reduces the likelihood of this possibility because the promoter region of the synapsin I gene also contains binding sites for EGR-1 (Thiel et al., 1994). These details open the prospect that AP-2␣ may be more significantly contributing to the observed DA receptor-mediated synapsin II expression changes, as AP-2␣ is a suggested promoter of synapsin II, but not synapsin I, expression (Thiel et al., 1994; Petersohn et al., 1995). Interestingly, chronic treatments with haloperidol and with SCH23390 respectively increased and decreased concentrations of this transcription factor in the striatum. Tendencies for a similar effect in the medial prefrontal cortex and nucleus accumbens were also observed, but were statistically insignificant. These findings strengthen the suggestion that AP-2␣ may participate in the observed DA receptor-mediated synapsin II expression changes, as the investigated dopaminergic modulators altered concentrations of the transcription factor in the same manner they changed synapsin II protein content in the rat brain. One may argue that the lack of significant effects observed for the haloperidoland SCH23390-induced AP-2␣ protein concentration changes in the medial prefrontal cortex and nucleus accumbens contradicts the proposal that this transcription factor is a significant contributor to DA-receptor-regulated synapsin II expression. However, these regions exhibited the least amount of synapsin II protein content changes following these treatments and therefore most likely experienced minute alterations in the expression of transcriptional regulators of the phosphoprotein. Unfortunately, the mechanism by which DA receptors modulate AP-2␣ concentrations via cAMP remains unclear, and comprehension of this control is needed to support the hypothesized model of DA receptor-mediated synapsin II expression. One way cAMP could be mediating AP-2␣ concentrations is through a positive autoregulatory pathway involving the cAMP-activated enzyme, protein kinase A (PKA) (Chin et al., 2002). This mechanism is plausible because AP-2␣ activity is stimulated by PKAmediated phosphorylation (Garcia et al., 1999), while the AP-2␣ gene can upregulate itself through the AP-2␣-binding sites in its regulatory upstream region (Bauer et al., 1994). This idea could explain how haloperidol-induced increases in cAMP concentrations and SCH23390-induced decreases in cAMP content could respectively elevate and reduce AP-2␣ protein concentrations in the brain. In fact,
preliminary studies from our laboratory demonstrate that haloperidol loses its ability to increase synapsin II protein concentrations when PKA activity or AP-2␣ expression is inhibited in cell cultures (K. Skoblenick, unpublished observations). These findings support our proposed PKA/AP2␣-dependent mechanism of DA receptor-regulated synapsin II expression. Alternatively, the observed synapsin II concentration changes could be occurring through DA receptor-mediated modulation of glutamate receptor activity, which can direct gene expression through calcium-dependent processes that may or may not involve AP-2␣ (McGinty, 1999). However, this possibility has yet to be investigated.
CONCLUSION In conclusion, our study may provide insight into the mode by which DA-D1 and -D2 receptors regulate synapsin II expression. Our investigation may therefore also contribute to our understanding of the mechanistic actions of haloperidol at the synaptic level. Acknowledgments—This work was supported by the Ontario Mental Health Foundation and National Institutes of Health (USA). R.K.M. is a recipient of the senior fellowship of the Ontario Mental Health Foundation. V.Z.C. is a recipient of the NSERC Canada Graduate Scholarship.
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(Accepted 19 November 2005) (Available online 18 January 2006)