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Repeated treatment with haloperidol, but not olanzapine, alters synaptic NMDA receptor composition in rat striatum
European Neuropsychopharmacology (2008) 18, 531–534
w w w. e l s e v i e r. c o m / l o c a t e / e u r o n e u r o
SHORT COMMUNICATION
Repeated treatment with haloperidol, but not olanzapine, alters synaptic NMDA receptor composition in rat striatum Fabrizio Gardoni, Angelisa Frasca, Elisa Zianni, Marco A. Riva, Monica Di Luca, Fabio Fumagalli ⁎ Department of Pharmacological Sciences and Centre of Excellence on Neurodegenerative Diseases, University of Milan, via Balzaretti 9, 20133 Milan, Italy
Received 13 June 2007; received in revised form 18 September 2007; accepted 23 October 2007
KEYWORDS Postsynaptic density; Striatum; CaMKII; Antipsychotic; NMDA receptor; Rat
Abstract We here show that repeated administration of the first generation antipsychotic haloperidol, but not of the second generation olanzapine, significantly reduced the expression of NMDA subunit NR2A at striatal synapses, whereas both drugs decreased αCaMKII protein levels and autophosphorylation degree. Given that alterations in the localization of NMDA receptor regulatory subunits at synapses have been described in experimental parkinsonism, the haloperidol-induced effect on NMDA subunit localization might contribute to drug-induced parkinsonism induced by haloperidol. © 2007 Elsevier B.V. and ECNP. All rights reserved.
1. Introduction Antipsychotic drugs (APDs) represent the mainstay of pharmacotherapy for schizophrenia. While first and second generation APDs are effective on psychotic symptoms, only second generation agents might produce some improvements on negative symptoms and cognitive deterioration (Keefe et al., 2006). Evidence exists that relates such differences to synaptic events, including receptor profiles and the modulation of neurotransmitter release. However, since therapeutic effects may develop over time, neuroadaptive changes taking
⁎ Corresponding author. Tel.: +39 0250318298; fax: +39 0250318278. E-mail address: [email protected] (F. Fumagalli).
place in selected brain regions might be relevant for functional improvement as shown in the experimental animal (Tarazi et al., 2003; Fumagalli et al., 2006). Besides clinical benefits, first and second generation APDs evoke distinct unwanted side effects that might also originate from adaptive mechanisms. Hence, although EPS are linked to the potent blockade of striatal dopaminergic D2 receptors, additional mechanisms originating from adaptive changes can not be ruled out, such as PKA activation (Dwivedi et al., 2002) or alterations in the expression of components of the glutamatergic synapse (Yoshida et al., 1994; Riva et al., 1997; De Bartolomeis et al., 2002). We have recently demonstrated that a key homeostatic issue for a physiological function of the glutamatergic synapse resides in the correct and balanced distribution of NMDA receptor subunits between synaptic and extrasynaptic membranes
0924-977X/$ - see front matter © 2007 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2007.10.004
532
F. Gardoni et al.
(Gardoni et al., 2006). Indeed, the biological consequences of NMDA receptor activation are a direct consequence of the location of the NMDA receptor signaling complex activated (Hardingham et al., 2002). Notably, alterations in the localization of NMDA receptor regulatory subunits at synapses have been described in experimental parkinsonism (Picconi et al., 2006). Thus, given the involvement of an abnormal synaptic localization of glutamate receptors in the manifestation of Parkinsonian-related motor symptoms, we investigated the possibility that prolonged treatment with haloperidol, a prototype first generation APD, or the second-generation agent olanzapine, differently affects the structural organization of the excitatory postsynaptic compartment in rat striatum.
2. Experimental procedures 2.1. Animal treatment and drug paradigms Male Sprague-Dawley rats (Charles River, Calco, Italy) weighing 225– 250 g were maintained under a 12 h light/12 h dark cycle with food and water available ad libitum. Animals were allowed to adapt to laboratory conditions for 2 weeks and handled 5 min a day during this period. Rats were subcutaneously injected for 14 days with haloperidol, (1 mg/kg, once a day) or olanzapine, (2 mg/kg, twice a day) and sacrificed 24 h after the last drug injection. Brain regions were immediately dissected, frozen on dry ice and stored at − 80 °C. Striatum, corresponding to the plates 11–14, was dissected from 2mm thick slices, according to the atlas of Paxinos and Watson (1996). Drug doses were chosen in accordance with published protocols (Bubser and Deutch, 2002; Kapur et al., 2003; Schotte et al., 1996).
Figure 2 Striatal homogenates (homo, left panels) and triton insoluble fractions (TIF, right panels) from control (C), Haloperidol (HA) and Olanzapine (OL) treated rats were analysed by western blot analysis with PSD-95, SAP97 and SAP102 antibodies. The same amount of protein was loaded per lane. Histograms show the quantification of western blotting for MAGUK proteins performed in homogenate and TIF fractions. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (Decreto Legislativo 116/92) and international laws (EEC Council Directive 86/609/1987).
2.2. Western Blot analysis
Figure 1 Effects of two week treatment with Haloperidol (HA) or Olanzapine (OL) on NMDA receptor subunit protein levels in rat striatum. Striatal homogenates (homo, left panels) and triton insoluble fractions (TIF, right panels) from control (C), Haloperidol and Olanzapine treated rats were analysed by western blot analysis with NR1, NR2A and NR2B antibodies. The same amount of protein was loaded per lane (⁎p = 0.002 haloperidol vs. controls).
Subcellular fractionation of striatal tissue was performed using a previously validated biochemical fractionating method (Gardoni et al., 2001, 2006). Striata were homogenized in ice-cold sucrose 0.32 M containing Hepes 1 mM, MgCl2 1 mM, EDTA 1 mM, NaHCO3 1 mM, PMSF 0.1 mM, at pH 7.4 in presence of a complete set of proteases inhibitors (Complete™, Roche Diagnostics, Basel, Switzerland) and phosphatases inhibitors (Sigma-Aldrich, St. Louis, MO). The homogenized tissue was centrifuged at 1,000× g for 10 min. The resulting supernatant was centrifuged at 13,000× g for 15 min to obtain a crude membrane fraction. The pellet was re-suspended in Hepes 1 mM + Complete™ and centrifuged at 100,000× g for 1 h. The pellet was re-suspended in buffer containing 75 mM KCl and 1% Triton-X 100 and centrifuged at 100,000× g for 1 h. The final pellet was homogenized in a glass–glass potter in 20 mM Hepes. Then, an equal volume of glycerol was added and this fraction, referred as Triton insoluble fraction (TIF), was stored at − 80 °C. The protein composition of this preparation was carefully tested for the absence of presynaptic markers (i.e. synaptophysin; see Gardoni et al., 2001). For each TIF preparation three independent western blotting experiments were run. Similar protein yield was obtained in TIF purified from striata of all experimental groups.
2.3. Antibodies NR1 (Pharmingen, San Diego, CA, USA), NR2A and NR2B (Zymed, San Francisco, CA, USA), PSD-95 and SAP102 (Affinity BioReagents, Golden, CO, USA), SAP97 (StressGen, Victoria, British Columbia,
Repeated treatment with Haloperidol alters synaptic NMDA receptor composition in rat striatum Canada), alphaCaMKII (Chemicon), p286-alphaCaMKII (Promega, Madison, WI, USA).
2.4. Data analysis and statistical evaluation Quantitation of western blot analysis was performed by means of computer assisted imaging (Quantity-OneR System; Biorad, CA, USA) and statistical evaluations were performed according to one-way analysis of variance (ANOVA) followed by Bonferroni as post hoc comparison test.
3. Results We initially examined NMDA receptor subunit protein levels by western blot analysis in purified triton insoluble postsynaptic fraction (TIF) enriched in PSD proteins (Gardoni et al., 2006) as compared to homogenate, prepared from striata of control, haloperidol and olanzapine treated rats. Repeated administration of haloperidol, but not olanzapine, produced a significant decrease of NR2A levels in the TIF fraction (Fig. 1, right panel; F2,27 = 13,802, p = 0.002 haloperidol vs. controls) without alterations in NR1 and NR2B levels with both drugs. Conversely, the expression of NMDA subunits in the homogenate fraction was not altered after chronic treatment (Fig. 1, left panels). Since it is known that MAGUK family members participate in NMDA receptors trafficking and clustering to synaptic sites (Sans et al., 2003; Mauceri et al., 2007), we tested whether haloperidolinduced NR2A reduction was correlated with changes in expression or localization of such proteins. Both drugs did not change the
Figure 3 Effects of 2 weeks treatment with Haloperidol (HA) or Olanzapine (OL) on total and p286-autophosphorylated αCaMKII protein levels in rat striatum. Striatal homogenates (homo, left panels) and triton insoluble fractions (TIF, right panels) from control, Haloperidol and Olanzapine treated rats were analysed by western blot analysis with αCaMKII and p286αCaMKII antibodies. The same amount of protein was loaded per lane (αCaMKII, ⁎p = 0.032 OL vs C, ⁎p = 0.029 HA vs. C; p286αCaMKII, §p = 0.01 OL vs. C, #p = 0.0008 HA vs. C).
533
levels of any MAGUK member in the homogenate samples (Fig. 2, left panels) as well as in the TIF fraction (Fig. 2, right panels), although haloperidol shows a trend toward a reduction of SAP-97 localization at synapses (F2,27 = 2501, p = 0.108, haloperidol vs. controls). Since we have recently demonstrated a specific role for αCaMKII in the regulation of NR2A subunit localization in the postsynaptic compartment (Mauceri et al., 2007) and given that phosphorylation processes regulate trafficking as well as synaptic anchoring of NMDA receptors (Prybylowski and Wenthold, 2004), we monitored the autophosphorylation-activation level of αCaMKII. As shown in Fig. 3, chronic treatment with haloperidol or olanzapine did not alter total and p286-autophosphorylated αCaMKII protein levels in the homogenate fraction (Fig. 3, left panels). Conversely, both drugs reduced total and p286-autophosphorylated αCaMKII protein levels in the TIF postsynaptic compartment (Fig. 3, right panel) (αCaMKII, F2,27 = 4844, p = 0.032 olanzapine vs controls, p = 0.029 haloperidol vs. controls; p286-αCaMKII, F2,27 = 9279, p = 0.01 olanzapine vs. controls, p = 0.0008 haloperidol vs. controls).
4. Discussion We show that repeated exposure to antipsychotics of first (haloperidol) or second (olanzapine) generation evokes changes in the subcellular distribution of crucial determinants of glutamatergic neurotransmission in rat striatum. In particular, both drugs similarly reduced synaptic localization and autophosphorylation of αCaMKII whereas haloperidol, but not olanzapine, reduced localization of NR2A at synaptic sites. Specific changes in the ionotropic glutamate receptor population at synapses may be due to several mechanisms, including differences in receptor trafficking, internalization and movement between synaptic and extrasynaptic regions (Groc and Choquet, 2006). In particular, the trafficking of NMDA receptors to the synapse and their organization in the postsynaptic membrane appear to depend on two major factors, phosphorylation of the receptor and its interaction with scaffolding proteins, i.e. MAGUK proteins (Prybylowski and Wenthold, 2004). Our results do not provide a clear cut response on this issue, since both antipsychotics reduce CaMKII autophosphorylation/distribution at striatal synapse without affecting the expression of MAGUK proteins, although a trend toward a decrease is observed for SAP97 after haloperidol treatment. Interestingly, the lack of changes in NR2A expression or distribution after acute exposure to haloperidol (data not shown) points to the reduction of this NMDA subunit as a (mal)adaptive mechanism set in motion by repeated exposure to the drug. Collectively, our data point to haloperidolinduced NR2A reduction, and consequent decrease of NR2A/ NR2B ratio, as a functionally relevant molecular dysregulation of glutamatergic synapse. Recent studies in rodents and primates suggested that altered NR2B/NR2A compartmentalization in synaptic membranes may be an important determinant of L-DOPA induced dyskinesia (Hallett et al., 2005; Gardoni et al., 2006). Accordingly, the evidence that haloperidol, but not olanzapine, reduces synaptic level of NR2A points to changes in NMDA receptor composition at synaptic sites as a relevant mechanism that could contribute
534 to the manifestation of motor symptoms produced by first generation APDs, although we can not rule out the possibility that, in other brain regions such as prefrontal cortex and hippocampus, such differences could be relevant for the therapeutic effects of the drugs.
Role of the funding source This work was supported by the Ministry of University and Research (PRIN2005 to MDL; PRIN 2005 to MAR) and by the Health Ministry (Ricerca Finalizzata 2005 to MAR). The Ministry of University and Research and the Health Ministry had no further role in study design, in the collection and interpretation of data, in the writing of the report and in the decision to submit the paper for publication.
Contributors Fabrizio Gardoni and Fabio Fumagalli coordinated the different phases of the experiments, undertook the statistical analysis, managed the literature searches and wrote the manuscript. Marco A. Riva and Monica Di Luca designed the study. Angelisa Frasca and Elisa Zianni performed all in vivo animal treatments and biochemical (western blotting) experiments.
Conflict of interest None of the authors has any potential conflict of interest nor financial interests to disclose.
Acknowledgments This work was supported by the Ministry of University and Research (PRIN2005 to MDL; PRIN 2005 to MAR) and by the Health Ministry (Ricerca Finalizzata 2005 to MAR).
References Bubser, M., Deutch, A.Y., 2002. Differential effects of typical and atypical antipsychotic drugs on striosome and matrix compartments of the striatum. Eur. J. Neurosci. 15, 713–720. De Bartolomeis, A., Aloj, L., Ambesi-Impiombato, A., Bravi, D., Caraco, C., Muscettola, G., Barone, P., 2002. Acute administration of antipsychotics modulates Homer striatal gene expression differentially. Mol. Brain Res. 98, 124–129. Dwivedi, Y., Rizavi, H.S., Pandey, G.N., 2002. Differential effects of Haloperidol and clozapine on [(3)H]cAMP binding, protein kinase A (PKA) activity, and mRNA and protein expression of selective regulatory and catalytic subunit isoforms of PKA in rat brain. J. Pharmacol. Exp. Ther. 300, 197–209. Fumagalli, F., Frasca, A., Sparta, M., Drago, F., Racagni, G., Riva, M.A., 2006. Long-term exposure to the atypical antipsychotic Olanzapine differently up-regulates extracellular signal-regulated kinases 1 and 2 phosphorylation in subcellular compartments of rat prefrontal cortex. Mol. Pharmacol. 69, 1366–1372. Gardoni, F., Schrama, L.H., Kamal, A., Gispen, W.H., Cattabeni, F., Di Luca, M., 2001. Hippocampal synaptic plasticity involves competition between Ca2+ /calmodulin-dependent protein kinase II and
F. Gardoni et al. postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor. J. Neurosci. 21, 1501–1509. Gardoni, F., Picconi, B., Ghiglieri, V., Polli, F., Bagetta, V., Bernardi, G., Cattabeni, F., Di Luca, M., Calabresi, P., 2006. A critical interaction between NR2B and MAGUK in L-DOPA induced dyskinesia. J. Neurosci. 26, 2914–2922. Groc, L., Choquet, D., 2006. AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse. Cell Tissue Res. 326, 423–438. Hallett, P.J., Dunah, A.W., Ravenscroft, P., Zhou, S., Bezard, E., Crossman, A.R., Brotchie, J.M., Standaert, D.G., 2005. Alterations of striatal NMDA receptor subunits associated with the development of dyskinesia in the MPTP-lesioned primate model of Parkinson's disease. Neuropharmacology 48, 503–516. Hardingham, G.E., Fukunaga, Y., Bading, H., 2002. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci. 5, 405–414. Kapur, S., VanderSpek, S.C., Brownlee, B.A., Nobrega, J.N., 2003. Antipsychotic dosing in preclinical models is often unrepresentative of the clinical condition: a suggested solution based on in vivo occupancy. J. Pharmacol. Exp. Ther. 305, 625–631. Keefe, R.S., Seidman, L.J., Christensen, B.K., Hamer, R.M., Sharma, T., Sitskoorn, M.M., Rock, S.L., Woolson, S., Tohen, M., Tollefson, G.D., Sanger, T.M., Lieberman, J.A., HGDH Research Group, 2006. Long-term neurocognitive effects of Olanzapine or low-dose Haloperidol in first-episode psychosis. Biol. Psychiatry 97–105. Mauceri, D., Gardoni, F., Marcello, E., Di Luca, M., 2007. Dual role of CaMKII-dependent SAP97 phosphorylation in mediating trafficking and insertion of NMDA receptor subunit NR2A. J. Neurochem. 100, 1032–1046. Paxinos, G., Watson, C., 1996. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Picconi, B., Tortiglione, A., Barone, I., Centone, D., Gardoni, F., Gubellini, P., Bonsi, P., Pisani, A., Bernardi, G., Di Luca, M., Calabresi, P., 2006. NR2B subunit exerts a critical role in postischemic synaptic plasticity. Stroke 37, 1895–1901. Prybylowski, K., Wenthold, R.J., 2004. N-methyl-D-aspartate receptors: subunit assembly and trafficking to the synapse. J. Biol. Chem. 279, 9673–9676. Riva, M.A., Tascedda, F., Lovati, E., Racagni, G., 1997. Regulation of NMDA receptor subunit messenger RNA levels in the rat brain following acute and chronic exposure to antipsychotic drugs. Brain Res. Mol. Brain Res. 50, 136–142. Sans, N., Prybylowski, K., Petralia, R.S., Chang, K., Wang, Y.X., Racca, C., Vicini, S., Wenthold, R.J., 2003. NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat. Cell Biol. 5, 520–530. Schotte, A., Janssen, P.F., Gommeren, W., Luyten, W.H., Van Gompel, P., Lesage, A.S., De Loore, K., Leysen, J.E., 1996. Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding. Psychopharmacology 124, 57–73. Tarazi, F.I., Baldessarini, R.J., Kula, N.S., Zhang, K., 2003. Long-term effects of Olanzapine, risperidone, and quetiapine on ionotropic glutamate receptor types: implications for antipsychotic drug treatment. J. Pharmacol. Exp. Ther. 306, 1145–1151. Yoshida, Y., Ono, T., Kawano, K., Miyagishi, T., 1994. Distinct sites of dopaminergic and glutamatergic regulation of Haloperidol-induced catalepsy within the rat caudate-putamen. Brain Res. 639, 139–148 124:57–73.
w w w. e l s e v i e r. c o m / l o c a t e / e u r o n e u r o
SHORT COMMUNICATION
Repeated treatment with haloperidol, but not olanzapine, alters synaptic NMDA receptor composition in rat striatum Fabrizio Gardoni, Angelisa Frasca, Elisa Zianni, Marco A. Riva, Monica Di Luca, Fabio Fumagalli ⁎ Department of Pharmacological Sciences and Centre of Excellence on Neurodegenerative Diseases, University of Milan, via Balzaretti 9, 20133 Milan, Italy
Received 13 June 2007; received in revised form 18 September 2007; accepted 23 October 2007
KEYWORDS Postsynaptic density; Striatum; CaMKII; Antipsychotic; NMDA receptor; Rat
Abstract We here show that repeated administration of the first generation antipsychotic haloperidol, but not of the second generation olanzapine, significantly reduced the expression of NMDA subunit NR2A at striatal synapses, whereas both drugs decreased αCaMKII protein levels and autophosphorylation degree. Given that alterations in the localization of NMDA receptor regulatory subunits at synapses have been described in experimental parkinsonism, the haloperidol-induced effect on NMDA subunit localization might contribute to drug-induced parkinsonism induced by haloperidol. © 2007 Elsevier B.V. and ECNP. All rights reserved.
1. Introduction Antipsychotic drugs (APDs) represent the mainstay of pharmacotherapy for schizophrenia. While first and second generation APDs are effective on psychotic symptoms, only second generation agents might produce some improvements on negative symptoms and cognitive deterioration (Keefe et al., 2006). Evidence exists that relates such differences to synaptic events, including receptor profiles and the modulation of neurotransmitter release. However, since therapeutic effects may develop over time, neuroadaptive changes taking
⁎ Corresponding author. Tel.: +39 0250318298; fax: +39 0250318278. E-mail address: [email protected] (F. Fumagalli).
place in selected brain regions might be relevant for functional improvement as shown in the experimental animal (Tarazi et al., 2003; Fumagalli et al., 2006). Besides clinical benefits, first and second generation APDs evoke distinct unwanted side effects that might also originate from adaptive mechanisms. Hence, although EPS are linked to the potent blockade of striatal dopaminergic D2 receptors, additional mechanisms originating from adaptive changes can not be ruled out, such as PKA activation (Dwivedi et al., 2002) or alterations in the expression of components of the glutamatergic synapse (Yoshida et al., 1994; Riva et al., 1997; De Bartolomeis et al., 2002). We have recently demonstrated that a key homeostatic issue for a physiological function of the glutamatergic synapse resides in the correct and balanced distribution of NMDA receptor subunits between synaptic and extrasynaptic membranes
0924-977X/$ - see front matter © 2007 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2007.10.004
532
F. Gardoni et al.
(Gardoni et al., 2006). Indeed, the biological consequences of NMDA receptor activation are a direct consequence of the location of the NMDA receptor signaling complex activated (Hardingham et al., 2002). Notably, alterations in the localization of NMDA receptor regulatory subunits at synapses have been described in experimental parkinsonism (Picconi et al., 2006). Thus, given the involvement of an abnormal synaptic localization of glutamate receptors in the manifestation of Parkinsonian-related motor symptoms, we investigated the possibility that prolonged treatment with haloperidol, a prototype first generation APD, or the second-generation agent olanzapine, differently affects the structural organization of the excitatory postsynaptic compartment in rat striatum.
2. Experimental procedures 2.1. Animal treatment and drug paradigms Male Sprague-Dawley rats (Charles River, Calco, Italy) weighing 225– 250 g were maintained under a 12 h light/12 h dark cycle with food and water available ad libitum. Animals were allowed to adapt to laboratory conditions for 2 weeks and handled 5 min a day during this period. Rats were subcutaneously injected for 14 days with haloperidol, (1 mg/kg, once a day) or olanzapine, (2 mg/kg, twice a day) and sacrificed 24 h after the last drug injection. Brain regions were immediately dissected, frozen on dry ice and stored at − 80 °C. Striatum, corresponding to the plates 11–14, was dissected from 2mm thick slices, according to the atlas of Paxinos and Watson (1996). Drug doses were chosen in accordance with published protocols (Bubser and Deutch, 2002; Kapur et al., 2003; Schotte et al., 1996).
Figure 2 Striatal homogenates (homo, left panels) and triton insoluble fractions (TIF, right panels) from control (C), Haloperidol (HA) and Olanzapine (OL) treated rats were analysed by western blot analysis with PSD-95, SAP97 and SAP102 antibodies. The same amount of protein was loaded per lane. Histograms show the quantification of western blotting for MAGUK proteins performed in homogenate and TIF fractions. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (Decreto Legislativo 116/92) and international laws (EEC Council Directive 86/609/1987).
2.2. Western Blot analysis
Figure 1 Effects of two week treatment with Haloperidol (HA) or Olanzapine (OL) on NMDA receptor subunit protein levels in rat striatum. Striatal homogenates (homo, left panels) and triton insoluble fractions (TIF, right panels) from control (C), Haloperidol and Olanzapine treated rats were analysed by western blot analysis with NR1, NR2A and NR2B antibodies. The same amount of protein was loaded per lane (⁎p = 0.002 haloperidol vs. controls).
Subcellular fractionation of striatal tissue was performed using a previously validated biochemical fractionating method (Gardoni et al., 2001, 2006). Striata were homogenized in ice-cold sucrose 0.32 M containing Hepes 1 mM, MgCl2 1 mM, EDTA 1 mM, NaHCO3 1 mM, PMSF 0.1 mM, at pH 7.4 in presence of a complete set of proteases inhibitors (Complete™, Roche Diagnostics, Basel, Switzerland) and phosphatases inhibitors (Sigma-Aldrich, St. Louis, MO). The homogenized tissue was centrifuged at 1,000× g for 10 min. The resulting supernatant was centrifuged at 13,000× g for 15 min to obtain a crude membrane fraction. The pellet was re-suspended in Hepes 1 mM + Complete™ and centrifuged at 100,000× g for 1 h. The pellet was re-suspended in buffer containing 75 mM KCl and 1% Triton-X 100 and centrifuged at 100,000× g for 1 h. The final pellet was homogenized in a glass–glass potter in 20 mM Hepes. Then, an equal volume of glycerol was added and this fraction, referred as Triton insoluble fraction (TIF), was stored at − 80 °C. The protein composition of this preparation was carefully tested for the absence of presynaptic markers (i.e. synaptophysin; see Gardoni et al., 2001). For each TIF preparation three independent western blotting experiments were run. Similar protein yield was obtained in TIF purified from striata of all experimental groups.
2.3. Antibodies NR1 (Pharmingen, San Diego, CA, USA), NR2A and NR2B (Zymed, San Francisco, CA, USA), PSD-95 and SAP102 (Affinity BioReagents, Golden, CO, USA), SAP97 (StressGen, Victoria, British Columbia,
Repeated treatment with Haloperidol alters synaptic NMDA receptor composition in rat striatum Canada), alphaCaMKII (Chemicon), p286-alphaCaMKII (Promega, Madison, WI, USA).
2.4. Data analysis and statistical evaluation Quantitation of western blot analysis was performed by means of computer assisted imaging (Quantity-OneR System; Biorad, CA, USA) and statistical evaluations were performed according to one-way analysis of variance (ANOVA) followed by Bonferroni as post hoc comparison test.
3. Results We initially examined NMDA receptor subunit protein levels by western blot analysis in purified triton insoluble postsynaptic fraction (TIF) enriched in PSD proteins (Gardoni et al., 2006) as compared to homogenate, prepared from striata of control, haloperidol and olanzapine treated rats. Repeated administration of haloperidol, but not olanzapine, produced a significant decrease of NR2A levels in the TIF fraction (Fig. 1, right panel; F2,27 = 13,802, p = 0.002 haloperidol vs. controls) without alterations in NR1 and NR2B levels with both drugs. Conversely, the expression of NMDA subunits in the homogenate fraction was not altered after chronic treatment (Fig. 1, left panels). Since it is known that MAGUK family members participate in NMDA receptors trafficking and clustering to synaptic sites (Sans et al., 2003; Mauceri et al., 2007), we tested whether haloperidolinduced NR2A reduction was correlated with changes in expression or localization of such proteins. Both drugs did not change the
Figure 3 Effects of 2 weeks treatment with Haloperidol (HA) or Olanzapine (OL) on total and p286-autophosphorylated αCaMKII protein levels in rat striatum. Striatal homogenates (homo, left panels) and triton insoluble fractions (TIF, right panels) from control, Haloperidol and Olanzapine treated rats were analysed by western blot analysis with αCaMKII and p286αCaMKII antibodies. The same amount of protein was loaded per lane (αCaMKII, ⁎p = 0.032 OL vs C, ⁎p = 0.029 HA vs. C; p286αCaMKII, §p = 0.01 OL vs. C, #p = 0.0008 HA vs. C).
533
levels of any MAGUK member in the homogenate samples (Fig. 2, left panels) as well as in the TIF fraction (Fig. 2, right panels), although haloperidol shows a trend toward a reduction of SAP-97 localization at synapses (F2,27 = 2501, p = 0.108, haloperidol vs. controls). Since we have recently demonstrated a specific role for αCaMKII in the regulation of NR2A subunit localization in the postsynaptic compartment (Mauceri et al., 2007) and given that phosphorylation processes regulate trafficking as well as synaptic anchoring of NMDA receptors (Prybylowski and Wenthold, 2004), we monitored the autophosphorylation-activation level of αCaMKII. As shown in Fig. 3, chronic treatment with haloperidol or olanzapine did not alter total and p286-autophosphorylated αCaMKII protein levels in the homogenate fraction (Fig. 3, left panels). Conversely, both drugs reduced total and p286-autophosphorylated αCaMKII protein levels in the TIF postsynaptic compartment (Fig. 3, right panel) (αCaMKII, F2,27 = 4844, p = 0.032 olanzapine vs controls, p = 0.029 haloperidol vs. controls; p286-αCaMKII, F2,27 = 9279, p = 0.01 olanzapine vs. controls, p = 0.0008 haloperidol vs. controls).
4. Discussion We show that repeated exposure to antipsychotics of first (haloperidol) or second (olanzapine) generation evokes changes in the subcellular distribution of crucial determinants of glutamatergic neurotransmission in rat striatum. In particular, both drugs similarly reduced synaptic localization and autophosphorylation of αCaMKII whereas haloperidol, but not olanzapine, reduced localization of NR2A at synaptic sites. Specific changes in the ionotropic glutamate receptor population at synapses may be due to several mechanisms, including differences in receptor trafficking, internalization and movement between synaptic and extrasynaptic regions (Groc and Choquet, 2006). In particular, the trafficking of NMDA receptors to the synapse and their organization in the postsynaptic membrane appear to depend on two major factors, phosphorylation of the receptor and its interaction with scaffolding proteins, i.e. MAGUK proteins (Prybylowski and Wenthold, 2004). Our results do not provide a clear cut response on this issue, since both antipsychotics reduce CaMKII autophosphorylation/distribution at striatal synapse without affecting the expression of MAGUK proteins, although a trend toward a decrease is observed for SAP97 after haloperidol treatment. Interestingly, the lack of changes in NR2A expression or distribution after acute exposure to haloperidol (data not shown) points to the reduction of this NMDA subunit as a (mal)adaptive mechanism set in motion by repeated exposure to the drug. Collectively, our data point to haloperidolinduced NR2A reduction, and consequent decrease of NR2A/ NR2B ratio, as a functionally relevant molecular dysregulation of glutamatergic synapse. Recent studies in rodents and primates suggested that altered NR2B/NR2A compartmentalization in synaptic membranes may be an important determinant of L-DOPA induced dyskinesia (Hallett et al., 2005; Gardoni et al., 2006). Accordingly, the evidence that haloperidol, but not olanzapine, reduces synaptic level of NR2A points to changes in NMDA receptor composition at synaptic sites as a relevant mechanism that could contribute
534 to the manifestation of motor symptoms produced by first generation APDs, although we can not rule out the possibility that, in other brain regions such as prefrontal cortex and hippocampus, such differences could be relevant for the therapeutic effects of the drugs.
Role of the funding source This work was supported by the Ministry of University and Research (PRIN2005 to MDL; PRIN 2005 to MAR) and by the Health Ministry (Ricerca Finalizzata 2005 to MAR). The Ministry of University and Research and the Health Ministry had no further role in study design, in the collection and interpretation of data, in the writing of the report and in the decision to submit the paper for publication.
Contributors Fabrizio Gardoni and Fabio Fumagalli coordinated the different phases of the experiments, undertook the statistical analysis, managed the literature searches and wrote the manuscript. Marco A. Riva and Monica Di Luca designed the study. Angelisa Frasca and Elisa Zianni performed all in vivo animal treatments and biochemical (western blotting) experiments.
Conflict of interest None of the authors has any potential conflict of interest nor financial interests to disclose.
Acknowledgments This work was supported by the Ministry of University and Research (PRIN2005 to MDL; PRIN 2005 to MAR) and by the Health Ministry (Ricerca Finalizzata 2005 to MAR).
References Bubser, M., Deutch, A.Y., 2002. Differential effects of typical and atypical antipsychotic drugs on striosome and matrix compartments of the striatum. Eur. J. Neurosci. 15, 713–720. De Bartolomeis, A., Aloj, L., Ambesi-Impiombato, A., Bravi, D., Caraco, C., Muscettola, G., Barone, P., 2002. Acute administration of antipsychotics modulates Homer striatal gene expression differentially. Mol. Brain Res. 98, 124–129. Dwivedi, Y., Rizavi, H.S., Pandey, G.N., 2002. Differential effects of Haloperidol and clozapine on [(3)H]cAMP binding, protein kinase A (PKA) activity, and mRNA and protein expression of selective regulatory and catalytic subunit isoforms of PKA in rat brain. J. Pharmacol. Exp. Ther. 300, 197–209. Fumagalli, F., Frasca, A., Sparta, M., Drago, F., Racagni, G., Riva, M.A., 2006. Long-term exposure to the atypical antipsychotic Olanzapine differently up-regulates extracellular signal-regulated kinases 1 and 2 phosphorylation in subcellular compartments of rat prefrontal cortex. Mol. Pharmacol. 69, 1366–1372. Gardoni, F., Schrama, L.H., Kamal, A., Gispen, W.H., Cattabeni, F., Di Luca, M., 2001. Hippocampal synaptic plasticity involves competition between Ca2+ /calmodulin-dependent protein kinase II and
F. Gardoni et al. postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor. J. Neurosci. 21, 1501–1509. Gardoni, F., Picconi, B., Ghiglieri, V., Polli, F., Bagetta, V., Bernardi, G., Cattabeni, F., Di Luca, M., Calabresi, P., 2006. A critical interaction between NR2B and MAGUK in L-DOPA induced dyskinesia. J. Neurosci. 26, 2914–2922. Groc, L., Choquet, D., 2006. AMPA and NMDA glutamate receptor trafficking: multiple roads for reaching and leaving the synapse. Cell Tissue Res. 326, 423–438. Hallett, P.J., Dunah, A.W., Ravenscroft, P., Zhou, S., Bezard, E., Crossman, A.R., Brotchie, J.M., Standaert, D.G., 2005. Alterations of striatal NMDA receptor subunits associated with the development of dyskinesia in the MPTP-lesioned primate model of Parkinson's disease. Neuropharmacology 48, 503–516. Hardingham, G.E., Fukunaga, Y., Bading, H., 2002. Extrasynaptic NMDARs oppose synaptic NMDARs by triggering CREB shut-off and cell death pathways. Nat. Neurosci. 5, 405–414. Kapur, S., VanderSpek, S.C., Brownlee, B.A., Nobrega, J.N., 2003. Antipsychotic dosing in preclinical models is often unrepresentative of the clinical condition: a suggested solution based on in vivo occupancy. J. Pharmacol. Exp. Ther. 305, 625–631. Keefe, R.S., Seidman, L.J., Christensen, B.K., Hamer, R.M., Sharma, T., Sitskoorn, M.M., Rock, S.L., Woolson, S., Tohen, M., Tollefson, G.D., Sanger, T.M., Lieberman, J.A., HGDH Research Group, 2006. Long-term neurocognitive effects of Olanzapine or low-dose Haloperidol in first-episode psychosis. Biol. Psychiatry 97–105. Mauceri, D., Gardoni, F., Marcello, E., Di Luca, M., 2007. Dual role of CaMKII-dependent SAP97 phosphorylation in mediating trafficking and insertion of NMDA receptor subunit NR2A. J. Neurochem. 100, 1032–1046. Paxinos, G., Watson, C., 1996. The Rat Brain in Stereotaxic Coordinates. Academic Press, New York. Picconi, B., Tortiglione, A., Barone, I., Centone, D., Gardoni, F., Gubellini, P., Bonsi, P., Pisani, A., Bernardi, G., Di Luca, M., Calabresi, P., 2006. NR2B subunit exerts a critical role in postischemic synaptic plasticity. Stroke 37, 1895–1901. Prybylowski, K., Wenthold, R.J., 2004. N-methyl-D-aspartate receptors: subunit assembly and trafficking to the synapse. J. Biol. Chem. 279, 9673–9676. Riva, M.A., Tascedda, F., Lovati, E., Racagni, G., 1997. Regulation of NMDA receptor subunit messenger RNA levels in the rat brain following acute and chronic exposure to antipsychotic drugs. Brain Res. Mol. Brain Res. 50, 136–142. Sans, N., Prybylowski, K., Petralia, R.S., Chang, K., Wang, Y.X., Racca, C., Vicini, S., Wenthold, R.J., 2003. NMDA receptor trafficking through an interaction between PDZ proteins and the exocyst complex. Nat. Cell Biol. 5, 520–530. Schotte, A., Janssen, P.F., Gommeren, W., Luyten, W.H., Van Gompel, P., Lesage, A.S., De Loore, K., Leysen, J.E., 1996. Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding. Psychopharmacology 124, 57–73. Tarazi, F.I., Baldessarini, R.J., Kula, N.S., Zhang, K., 2003. Long-term effects of Olanzapine, risperidone, and quetiapine on ionotropic glutamate receptor types: implications for antipsychotic drug treatment. J. Pharmacol. Exp. Ther. 306, 1145–1151. Yoshida, Y., Ono, T., Kawano, K., Miyagishi, T., 1994. Distinct sites of dopaminergic and glutamatergic regulation of Haloperidol-induced catalepsy within the rat caudate-putamen. Brain Res. 639, 139–148 124:57–73.