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Increased N-acetylaspartate in rat striatum following long-term administration of haloperidol
Schizophrenia Research 75 (2005) 303 – 308 www.elsevier.com/locate/schres
Increased N-acetylaspartate in rat striatum following long-term administration of haloperidol M.K. Hartea,*, S.B. Bachusb, G.P. Reynoldsa,c b
a Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, United Kingdom Department of Psychology and Krasnow Institute, George Mason University, Fairfax, Virginia, 22030, USA c Division of Psychiatry and Neuroscience, Queen’s University Belfast, BT9 7BL, United Kingdom
Received 18 October 2004; received in revised form 25 October 2004; accepted 3 November 2004 Available online 1 December 2004
Abstract N-acetylaspartate (NAA) is present in high concentrations in the CNS and is found primarily in neurons. NAA is considered to be a marker of neuronal viability. Numerous magnetic resonance spectroscopy (MRS) and postmortem studies have shown reductions of NAA in different brain regions in schizophrenia. Most of these studies involved patients chronically treated with antipsychotic drugs. However, the effect of chronic antipsychotic treatment on NAA remains unclear. In the present study, we measured NAA in brain tissue taken from 43 male Long-Evans rats receiving 28.5 mg/kg haloperidol decanoate i.m. every 3 weeks for 24 weeks and from 21 controls administered with vehicle. Determination of tissue concentrations of NAA was achieved by HPLC of sections of frozen tissue from several brain regions with relevance to schizophrenia. Chronic administration of haloperidol was associated with a significant increase (+23%) in NAA in the striatum ( pb0.05) when compared to controls, with no significant changes in the other regions investigated (frontal and temporal cortex, thalamus, hippocampus, amygdala, and nucleus accumbens). NAA appears to be selectively increased in the striatum of rats chronically receiving haloperidol. This increase may reflect a hyperfunction of striatal neurons and relate to the reported increase in somal size of these cells and/or the increase in synaptic density seen in this region following antipsychotic administration. The lack of effect in other regions indicates that the welldocumented NAA deficits seen in chronically treated schizophrenia patients is not an effect of antipsychotic medication and may in fact be related to the disease process. D 2004 Elsevier B.V. All rights reserved. Keywords: N-acetylaspartate; Antipsychotics; Postmortem; Schizophrenia
1. Introduction * Corresponding author. Tel.: +44 114 2222313; fax: +44 114 2765413. E-mail address: [email protected] (M.K. Harte). 0920-9964/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2004.11.001
N-acetylaspartate (NAA) was first identified as a major amino acid derivative in the mammalian brain
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M.K. Harte et al. / Schizophrenia Research 75 (2005) 303–308
by Tallan et al. (1956). It is present in high concentrations in the CNS and is the second most abundant amino acid after glutamate, with intraneuronal concentrations between 10 and 14 mM. NAA is synthesised in neuronal mitochondria from acetyl-coenzyme A and aspartate by the enzyme NAA transferase. In the adult brain, NAA is considered a neuron-specific metabolite (Moffett et al., 1991) and its reduction a marker of neuronal loss. However, recent studies demonstrating the reversibility of NAA deficits following acute brain injury (De Stefano et al., 1995; Kalra et al., 1998) indicate that NAA may be a marker of both neuronal loss and cellular dysfunction (Demougeot et al., 2001). Although the biological function of NAA is not fully understood, it has been shown to be involved in myelin synthesis (Chakraborty et al., 2001), osmoregulation (Baslow, 2003; Taylor et al., 1994), and a recent study showed a possible role as an antiinflammatory (Rael et al., 2004). NAA was also shown to reflect glutamate concentrations and thus may provide a marker of glutamatergic neuronal function (Petroff et al., 2002). In schizophrenia, frontal lobe NAA deficits were shown to correlate with psychopathology and thus may be useful as an indicator of disease severity (Sigmundsson et al., 2003). As it is a noninvasive technique, magnetic resonance spectroscopy (MRS) has become an important tool in monitoring disease progression and in therapy evaluation in patients with neurodegenerative disorders. Of particular interest are the numerous MRS studies that measured brain NAA in discrete brain regions in schizophrenia patients. The majority of these studies have identified reductions in NAA in brain regions implicated in the disease (Rowland et al., 2001). These results are substantiated by recent postmortem studies from this laboratory that have also shown regionally specific reductions in NAA in the temporal cortex, cingulate cortex, hippocampus, amygdala, and caudate nucleus in schizophrenia patients (Nudmamud et al., 2003; Reynolds and Reynolds, 2004). We have also shown regionally specific temporal but not frontal cortical deficits of NAA in two different animal models of schizophrenia (Harte et al., 2004; Reynolds et al., 2005). However, the effect of chronic treatment with antipsychotic drugs on NAA measures remains far from clear.
The importance of examining neuroleptic-naive first-episode patients has been recognized in neurochemical studies of schizophrenia. However, drugnaive and unequivocally diagnosed cases are rare and it is difficult to acquire this patient population. This has lead to investigations of animals administered antipsychotic drugs. Previous studies of chronic antipsychotic administration in rats have demonstrated various behavioural and structural changes comparable to those seen in humans following antipsychotic treatment (Harrison, 1999). Two previous animal studies have attempted to assess the effect of exposure to antipsychotic agents on NAA measures in rat brain. The first was a 1-week study in which rats were given antipsychotics and NAA was measured using MRS. No significant differences were seen between rats receiving haloperidol, clozapine, or olanzapine when compared to vehicle-treated controls (Lindquist et al., 2000). The second was a 6-week study in which NAA in various regions of rat brain was measured by HPLC, following treatment with haloperidol or clozapine. Again there was no significant difference between animals receiving antipsychotics and vehicletreated controls (Bustillo et al., 2004). Although some structural changes are apparent in rats following 6-week exposure to antipsychotics (Harrison, 1999), the NAA reductions in patients treated with antipsychotic agents have been described after months or years of treatment. Therefore, in the current study, we investigated the effects of a more prolonged exposure (24 weeks) to the typical antipsychotic haloperidol, on NAA concentrations in various brain regions implicated in schizophrenia.
2. Methods 2.1. Animals A total of 64 Long–Evans rats, with an initial weight of 90–150 g, were used in this experiment. All rats were kept on a 12-h light–dark cycle (lights on from 0700 to 1900) in a colony room with temperature and humidity remaining constant. Food and water were available ad libitum. Group housed Long–Evans rats were given haloperidol decanoate (28.5 mg/kg/ml, i.m., at 3-week intervals for 24 weeks: n=43) or vehicle (sesame oil: n=21) injections
M.K. Harte et al. / Schizophrenia Research 75 (2005) 303–308
occurring every third week. Experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. All efforts were made to minimize the number of animals used and the suffering of animals in these experiments. 2.2. Tissue preparation and HPLC Animals were killed by decapitation, brains removed and immediately frozen on dry ice. About 16-Am sections were cut using a cryostat and then stored frozen at 70 8C until analysis. NAA was measured in the frontal cortex (Bregma +1, included the cingulate, primary and secondary cortices), temporal cortex (Bregma 3.3, included the primary and secondary auditory cortex and the temporal association cortex), hippocampus (Bregma 3.3 included the dentate gyrus and CA regions), amygdala (Bregma 3.3 included the basolateral, central, lateral, and medial amygdaloid nuclei), thalamus (Bregma 3.3 included nuclei from the anterior, medial and lateral groups), striatum (Bregma +1), and the nucleus accumbens (Bregma +1, included the core and shell regions; Paxinos and Watson, 1998) from these animals. NAA was determined by an established HPLC method (Harte et al., 2004). Briefly, tissue was scraped and pooled from two adjacent frozen sections. The tissue was treated with 0.1 M perchloric acid to precipitate the protein and NAA was extracted from the supernatant using SAX anion columns. The extracted sample was analysed for Table 1 Levels (meanFS.D.) of NAA from rats chronically treated with haloperidol (28.5 mg/kg/ml, i.m., at 3-week intervals for 24 weeks: n=43) and in their vehicle (sesame oil: n=21)-treated controls NAA (nmol/mg protein) Region
Vehicle
Haloperidol
Frontal cortex Temporal cortex Thalamus Hippocampus Amygdala Striatum Nucleus accumbens
89.08F19.24 86.53F12.99 76.52F7.31 76.28F7.93 70.45F10.21 61.85F16.12 83.98F34.21
87.28F19.48 82.37F13.01 79.57F12.32 77.39F10.29 68.02F12.38 74.11F19.73* 89.27F35.54
* pb0.05 vs. control.
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NAA by HPLC (Genesis, C18, 4 Am, 4.6 mm250 mm) using a mobile phase of 0.1% phosphoric acid at a flow rate of 0.5 ml/min. UV detection was at 215 nm. The NAA peak in the samples was identified by its retention time when compared to external standards; samples typically gave peak heights over 100-fold above the limits of sensitivity. NAA gave a linear standard curve (0–50 AM) and was quantified by using relative peak height measurements. Protein concentration in the sample was determined by the Coomassie Blue method and NAA concentrations reported as nmol/mg protein. Statistical analysis (one-way ANOVA) was undertaken using SPSS v10.
3. Results There was a main effect of drug on NAA in the striatum [ F(1,53)=4.31, pb0.05], with rats administered with haloperidol showing increased NAA (+23%) compared to those rats receiving vehicle, after 24 weeks of treatment (Vehicle 61.85F3.51 haloperidol 74.11F3.08 nmol/mg protein). However, NAA concentrations were not significantly different from controls in any of the other regions investigated. NAA concentrations found in the various brain regions from animals receiving vehicle or haloperidol are shown in Table 1.
4. Discussion The main finding of the present study was a robust increase (+23%) in NAA, specific to the striatum, in animals following long-term administration of the typical antipsychotic haloperidol. The striatum, in particular, has been shown to be affected both metabolically (Buchsbaum et al., 1992; Holcomb et al., 1996) and structurally (Chakos et al., 1995; Gur et al., 1998; Harrison, 1999) by treatment with typical antipsychotics. In the adult rat brain, NAA is present in the soma, axon, and dendritic processes. The increase in NAA observed in the present study may reflect a hyperfunction of striatal neurons, relate to the reported increase in somal size of these cells, and/or an increase in synaptic density in this region. These findings may, in turn, be a correlate of striatal (i.e.,
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extrapyramidal motor) side effects of treatment with typical antipsychotic drugs. In support of this are numerous ultrastructural studies looking at the effect of antipsychotics on neuronal processes in the striatum of the rat. These studies have shown an increase in the size of axon terminals (Benes et al., 1985), an increase in synapses with perforated postsynaptic densities, which is an indicator of newly formed synapses and thus a marker of synaptic plasticity (Meshul and Casey, 1989; Meshul et al., 1992; See et al., 1992), an increase in axodendritic and axospinous synapses, with the axospinous synapses showing larger axon terminals with more mitochondria (Uranova et al., 1991), and an increase in various synaptic markers in the striatum (Eastwood et al., 2000; Kroesen et al., 1995). The observation that most of these changes reverse within weeks of stopping the drugs and that typical and atypical antipsychotics may have different effects are further evidences of a medication effect and are not indicative of a disease related abnormality in this region. In addition, an equally important finding, in terms of MRS and postmortem work in schizophrenia, was the lack of effect of haloperidol on NAA in the other brain regions investigated. Previous studies have reported deficits in NAA in the frontal cortex, temporal cortex, hippocampus, thalamus, and dorsolateral prefrontal cortex of schizophrenic patients; however, some studies have failed to identify NAA abnormalities in these regions (Bertolino et al., 1998; Buckley et al., 1994; Deicken et al., 1998; Fukuzako et al., 1995; Kegeles et al., 2000; Omori et al., 2000; Renshaw et al., 1995). However, the majority of these studies involved patients chronically treated with antipsychotic drugs. In a recent longitudinal study by Bustillo et al. (2002), schizophrenia patients, with history of minimal previous treatment (b3 weeks), showed reductions in NAA in the frontal cortex during the first year of treatment with antipsychotic medications (NAA levels at baseline were similar to controls). Patients had a clear clinical response to treatment but changes in frontal NAA were not correlated with symptom improvement. The authors concluded that the reduced frontal NAA reported in schizophrenia may not be a trait of the illness but may represent either a medication effect or progression of the disease.
However, evidence against a medication effect comes from other MRS studies which have demonstrated lower frontal NAA in neuroleptic-naive and drug-free chronic schizophrenia patients (Cecil et al., 1999; Choe et al., 1994). Another recent report arguing against a medication effect comes from a study where NAA deficits were found in the hippocampus of antipsychotic-naive patients (Fannon et al., 2003). MRS studies have consistently revealed lower NAA concentrations in chronic schizophrenic patients, while the results in recent onset patients are contradictory. In a recent study by Molina et al. (2005), where chronic patients were distinguished form recent onset patients, a significant relationship between NAA and disease duration was reported. If NAA is a marker of neuronal loss/viability, then such findings are consistent with a neurodegenerative hypothesis of schizophrenia. However, the majority of evidence does not support antipsychotic-induced neuronal death or permanent damage. In a recent study by Selemon et al. (1999), there was no evidence of neuronal loss or gliosis in monkeys exposed (6 months) to various typical and atypical drugs. Furthermore, postmortem neurodegenerative changes in schizophrenia due to a medication effect are not supported in the literature (Baldessarini et al., 1997). Taken together, the results of the present study suggest that the deficits of NAA reported in MRS and postmortem studies in schizophrenia are not likely to be caused by long term treatment with typical antipsychotics. Whether the same holds true for the newer atypical antipsychotic, a class of drugs with a different receptor profile needs to be further investigated.
Acknowledgements M.K. Harte is supported by a Merck Sharp and Dohme Pharmacology Fellowship.
References Baldessarini, R.J., Hegarty, J.D., Bird, E.D., Benes, F.M., 1997. Meta-analysis of post-mortem studies of Alzheimer’s diseaselike neuropathology in schizophrenia. Am. J. Psychiatry 154, 861 – 863. Baslow, M.H., 2003. N-acetylaspartate in the vertebrate brain: metabolism and function. Neurochem. Res. 28, 941 – 953.
M.K. Harte et al. / Schizophrenia Research 75 (2005) 303–308 Benes, F.M., Paskevich, P.A., Davidson, J., Domesick, V.B., 1985. The effects of haloperidol on synaptic patterns in the rat striatum. Brain Res. 329, 265 – 274. Bertolino, A., Callicott, J.H., Elman, I., Mattay, V.S., Tedeschi, G., Frank, J.A., 1998. Regionally specific neuronal pathology in untreated patients with schizophrenia: a proton magnetic resonance spectroscopic imaging study. Biol. Psychiatry 43, 641 – 648. Buchsbaum, M.S., Potkin, S.G., Siegel Jr., B.V., Lohr, J., Katz, M., Gottschalk, L.A., Gulasekaram, B., Marshall, J.F., Lottenberg, S., Teng, C.Y., 1992. Striatal metabolic rate and clinical response to neuroleptics in schizophrenia. Arch. Gen. Psychiatry 49, 966 – 974. Buckley, P.F., Moore, C., Long, H., Larkin, C., Thompson, P., Mulvany, F., 1994. 1H-magnetic resonance spectroscopy of the left frontal lobes in schizophrenia: clinical, neurodevelopmental, and cognitive correlates. Biol. Psychiatry 36, 792 – 800. Bustillo, J.R., Lauriello, J., Rowland, L.M., Thomson, L.M., Petropoulos, H., Hammond, R., Hart, B., Brooks, W.M., 2002. Longitudinal follow-up of neurochemical changes during the first year of antipsychotic treatment in schizophrenia patients with minimal previous medication exposure. Schizophr. Res. 58, 313 – 321. Bustillo, J.R., Wolff, C., Gutierrez, A.M., Dettmer, T.S., Cooper, T.B., Allan, A., Lauriello, J., Valenzuela, C.F., 2004. Treatment of rats with antipsychotic drugs: lack of an effect on brain Nacetyl aspartate levels. Schizophr. Res. 66, 31 – 39. Cecil, K.M., Lenkinski, R.E., Gur, R.E., Gur, R.C., 1999. Proton magnetic resonance spectroscopy in the frontal and temporal lobes of neuroleptic naive patients with schizophrenia. Neuropsychopharmacology 20, 131 – 140. Chakos, M.H., Lieberman, J.A., Alvir, J., Bilder, R., Ashtari, M., 1995. Caudate nuclei volumes in schizophrenic patients treated with typical antipsychotics or clozapine. Lancet 345, 456 – 457. Chakraborty, G., Mekala, P., Yahya, D., Wu, G., Ledeen, R.W., 2001. Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase. J. Neurochem. 78, 736 – 745. Choe, B., Kim, K., Suh, T., Lee, C., Paik, I., Bahk, Y., Shinn, K., Lenkinski, R., 1994. H-magnetic resonance spectroscopy characterization of neuronal dysfunction in drug-naRve, chronic schizophrenia. Acad. Radiol. 1, 211 – 216. De Stefano, N., Matthews, P.M., Arnold, D.L., 1995. Reversible decrease in N-acetylaspartate after acute brain injury. Magn. Reson. Med. 34, 721 – 727. Deicken, R.F., Zhou, L., Schuff, N., Fein, G., Weiner, M.W., 1998. Hippocampal neuronal dysfunction in schizophrenia as measured by proton magnetic resonance spectroscopy. Biol. Psychiatry 43, 483 – 488. Demougeot, C., Garnier, P., Mossiat, C., Bertrand, N., Giroud, M., Beley, A., Marie, C., 2001. N-acetylaspartate, a marker of both cellular dysfunction and neuronal loss: its relevance to studies of acute brain injury. J. Neurochem. 77, 408 – 415. Eastwood, S.L., Burnet, P.W., Harrison, P.J., 2000. Expression of complexin I and II mRNAs and their regulation by antipsychotic drugs in the rat forebrain. Synapse 36, 167 – 177.
307
Fannon, D., Simmons, A., Tennakoon, L., O’Ceallaigh, S., Sumich, A., Doku, V., Shew, C., Sharma, T., 2003. Selective deficit of hippocampal N-acetylaspartate in antipsychotic-naive patients with schizophrenia. Biol. Psychiatry 54, 587 – 598. Fukuzako, H., Takeuchi, K., Hokazono, Y., Fukuzako, T., Yamada, K., Hashiguchi, T., 1995. Proton magnetic resonance spectroscopy of the left medial temporal and frontal lobes in chronic schizophrenia: preliminary report. Psychiatry Res. Neuroimaging 61, 193 – 200. Gur, R.E., Maany, V., Mozley, P.D., Swanson, C., Bilker, W., Gur, R.C., 1998. Subcortical MRI volumes in neuroleptic-naive and treated patients with schizophrenia. Am. J. Psychiatry 155, 1711 – 1717. Harrison, P., 1999. The neuropathological effects of antipsychotic drugs. Schizophr. Res. 40, 87 – 99. Harte, M.K., Powell, S.B., Reynolds, L.M., Swerdlow, N.R., Geyer, M.A., Reynolds, G.P., 2004. Reduced n-acetylaspartate in the temporal cortex of rats reared in isolation. Biol. Psychiatry 56, 296 – 299. Holcomb, H.H., Cascella, N.G., Thaker, G.K., Medoff, D.R., Dannals, R.F., Tamminga, C.A., 1996. Functional sites of neuroleptic drug action in the human brain: PET/FDG studies with and without haloperidol. Am. J. Psychiatry 153, 41 – 49. Kalra, S., Cashman, S.H., Genge, A., Arnold, D.L., 1998. Recovery of N-acetylaspartate in corticomotor neurons of patients with ALS after riluzole therapy. NeuroReport 9, 1757 – 1761. Kegeles, L.S., Shungu, D.C., Anjilvel, S., Chan, S., Ellis, S.P., Xanthopoulos, E., 2000. Hippocampal pathology in schizophrenia: magnetic resonance imaging and spectroscopy studies. Psychiatry Res. 98, 163 – 175. Kroesen, S., Marksteiner, J., Mahata, S.K., Mahata, M., FischerColbrie, R., Saria, A., Kapeller, I., Winkler, H., 1995. Effects of haloperidol, clozapine and citalopram on messenger RNA levels of chromogranins A and B and secretogranin II in various regions of rat brain. Neuroscience 69, 881 – 891. Lindquist, D.M., Hawk, R.M., Karson, C.N., Komoroski, R.A., 2000. Effects of antipsychotic drugs on metabolite ratios in rat brain in vivo. Magn. Reson. Med. 43, 355 – 358. Meshul, C.K., Casey, D.E., 1989. Regional, reversible ultrastructural changes in rat brain with chronic neuroleptic treatment. Brain Res. 489, 338 – 346. Meshul, C.K., Janowsky, A., Casey, D.E., Stallbaumer, R.K., Taylor, B., 1992. Coadministration of haloperidol and SCH23390 prevents the increase in dperforatedT synapses due to either drug alone. Neuropsychopharmacology 7, 285 – 293. Moffett, J.R., Namboodiri, M.A., Cangro, C.B., Neale, J.H., 1991. Immunohistochemical localisation of N-acetylaspartate in rat brain. NeuroReport 2, 131 – 134. Molina, V., Sa´nchez, J., Reig, S., Sanz, J., Benito, C., Santamarta, C., Pascau, J., Sarramea, F., Gispert, J.D., Misiego, J.M., 2005. N-acetyl-aspartate levels in the dorsolateral prefrontal cortex in the early years of schizophrenia are inversely related to disease duration. Schizophr. Res. 73. Nudmamud, S., Reynolds, L.M., Reynolds, G.P., 2003. N-acetylaspartate and N-acetylaspartylglutamate deficits in superior temporal cortex in schizophrenia and bipolar disorder. Biol. Psychiatry 53, 1138 – 1141.
308
M.K. Harte et al. / Schizophrenia Research 75 (2005) 303–308
Omori, M., Murata, T., Kimura, H., Koshimoto, Y., Kado, H., Ishimori, Y., Ito, H., Wada, Y., 2000. Thalamic abnormalities in patients with schizophrenia revealed by proton magnetic resonance spectroscopy. Psychiatry Res. 98, 155 – 162. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates (fourth edition). Academic Press, Orlando, FL. Petroff, O.A, Errante, L.D., Rothman, D.L, Kim, J.H., Spencer, D.D., 2002. Neuronal and glial metabolite content of the epileptogenic human hippocampus. Ann. Neurol. 52, 635 – 642. Rael, L.T., Thomas, G.W., Bar-Or, R., Craun, M.L., Bar-Or, D., 2004. An anti-inflammatory role for N-acetyl aspartate in stimulated human astroglial cells. Biochem. Biophys. Res. Commun. 2, 847 – 853. Renshaw, P.F., Yurgelun-Todd, D.A., Tohen, G.S., Cohen, B.M., 1995. Temporal lobe proton magnetic resonance spectroscopy of patients with first-episode psychosis. Am. J. Psychiatry 152, 444 – 446. Reynolds, L.M., Reynolds, G.P., 2004. Post-mortem N-acetylaspartate in schizophrenia, bipolar disorder and depression. J. Psychopharmacol. 18, A66. (Supl.). Reynolds, L.M., Cochran, S.M., Morris, B.J., Pratt, J.A., Reynolds, G.P., 2005. Chronic phencyclidine administration induces schizophrenia-like changes in N-acetylaspartate and N-acetylaspartylglutamate in rat brain. Schizophr. Res. 73. Rowland, L., Bustillo, J., Lauriello, J., 2001. Proton magnetic resonance spectroscopy (1H-MRS) studies of schizophrenia. Semin. Clin. Neuropsychiatry 6, 121 – 130.
See, R.E., Chapman, M.A., Meshul, C.K., 1992. Comparison of chronic intermittent haloperidol and raclopride effects on striatal dopamine release and synaptic ultrastructure in rats. Synapse 12, 147 – 154. Selemon, L.D., Lidow, M.S., Goldman-Rakic, P.S., 1999. Increased volume and glial density in primate prefrontal cortex associated with chronic antipsychotic drug exposure. Biol. Psychiatry 46, 161 – 172. Sigmundsson, T., Maier, M., Toone, B.K., Williams, S.C., Simmons, A., Greenwood, K., Ron, M.A., 2003. Frontal lobe N-acetylaspartate correlates with psychopathology in schizophrenia: a proton magnetic resonance spectroscopy study. Schizophr. Res. 64, 63 – 71. Tallan, H.H., Moore, S., Stein, W.H., 1956. N-acetyl-l-aspartic acid in brain. J. Biol. Chem. 219, 257 – 264. Taylor, D.L., Davies, S.E., Obrenovitch, T.P., Urenjak, J., Richards, D.A., Clarck, J.B., Symon, L., 1994. Extracellular N-acetyl-aspartate in the rat brain: in vivo determination of basal levels and changes evoked by high K+. J. Neurochem. 62, 2349 – 2355. Uranova, N.A., Orlovskaya, D.D., Apel, K., Klintzova, A.J., Haselhfrst, U., Schenk, H., 1991. Morphometric study of synaptic patterns in the rat caudate nucleus and hippocampus under haloperidol treatment. Synapse 7, 253 – 259.
Increased N-acetylaspartate in rat striatum following long-term administration of haloperidol M.K. Hartea,*, S.B. Bachusb, G.P. Reynoldsa,c b
a Department of Biomedical Science, University of Sheffield, Sheffield, S10 2TN, United Kingdom Department of Psychology and Krasnow Institute, George Mason University, Fairfax, Virginia, 22030, USA c Division of Psychiatry and Neuroscience, Queen’s University Belfast, BT9 7BL, United Kingdom
Received 18 October 2004; received in revised form 25 October 2004; accepted 3 November 2004 Available online 1 December 2004
Abstract N-acetylaspartate (NAA) is present in high concentrations in the CNS and is found primarily in neurons. NAA is considered to be a marker of neuronal viability. Numerous magnetic resonance spectroscopy (MRS) and postmortem studies have shown reductions of NAA in different brain regions in schizophrenia. Most of these studies involved patients chronically treated with antipsychotic drugs. However, the effect of chronic antipsychotic treatment on NAA remains unclear. In the present study, we measured NAA in brain tissue taken from 43 male Long-Evans rats receiving 28.5 mg/kg haloperidol decanoate i.m. every 3 weeks for 24 weeks and from 21 controls administered with vehicle. Determination of tissue concentrations of NAA was achieved by HPLC of sections of frozen tissue from several brain regions with relevance to schizophrenia. Chronic administration of haloperidol was associated with a significant increase (+23%) in NAA in the striatum ( pb0.05) when compared to controls, with no significant changes in the other regions investigated (frontal and temporal cortex, thalamus, hippocampus, amygdala, and nucleus accumbens). NAA appears to be selectively increased in the striatum of rats chronically receiving haloperidol. This increase may reflect a hyperfunction of striatal neurons and relate to the reported increase in somal size of these cells and/or the increase in synaptic density seen in this region following antipsychotic administration. The lack of effect in other regions indicates that the welldocumented NAA deficits seen in chronically treated schizophrenia patients is not an effect of antipsychotic medication and may in fact be related to the disease process. D 2004 Elsevier B.V. All rights reserved. Keywords: N-acetylaspartate; Antipsychotics; Postmortem; Schizophrenia
1. Introduction * Corresponding author. Tel.: +44 114 2222313; fax: +44 114 2765413. E-mail address: [email protected] (M.K. Harte). 0920-9964/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2004.11.001
N-acetylaspartate (NAA) was first identified as a major amino acid derivative in the mammalian brain
304
M.K. Harte et al. / Schizophrenia Research 75 (2005) 303–308
by Tallan et al. (1956). It is present in high concentrations in the CNS and is the second most abundant amino acid after glutamate, with intraneuronal concentrations between 10 and 14 mM. NAA is synthesised in neuronal mitochondria from acetyl-coenzyme A and aspartate by the enzyme NAA transferase. In the adult brain, NAA is considered a neuron-specific metabolite (Moffett et al., 1991) and its reduction a marker of neuronal loss. However, recent studies demonstrating the reversibility of NAA deficits following acute brain injury (De Stefano et al., 1995; Kalra et al., 1998) indicate that NAA may be a marker of both neuronal loss and cellular dysfunction (Demougeot et al., 2001). Although the biological function of NAA is not fully understood, it has been shown to be involved in myelin synthesis (Chakraborty et al., 2001), osmoregulation (Baslow, 2003; Taylor et al., 1994), and a recent study showed a possible role as an antiinflammatory (Rael et al., 2004). NAA was also shown to reflect glutamate concentrations and thus may provide a marker of glutamatergic neuronal function (Petroff et al., 2002). In schizophrenia, frontal lobe NAA deficits were shown to correlate with psychopathology and thus may be useful as an indicator of disease severity (Sigmundsson et al., 2003). As it is a noninvasive technique, magnetic resonance spectroscopy (MRS) has become an important tool in monitoring disease progression and in therapy evaluation in patients with neurodegenerative disorders. Of particular interest are the numerous MRS studies that measured brain NAA in discrete brain regions in schizophrenia patients. The majority of these studies have identified reductions in NAA in brain regions implicated in the disease (Rowland et al., 2001). These results are substantiated by recent postmortem studies from this laboratory that have also shown regionally specific reductions in NAA in the temporal cortex, cingulate cortex, hippocampus, amygdala, and caudate nucleus in schizophrenia patients (Nudmamud et al., 2003; Reynolds and Reynolds, 2004). We have also shown regionally specific temporal but not frontal cortical deficits of NAA in two different animal models of schizophrenia (Harte et al., 2004; Reynolds et al., 2005). However, the effect of chronic treatment with antipsychotic drugs on NAA measures remains far from clear.
The importance of examining neuroleptic-naive first-episode patients has been recognized in neurochemical studies of schizophrenia. However, drugnaive and unequivocally diagnosed cases are rare and it is difficult to acquire this patient population. This has lead to investigations of animals administered antipsychotic drugs. Previous studies of chronic antipsychotic administration in rats have demonstrated various behavioural and structural changes comparable to those seen in humans following antipsychotic treatment (Harrison, 1999). Two previous animal studies have attempted to assess the effect of exposure to antipsychotic agents on NAA measures in rat brain. The first was a 1-week study in which rats were given antipsychotics and NAA was measured using MRS. No significant differences were seen between rats receiving haloperidol, clozapine, or olanzapine when compared to vehicle-treated controls (Lindquist et al., 2000). The second was a 6-week study in which NAA in various regions of rat brain was measured by HPLC, following treatment with haloperidol or clozapine. Again there was no significant difference between animals receiving antipsychotics and vehicletreated controls (Bustillo et al., 2004). Although some structural changes are apparent in rats following 6-week exposure to antipsychotics (Harrison, 1999), the NAA reductions in patients treated with antipsychotic agents have been described after months or years of treatment. Therefore, in the current study, we investigated the effects of a more prolonged exposure (24 weeks) to the typical antipsychotic haloperidol, on NAA concentrations in various brain regions implicated in schizophrenia.
2. Methods 2.1. Animals A total of 64 Long–Evans rats, with an initial weight of 90–150 g, were used in this experiment. All rats were kept on a 12-h light–dark cycle (lights on from 0700 to 1900) in a colony room with temperature and humidity remaining constant. Food and water were available ad libitum. Group housed Long–Evans rats were given haloperidol decanoate (28.5 mg/kg/ml, i.m., at 3-week intervals for 24 weeks: n=43) or vehicle (sesame oil: n=21) injections
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occurring every third week. Experiments were carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) revised 1996. All efforts were made to minimize the number of animals used and the suffering of animals in these experiments. 2.2. Tissue preparation and HPLC Animals were killed by decapitation, brains removed and immediately frozen on dry ice. About 16-Am sections were cut using a cryostat and then stored frozen at 70 8C until analysis. NAA was measured in the frontal cortex (Bregma +1, included the cingulate, primary and secondary cortices), temporal cortex (Bregma 3.3, included the primary and secondary auditory cortex and the temporal association cortex), hippocampus (Bregma 3.3 included the dentate gyrus and CA regions), amygdala (Bregma 3.3 included the basolateral, central, lateral, and medial amygdaloid nuclei), thalamus (Bregma 3.3 included nuclei from the anterior, medial and lateral groups), striatum (Bregma +1), and the nucleus accumbens (Bregma +1, included the core and shell regions; Paxinos and Watson, 1998) from these animals. NAA was determined by an established HPLC method (Harte et al., 2004). Briefly, tissue was scraped and pooled from two adjacent frozen sections. The tissue was treated with 0.1 M perchloric acid to precipitate the protein and NAA was extracted from the supernatant using SAX anion columns. The extracted sample was analysed for Table 1 Levels (meanFS.D.) of NAA from rats chronically treated with haloperidol (28.5 mg/kg/ml, i.m., at 3-week intervals for 24 weeks: n=43) and in their vehicle (sesame oil: n=21)-treated controls NAA (nmol/mg protein) Region
Vehicle
Haloperidol
Frontal cortex Temporal cortex Thalamus Hippocampus Amygdala Striatum Nucleus accumbens
89.08F19.24 86.53F12.99 76.52F7.31 76.28F7.93 70.45F10.21 61.85F16.12 83.98F34.21
87.28F19.48 82.37F13.01 79.57F12.32 77.39F10.29 68.02F12.38 74.11F19.73* 89.27F35.54
* pb0.05 vs. control.
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NAA by HPLC (Genesis, C18, 4 Am, 4.6 mm250 mm) using a mobile phase of 0.1% phosphoric acid at a flow rate of 0.5 ml/min. UV detection was at 215 nm. The NAA peak in the samples was identified by its retention time when compared to external standards; samples typically gave peak heights over 100-fold above the limits of sensitivity. NAA gave a linear standard curve (0–50 AM) and was quantified by using relative peak height measurements. Protein concentration in the sample was determined by the Coomassie Blue method and NAA concentrations reported as nmol/mg protein. Statistical analysis (one-way ANOVA) was undertaken using SPSS v10.
3. Results There was a main effect of drug on NAA in the striatum [ F(1,53)=4.31, pb0.05], with rats administered with haloperidol showing increased NAA (+23%) compared to those rats receiving vehicle, after 24 weeks of treatment (Vehicle 61.85F3.51 haloperidol 74.11F3.08 nmol/mg protein). However, NAA concentrations were not significantly different from controls in any of the other regions investigated. NAA concentrations found in the various brain regions from animals receiving vehicle or haloperidol are shown in Table 1.
4. Discussion The main finding of the present study was a robust increase (+23%) in NAA, specific to the striatum, in animals following long-term administration of the typical antipsychotic haloperidol. The striatum, in particular, has been shown to be affected both metabolically (Buchsbaum et al., 1992; Holcomb et al., 1996) and structurally (Chakos et al., 1995; Gur et al., 1998; Harrison, 1999) by treatment with typical antipsychotics. In the adult rat brain, NAA is present in the soma, axon, and dendritic processes. The increase in NAA observed in the present study may reflect a hyperfunction of striatal neurons, relate to the reported increase in somal size of these cells, and/or an increase in synaptic density in this region. These findings may, in turn, be a correlate of striatal (i.e.,
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extrapyramidal motor) side effects of treatment with typical antipsychotic drugs. In support of this are numerous ultrastructural studies looking at the effect of antipsychotics on neuronal processes in the striatum of the rat. These studies have shown an increase in the size of axon terminals (Benes et al., 1985), an increase in synapses with perforated postsynaptic densities, which is an indicator of newly formed synapses and thus a marker of synaptic plasticity (Meshul and Casey, 1989; Meshul et al., 1992; See et al., 1992), an increase in axodendritic and axospinous synapses, with the axospinous synapses showing larger axon terminals with more mitochondria (Uranova et al., 1991), and an increase in various synaptic markers in the striatum (Eastwood et al., 2000; Kroesen et al., 1995). The observation that most of these changes reverse within weeks of stopping the drugs and that typical and atypical antipsychotics may have different effects are further evidences of a medication effect and are not indicative of a disease related abnormality in this region. In addition, an equally important finding, in terms of MRS and postmortem work in schizophrenia, was the lack of effect of haloperidol on NAA in the other brain regions investigated. Previous studies have reported deficits in NAA in the frontal cortex, temporal cortex, hippocampus, thalamus, and dorsolateral prefrontal cortex of schizophrenic patients; however, some studies have failed to identify NAA abnormalities in these regions (Bertolino et al., 1998; Buckley et al., 1994; Deicken et al., 1998; Fukuzako et al., 1995; Kegeles et al., 2000; Omori et al., 2000; Renshaw et al., 1995). However, the majority of these studies involved patients chronically treated with antipsychotic drugs. In a recent longitudinal study by Bustillo et al. (2002), schizophrenia patients, with history of minimal previous treatment (b3 weeks), showed reductions in NAA in the frontal cortex during the first year of treatment with antipsychotic medications (NAA levels at baseline were similar to controls). Patients had a clear clinical response to treatment but changes in frontal NAA were not correlated with symptom improvement. The authors concluded that the reduced frontal NAA reported in schizophrenia may not be a trait of the illness but may represent either a medication effect or progression of the disease.
However, evidence against a medication effect comes from other MRS studies which have demonstrated lower frontal NAA in neuroleptic-naive and drug-free chronic schizophrenia patients (Cecil et al., 1999; Choe et al., 1994). Another recent report arguing against a medication effect comes from a study where NAA deficits were found in the hippocampus of antipsychotic-naive patients (Fannon et al., 2003). MRS studies have consistently revealed lower NAA concentrations in chronic schizophrenic patients, while the results in recent onset patients are contradictory. In a recent study by Molina et al. (2005), where chronic patients were distinguished form recent onset patients, a significant relationship between NAA and disease duration was reported. If NAA is a marker of neuronal loss/viability, then such findings are consistent with a neurodegenerative hypothesis of schizophrenia. However, the majority of evidence does not support antipsychotic-induced neuronal death or permanent damage. In a recent study by Selemon et al. (1999), there was no evidence of neuronal loss or gliosis in monkeys exposed (6 months) to various typical and atypical drugs. Furthermore, postmortem neurodegenerative changes in schizophrenia due to a medication effect are not supported in the literature (Baldessarini et al., 1997). Taken together, the results of the present study suggest that the deficits of NAA reported in MRS and postmortem studies in schizophrenia are not likely to be caused by long term treatment with typical antipsychotics. Whether the same holds true for the newer atypical antipsychotic, a class of drugs with a different receptor profile needs to be further investigated.
Acknowledgements M.K. Harte is supported by a Merck Sharp and Dohme Pharmacology Fellowship.
References Baldessarini, R.J., Hegarty, J.D., Bird, E.D., Benes, F.M., 1997. Meta-analysis of post-mortem studies of Alzheimer’s diseaselike neuropathology in schizophrenia. Am. J. Psychiatry 154, 861 – 863. Baslow, M.H., 2003. N-acetylaspartate in the vertebrate brain: metabolism and function. Neurochem. Res. 28, 941 – 953.
M.K. Harte et al. / Schizophrenia Research 75 (2005) 303–308 Benes, F.M., Paskevich, P.A., Davidson, J., Domesick, V.B., 1985. The effects of haloperidol on synaptic patterns in the rat striatum. Brain Res. 329, 265 – 274. Bertolino, A., Callicott, J.H., Elman, I., Mattay, V.S., Tedeschi, G., Frank, J.A., 1998. Regionally specific neuronal pathology in untreated patients with schizophrenia: a proton magnetic resonance spectroscopic imaging study. Biol. Psychiatry 43, 641 – 648. Buchsbaum, M.S., Potkin, S.G., Siegel Jr., B.V., Lohr, J., Katz, M., Gottschalk, L.A., Gulasekaram, B., Marshall, J.F., Lottenberg, S., Teng, C.Y., 1992. Striatal metabolic rate and clinical response to neuroleptics in schizophrenia. Arch. Gen. Psychiatry 49, 966 – 974. Buckley, P.F., Moore, C., Long, H., Larkin, C., Thompson, P., Mulvany, F., 1994. 1H-magnetic resonance spectroscopy of the left frontal lobes in schizophrenia: clinical, neurodevelopmental, and cognitive correlates. Biol. Psychiatry 36, 792 – 800. Bustillo, J.R., Lauriello, J., Rowland, L.M., Thomson, L.M., Petropoulos, H., Hammond, R., Hart, B., Brooks, W.M., 2002. Longitudinal follow-up of neurochemical changes during the first year of antipsychotic treatment in schizophrenia patients with minimal previous medication exposure. Schizophr. Res. 58, 313 – 321. Bustillo, J.R., Wolff, C., Gutierrez, A.M., Dettmer, T.S., Cooper, T.B., Allan, A., Lauriello, J., Valenzuela, C.F., 2004. Treatment of rats with antipsychotic drugs: lack of an effect on brain Nacetyl aspartate levels. Schizophr. Res. 66, 31 – 39. Cecil, K.M., Lenkinski, R.E., Gur, R.E., Gur, R.C., 1999. Proton magnetic resonance spectroscopy in the frontal and temporal lobes of neuroleptic naive patients with schizophrenia. Neuropsychopharmacology 20, 131 – 140. Chakos, M.H., Lieberman, J.A., Alvir, J., Bilder, R., Ashtari, M., 1995. Caudate nuclei volumes in schizophrenic patients treated with typical antipsychotics or clozapine. Lancet 345, 456 – 457. Chakraborty, G., Mekala, P., Yahya, D., Wu, G., Ledeen, R.W., 2001. Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase. J. Neurochem. 78, 736 – 745. Choe, B., Kim, K., Suh, T., Lee, C., Paik, I., Bahk, Y., Shinn, K., Lenkinski, R., 1994. H-magnetic resonance spectroscopy characterization of neuronal dysfunction in drug-naRve, chronic schizophrenia. Acad. Radiol. 1, 211 – 216. De Stefano, N., Matthews, P.M., Arnold, D.L., 1995. Reversible decrease in N-acetylaspartate after acute brain injury. Magn. Reson. Med. 34, 721 – 727. Deicken, R.F., Zhou, L., Schuff, N., Fein, G., Weiner, M.W., 1998. Hippocampal neuronal dysfunction in schizophrenia as measured by proton magnetic resonance spectroscopy. Biol. Psychiatry 43, 483 – 488. Demougeot, C., Garnier, P., Mossiat, C., Bertrand, N., Giroud, M., Beley, A., Marie, C., 2001. N-acetylaspartate, a marker of both cellular dysfunction and neuronal loss: its relevance to studies of acute brain injury. J. Neurochem. 77, 408 – 415. Eastwood, S.L., Burnet, P.W., Harrison, P.J., 2000. Expression of complexin I and II mRNAs and their regulation by antipsychotic drugs in the rat forebrain. Synapse 36, 167 – 177.
307
Fannon, D., Simmons, A., Tennakoon, L., O’Ceallaigh, S., Sumich, A., Doku, V., Shew, C., Sharma, T., 2003. Selective deficit of hippocampal N-acetylaspartate in antipsychotic-naive patients with schizophrenia. Biol. Psychiatry 54, 587 – 598. Fukuzako, H., Takeuchi, K., Hokazono, Y., Fukuzako, T., Yamada, K., Hashiguchi, T., 1995. Proton magnetic resonance spectroscopy of the left medial temporal and frontal lobes in chronic schizophrenia: preliminary report. Psychiatry Res. Neuroimaging 61, 193 – 200. Gur, R.E., Maany, V., Mozley, P.D., Swanson, C., Bilker, W., Gur, R.C., 1998. Subcortical MRI volumes in neuroleptic-naive and treated patients with schizophrenia. Am. J. Psychiatry 155, 1711 – 1717. Harrison, P., 1999. The neuropathological effects of antipsychotic drugs. Schizophr. Res. 40, 87 – 99. Harte, M.K., Powell, S.B., Reynolds, L.M., Swerdlow, N.R., Geyer, M.A., Reynolds, G.P., 2004. Reduced n-acetylaspartate in the temporal cortex of rats reared in isolation. Biol. Psychiatry 56, 296 – 299. Holcomb, H.H., Cascella, N.G., Thaker, G.K., Medoff, D.R., Dannals, R.F., Tamminga, C.A., 1996. Functional sites of neuroleptic drug action in the human brain: PET/FDG studies with and without haloperidol. Am. J. Psychiatry 153, 41 – 49. Kalra, S., Cashman, S.H., Genge, A., Arnold, D.L., 1998. Recovery of N-acetylaspartate in corticomotor neurons of patients with ALS after riluzole therapy. NeuroReport 9, 1757 – 1761. Kegeles, L.S., Shungu, D.C., Anjilvel, S., Chan, S., Ellis, S.P., Xanthopoulos, E., 2000. Hippocampal pathology in schizophrenia: magnetic resonance imaging and spectroscopy studies. Psychiatry Res. 98, 163 – 175. Kroesen, S., Marksteiner, J., Mahata, S.K., Mahata, M., FischerColbrie, R., Saria, A., Kapeller, I., Winkler, H., 1995. Effects of haloperidol, clozapine and citalopram on messenger RNA levels of chromogranins A and B and secretogranin II in various regions of rat brain. Neuroscience 69, 881 – 891. Lindquist, D.M., Hawk, R.M., Karson, C.N., Komoroski, R.A., 2000. Effects of antipsychotic drugs on metabolite ratios in rat brain in vivo. Magn. Reson. Med. 43, 355 – 358. Meshul, C.K., Casey, D.E., 1989. Regional, reversible ultrastructural changes in rat brain with chronic neuroleptic treatment. Brain Res. 489, 338 – 346. Meshul, C.K., Janowsky, A., Casey, D.E., Stallbaumer, R.K., Taylor, B., 1992. Coadministration of haloperidol and SCH23390 prevents the increase in dperforatedT synapses due to either drug alone. Neuropsychopharmacology 7, 285 – 293. Moffett, J.R., Namboodiri, M.A., Cangro, C.B., Neale, J.H., 1991. Immunohistochemical localisation of N-acetylaspartate in rat brain. NeuroReport 2, 131 – 134. Molina, V., Sa´nchez, J., Reig, S., Sanz, J., Benito, C., Santamarta, C., Pascau, J., Sarramea, F., Gispert, J.D., Misiego, J.M., 2005. N-acetyl-aspartate levels in the dorsolateral prefrontal cortex in the early years of schizophrenia are inversely related to disease duration. Schizophr. Res. 73. Nudmamud, S., Reynolds, L.M., Reynolds, G.P., 2003. N-acetylaspartate and N-acetylaspartylglutamate deficits in superior temporal cortex in schizophrenia and bipolar disorder. Biol. Psychiatry 53, 1138 – 1141.
308
M.K. Harte et al. / Schizophrenia Research 75 (2005) 303–308
Omori, M., Murata, T., Kimura, H., Koshimoto, Y., Kado, H., Ishimori, Y., Ito, H., Wada, Y., 2000. Thalamic abnormalities in patients with schizophrenia revealed by proton magnetic resonance spectroscopy. Psychiatry Res. 98, 155 – 162. Paxinos, G., Watson, C., 1998. The Rat Brain in Stereotaxic Coordinates (fourth edition). Academic Press, Orlando, FL. Petroff, O.A, Errante, L.D., Rothman, D.L, Kim, J.H., Spencer, D.D., 2002. Neuronal and glial metabolite content of the epileptogenic human hippocampus. Ann. Neurol. 52, 635 – 642. Rael, L.T., Thomas, G.W., Bar-Or, R., Craun, M.L., Bar-Or, D., 2004. An anti-inflammatory role for N-acetyl aspartate in stimulated human astroglial cells. Biochem. Biophys. Res. Commun. 2, 847 – 853. Renshaw, P.F., Yurgelun-Todd, D.A., Tohen, G.S., Cohen, B.M., 1995. Temporal lobe proton magnetic resonance spectroscopy of patients with first-episode psychosis. Am. J. Psychiatry 152, 444 – 446. Reynolds, L.M., Reynolds, G.P., 2004. Post-mortem N-acetylaspartate in schizophrenia, bipolar disorder and depression. J. Psychopharmacol. 18, A66. (Supl.). Reynolds, L.M., Cochran, S.M., Morris, B.J., Pratt, J.A., Reynolds, G.P., 2005. Chronic phencyclidine administration induces schizophrenia-like changes in N-acetylaspartate and N-acetylaspartylglutamate in rat brain. Schizophr. Res. 73. Rowland, L., Bustillo, J., Lauriello, J., 2001. Proton magnetic resonance spectroscopy (1H-MRS) studies of schizophrenia. Semin. Clin. Neuropsychiatry 6, 121 – 130.
See, R.E., Chapman, M.A., Meshul, C.K., 1992. Comparison of chronic intermittent haloperidol and raclopride effects on striatal dopamine release and synaptic ultrastructure in rats. Synapse 12, 147 – 154. Selemon, L.D., Lidow, M.S., Goldman-Rakic, P.S., 1999. Increased volume and glial density in primate prefrontal cortex associated with chronic antipsychotic drug exposure. Biol. Psychiatry 46, 161 – 172. Sigmundsson, T., Maier, M., Toone, B.K., Williams, S.C., Simmons, A., Greenwood, K., Ron, M.A., 2003. Frontal lobe N-acetylaspartate correlates with psychopathology in schizophrenia: a proton magnetic resonance spectroscopy study. Schizophr. Res. 64, 63 – 71. Tallan, H.H., Moore, S., Stein, W.H., 1956. N-acetyl-l-aspartic acid in brain. J. Biol. Chem. 219, 257 – 264. Taylor, D.L., Davies, S.E., Obrenovitch, T.P., Urenjak, J., Richards, D.A., Clarck, J.B., Symon, L., 1994. Extracellular N-acetyl-aspartate in the rat brain: in vivo determination of basal levels and changes evoked by high K+. J. Neurochem. 62, 2349 – 2355. Uranova, N.A., Orlovskaya, D.D., Apel, K., Klintzova, A.J., Haselhfrst, U., Schenk, H., 1991. Morphometric study of synaptic patterns in the rat caudate nucleus and hippocampus under haloperidol treatment. Synapse 7, 253 – 259.