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Treatment of rats with antipsychotic drugs: lack of an effect on brain N-acetyl aspartate levels
Schizophrenia Research 66 (2004) 31 – 39 www.elsevier.com/locate/schres
Treatment of rats with antipsychotic drugs: lack of an effect on brain N-acetyl aspartate levels Juan Bustillo a,b,1,2, Christina Wolff a, Adan Myers-y-Gutierrez c, Todd S. Dettmer b,3, Tom B. Cooper d,e, Andrea Allan b, John Lauriello a,1, C. Fernando Valenzuela b,*,4 a
b
Department of Psychiatry, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA Department of Neurosciences, University of New Mexico Health Sciences Center, Basic Medical Sciences Building, Room 145, Albuquerque, NM 87131, USA c Mental Illness Neuroscience Discovery Institute, USA d Nathan Kline Institute, USA e College of P&S Columbia University, USA Received 6 June 2002; received in revised form 2 December 2002; accepted 12 December 2002
Abstract Background: Proton magnetic resonance spectroscopy (1H-MRS) studies of schizophrenia suggest an effect of the disease or of antipsychotic medications on brain N-acetyl aspartate (NAA), a marker of neuronal viability. We studied in the rat the effect of antipsychotic drugs on NAA in several brain regions where NAA reductions have been reported in chronically medicated patients with schizophrenia. Methods: Three groups of nine rats each were treated with haloperidol (6 mg/kg/day), clozapine (70 mg/kg/day) and vehicle for 6 weeks and were sacrificed. Concentrations of NAA were determined by high-performance liquid chromatography (HPLC) from the following brain regions: cortex, striatum, thalamus, hippocampus and cerebellum. Results: Mixed-factorial ANOVA of NAA concentrations revealed no significant effect of drug group [ F(2, 24) = 0.034; p = 0.966] or a group by brain region interaction [ F(8, 44) = 0.841; p = 0.572]. There was a significant main effect of region [ F(4, 21) = 6.104; p = 0.002] with higher NAA in the cortex. Conclusions: These results are consistent with the only other study of the effect of typical and atypical antipsychotic drugs on NAA in the rat brain. The well-documented lower NAA in chronically treated schizophrenia patients is probably not a simple effect of antipsychotic medications. D 2003 Elsevier Science B.V. All rights reserved. Keywords: N-acetyl aspartate; Clozapine; Haloperidol; Antipsychotic; Schizophrenia; HPLC
* Corresponding author. Tel.: +1-505-272-3128; fax: +1-505-272-8082. E-mail address: [email protected] (C.F. Valenzuela). 1 Financial support: State of New Mexico Antipsychotic Algorithm Project Consultation Grant from New Mexico Department of Health. 2 Financial support: Mental Illness Neuroscience Discovery Institute. 3 Financial support: UNM-Minority Biomedical Research Support Program funded by NIH Grant R25 GM 60201-2. 4 Financial support: NIH Grants MH63126 and AA12684. 0920-9964/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0920-9964(02)00528-5
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1. Introduction Proton magnetic resonance spectroscopy ( 1HMRS) studies of schizophrenia have reported reduced N-acetyl aspartate (NAA) mainly in frontal and mesial temporal, but also in thalamic and cerebellar regions (see Rowland et al., 2001, for review). The predominant view regarding NAA is that it is produced in the neuronal mitochondria, found almost exclusively in the neuron and is more concentrated in gray than white matter (Tsai and Coyle, 1995; Moffet and Namdoori, 1995; Pfefferbaum et al., 1999). NAA may play a role in osmoregulation (Taylor et al., 1994), myelination (Bhakoo and Pearce, 2000), glial cell signaling (Baslow, 2000) and neuronal function (Daughtry et al., 2000), but its specific functions are far from clear. Once thought to be a relatively stable marker of neuronal density, NAA is now also considered a sensitive marker of neuronal viability and function. In a longitudinal study of Alzheimer’s dementia and healthy subjects, NAA became globally reduced in the demented group before cortical atrophy was apparent (Adalsteinsson et al., 2000). Finally, NAA is related to cognitive functioning. For example, a direct relationship between cognitive performance and NAA levels has been described in: systemic lupus erythematosus (Friedman et al., 1998), traumatic brain injury (Ross et al., 1998; Brooks et al., 1999), HIV encephalopathy (Lopez-Villegas et al., 1997), temporal lobe epilepsy (Martin et al., 1999; Pauli et al., 2000; Kikuchi et al., 2001), schizophrenia (Bertolino et al., 2000; Bustillo et al., 2001; Braus et al., 2002) and in healthy volunteers (Jung et al., 1999). The majority of schizophrenia studies that have documented NAA reductions involved patients chronically treated with antipsychotic medications. The few studies assessing antipsychotic-naı¨ve patients have been inconclusive, perhaps due to methodological differences (Rowland et al., 2001). We have presented preliminary longitudinal data in schizophrenia patients with history of minimal previous treatment that documents reductions in frontal NAA during the first year of treatment with antipsychotic medications (Bustillo et al., 2002). Antipsychotic drugs exert both their therapeutic and deleterious extrapyramidal side effects by blocking dopamine D2 receptors and have been shown to lead to both structural and metabolic changes in the brain (Chakos et al., 1995; Keshavan
et al., 1994; Holcomb et al., 1996). Hence, it is possible that NAA reductions in schizophrenia are the result of antipsychotic medications, but separating the impact of the disease from the treatment is problematic in clinical populations. One study that assessed the effect of 1 week exposure to antipsychotic agents on NAA measures in rat brain found negative results (Lindquist et al., 2000). In the current study, we investigated the effects of a more prolonged exposure (6 weeks) to two antipsychotic medications, haloperidol and clozapine, on NAA levels on various brain regions in rats.
2. Methods All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of New Mexico (UNM) Health Sciences Center and were performed according to the guidelines of the National Institute of Health. Two-monthold male Sprague –Dawley rats weighing about 200 g each were purchased from Harlan (Indianapolis, IN), housed in pairs and allowed to acclimatize for 2 weeks before the start of the study. Rats were housed at the UNM animal facility under a 12:12 light/dark cycle (lights on at 7 am) and received Purina rat chow and tap water ad libitum. After a training period of about a week, rats were administered by gastric intubation 70 mg/kg/day of clozapine (n = 9; kindly provided by Anna-Maria Sutter, Novartis Pharma, Basel, Switzerland) and 6 mg/kg/day of haloperidol (n = 9; Sigma, St. Louis, MO). These dosages are significantly higher than those used in other studies in rats. However, pilot studies in our laboratory confirmed their usefulness in achieving blood levels comparable to those considered therapeutic in humans (Palao et al., 1994; VanderZwaag et al., 1996). Dosages were given twice a day (9 am and 5 pm) for 39 – 40 days. The drugs were dissolved in 1 M ascorbic acid as 23 and 1.8 mg/ml stock solutions for clozapine and haloperidol, respectively. Control rats (n = 9) received equivalent amounts of this vehicle. At the end of the treatment, rats belonging to the control, haloperidol and clozapine groups weighed 374 F 7, 344 F 13 and 351 F 9 g, respectively. These values were found not to be statistically significantly different by ANOVA ( p>0.09).
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Rats were euthanized by decapitation with a guillotine under halothane anesthesia 4 – 6 h after the morning gavages. Trunk blood was collected in heparinized tubes and centrifuged to obtain plasma to assay for drug levels. Clozapine was quantified as described by Simpson and Cooper (1978) with two modifications: norclozapine was measured using a capillary column and a nitrogen detector was substituted for the electron capture detector originally described. This latter modification gives clean chromatography with excellent stability over time. Interassay coefficient of variation (CV) is 4.5% for clozapine and 6.2% for norclozapine at 300 ng/ml. Haloperidol was determined in plasma using a gas chromatography procedure as described by Bianchetti and Moselli (1978) with two modifications: the use of chlorohaloperidol as an internal standard and the simultaneous quantitation of reduced haloperidol. The interassay CVs for haloperidol and reduced haloperidol were both < 6%. Brains were rapidly removed and regions of interest (cortex, striatum, thalamus, hippocampus and cerebellum, all bilateral) were dissected out on ice and placed in plastic tubes that had been precooled on dry ice. Samples were stored at 80 jC until ready to use. Samples were prepared essentially as described by Florian et al. (1996). Twenty milligrams of thawed tissue from each region was homogenized by sonication in 400 Al of 12% perchloric acid and incubated on ice for 30 min. Samples were centrifuged at 3000 g for 10 min and the supernatant was transferred to a new tube. Supernatants were neutralized to pH 5– 6 with KOH and centrifuged again as described above. Supernatants were then stored at 20 jC. Concentrations of NAA were determined by highperformance liquid chromatography (HPLC) using a Partisil-10 SAX anion exchange column (Whatman, Clifton, NJ). A HPXL solvent delivery HPLC system (Rainin, Woburn, MA) controlled by a Macintosh IISi computer was used for these determinations. This system is equipped with a Dynamax absorbance detector Model UV-DII. The sampling interval was 0.5 s and the detector wavelength was 214 nm. The mobile phase was 0.1 M potassium phosphate monobasic and 0.025 M potassium chloride at pH 4.5. Buffer was passed through a 0.22-Am filter and degassed under vacuum and bath sonication for 30 min. The column was equilibrated with this buffer for
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40 min at a flow rate of 1.5 ml/min. NAA standard (Sigma) was diluted in this buffer. To analyze the samples from the brain regions of interest, an aliquot of the supernatants corresponding to 2 mg of wet tissue was injected in a total volume of 500 Al of the mobile phase buffer. Samples were injected manually. Area under the curve for the peaks of interest was determined with Dynamax HPLC Method Manager software (Rainin). Concentrations of NAA were determined by extrapolation into a standard curve measured on the same day as unknowns. The total run time for each sample was 9 – 10 min. NAA levels were analyzed with a 3 (group) by 5 (region) mixed-factorial ANOVA with repeated measures for region using SPSS software. Data are presented as mean F S.E.M. in all cases.
3. Results We first injected increasing amounts of NAA standard dissolved in mobile phase buffer. These standards eluted as single peaks with retention times ranging between 5.4 and 5.6 min (data not shown). These values are in agreement with previous reports (Koller et al., 1984). Quantification of these peaks revealed that the area under the curve increases linearly with respect to the NAA amount injected (data not shown). These values were used to generate standard curves on each of the different days the assay was performed. The concentration of NAA in the five
Fig. 1. Sample chromatogram obtained by injection of an amount of cortical extract from a clozapine-treated rat corresponding to 0.66 mg of wet tissue. The injection volume was 500 Al. The dotted lines represent the baseline used by the computer program to calculate peak area. The flow rate was 1.5 ml/min and sampling interval was 0.5 s. The arrow indicates the time of injection.
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regions of the rat brain was then quantified using the respective standard curve acquired on a given day. Fig. 1 illustrates a typical chromatogram obtained by injected extracts from the rat cortex. Similar chromatograms were obtained from other brain regions. As in the case of the standards, NAA eluted as peaks with retention times ranging between 5.4 and 5.6 min. The quantification of NAA levels in the cortex, striatum, thalamus, hippocampus and cerebellum from control, haloperidol and clozapine-treated rats is shown in Fig. 2. The mixed-factorial ANOVA revealed no significant group by region interaction [Wilk’s k = 0.734, F(8, 44) = 0.841, p = 0.572] and no main effect of group [ F(2, 24) = 0.034, p = 0.966]. These results do not support an effect of antipsychotic drug exposure on NAA levels in any of the regions studied. However, there was a significant main effect of region [Wilk’s k = 0.462, F(4, 21) = 6.104, p = 0.002] with follow-up comparisons revealing significantly higher NAA in cortex compared to striatum ( p = 0.01), thalamus ( p = 0.01) and hippocampus ( p = 0.01). These higher concentrations in cortex are consistent with previous rat studies using HPLC methods (Koller et al., 1984; Florian et al., 1996). The plasma concentrations of clozapine and norclozapine were 242.9 (S.E. = 51.1) and 980.2
Fig. 2. Distribution of NAA levels in the cortex (CTX), striatum (Stri), thalamus (Thal), hippocampus (Hipp) and cerebellum (Cer). Samples were injected as described in the legend for Fig. 1. NAA levels were measured in samples from rats belonging to the control (C), haloperidol (H) and clozapine (Cl) groups. Each bar represents the mean F S.E.M. NAA level obtained in each region (n = 9 separate rats per group).
(S.E. = 92.6) ng/ml, respectively. The concentration of haloperidol was 31.8 (S.E. = 4.1) ng/ml.
4. Discussion In rats exposed to clozapine or haloperidol, we found no differences in NAA concentrations in cortex, striatum, thalamus, hippocampus or cerebellum, compared to animals treated with vehicle. We are aware of only one previous study assessing the impact of antipsychotic drugs on NAA brain levels in animals. Lindquist et al. (2000) treated rats with intraperitoneal daily injections of olanzapine (1 mg/kg/day; n = 8), clozapine (10 mg/kg/day; n = 8), haloperidol (0.2 mg/ kg/day; n = 8) and vehicle (n = 8) for 1 week. Ratios of NAA to creatine (NAA/Cre) were calculated from spectra measured at 4.7 T with single-voxel 1H-MRS before, after 1 day and at the end of treatment. The voxel studied was 0.216 cm3 and located ‘‘. . . in front of the cerebellum, and within the volume defined by the left and right ventricle’’ (Lindquist et al., 2000). There were no differences in NAA/Cre between the four groups at the end of treatment. There was a small reduction in NAA/Cre between baseline and 1 week values only in the olanzapine-treated group (1.16 vs. 1.12; p < 0.04). However, this was not a predicted result, uncorrected for multiple comparisons and the authors interpreted the overall findings as not supportive of a medication effect on NAA/Cre ratios. In contrast to the shortage of animal studies, there are many reports using 1H-MRS to study NAA in schizophrenia populations treated with antipsychotic medications. Most 1H-MRS schizophrenia studies involve cross-sectional assessments of patients chronically treated with mainly typical antipsychotics (like haloperidol, potent blockers of dopamine D2 receptors more likely to induce extrapyramidal side effects— EPS). These studies report lower NAA or NAA/Cre in several brain structures like: mesial temporal lobe, which include the hippocampus (Nasrallah et al., 1994; Fukuzako et al., 1995; Maier et al., 1995; Bertolino et al., 1996; Yurgelun-Todd et al., 1996; Deicken et al., 1998; but not Buckley et al., 1994; or Heimberg et al., 1998); anterior cingulate cortex (Deicken et al., 1997; Thomas et al., 1998; Ende et al., 2000); dorsolateral prefrontal cortex (DLPFC; Bertolino et al., 1996; but not Kegeles et al., 2000); larger
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(8– 12 cm3) prefrontal locations (Buckley et al., 1994; Lim et al., 1998; Bustillo et al., 2001; but not Fukuzako et al., 1995; or Omori et al., 2000); thalamus (Deicken et al., 2000; Omori et al., 2000; but not Bertolino et al., 1996); and the cerebellum (Deicken et al., 2001; but not Eluri et al., 1998). Three cross-sectional studies specifically investigated differential effects between typical and atypical medications (drugs with a lower EPS profile, like clozapine). In two studies, schizophrenia patients treated with typical agents had lower frontal NAA than normal controls while patients receiving atypical medications had intermediate, but not significantly different levels (Ende et al., 2000; Bustillo et al., 2001). A recent report found higher anterior cingulate NAA in patients treated with atypicals (mostly clozapine) compared to a group receiving typical agents (Braus et al., 2001). Interestingly, these authors also found a direct relationship between length of time on atypical and NAA that was interpreted as a restorative effect of atypical drugs on the metabolite. The few studies that investigated NAA early in schizophrenia and before treatment have produced inconsistent results. Renshaw et al. (1995) and Cecil et al. (1999) found lower NAA/Cre in mesial temporal regions but Bartha et al. (1999) found normal NAA concentrations. Choe et al. (1994) and Cecil et al. (1999) reported lower frontal NAA/Cre, whereas Stanley et al. (1996), Bartha et al. (1997) and Bustillo et al. (2002) found frontal NAA concentrations similar to healthy controls. These contradicting results are probably due to methodological differences, like large voxels with varied tissue composition and use of metabolite ratios. Because NAA, Cho and Cre can vary independently in the human brain (Pfefferbaum et al., 1999), metabolite concentrations may be a more valid measure than ratios. Few longitudinal studies of schizophrenia have assessed NAA in the context of antipsychotic drug treatment. Choe et al. (1996) found no changes in left frontal NAA/Cre during naturalistic treatment with typical and atypical agents. Bertolino et al. (2001), in a retrospective study, reported higher NAA/Cre in the DLPFC in patients while treated with antipsychotic medication as compared to when they were medication-free. We found reductions in left frontal NAA in patients with minimal prior lifetime antipsychotic exposure (less than 3 weeks)
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during the first year of randomized treatment with either haloperidol or quetiapine (Bustillo et al., 2002). Williamson et al. (2001) studied 10 antipsychotic-naı¨ve patients before and after treatment with atypical antipsychotic drugs. They report normal NAA in the anterior cingulate and thalamus before medications, with subsequent reductions within 1 year of treatment in both regions. Again, inconsistent findings in these longitudinal studies likely reflect methodological differences, but ours (Bustillo et al., 2002) and the preliminary results of Williamson et al. (2001) suggest frontal NAA reductions early in schizophrenia in the context of initial antipsychotic exposure and regardless of the use of atypical agents. Results from the present study do not support a simple medication effect. Progression of the disease or an interaction between disease and antipsychotic medications are alternative explanations. Antipsychotic drugs clearly have metabolic, macroand microstructural brain effects that go beyond their well-described receptor blocking properties. The best documented macrostructural effect in persons with schizophrenia is the enlargement of striatal volumes with typical agents, which reverses when subjects are switched to treatment with the atypical drug clozapine (Keshavan et al., 1994; Chakos et al., 1995). Several reports of first-episode schizophrenia patients longitudinally followed with magnetic resonance imaging (MRI) demonstrate subtle global brain volume reductions over the first few years of treatment (DeLisi et al., 1997; Gur et al., 1998; Giedd et al., 1999; Lieberman et al., 2001). Because antipsychotic medications can cause neurotoxic effects (e.g. tardive dyskinesia), their potential impact on these structural findings has not been excluded. However, the preponderance of evidence does not support antipsychotic-induced neuronal death (Baldessarini et al., 1997; Selemon et al., 1999; Lidow et al., 2001). Lower frontal glucose metabolism and cerebral blood flow have been found in positron emission tomography (PET) studies of schizophrenia patients treated with antipsychotic medications compared to a drug-free state (Holcomb et al., 1996; Miller et al., 2001). Also, a single dose of haloperidol resulted in a similar reduction in cortical metabolism in healthy volunteers (Bartlett et al., 1994). This hypometabolic frontal effect appears to be more severe with typical than with atypical antipsychotics (Miller et al., 2001),
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but it is not established if it relates to the therapeutic and/or the deleterious effects of the medications. A variety of microstructural effects have been described in studies of rodents exposed to antipsychotics drugs (Nielsen and Lyon, 1978; Benes et al., 1985; Roberts et al., 1995; Meshul et al., 1996; Kelley et al., 1997). These mainly involve an increase in the proportion of symmetric synapses in the striatum (see Harrison, 1999, for review). In nonhuman primates, prolonged exposure to both typical and atypical agents results in up-regulation of dopamine D2 and down-regulation of D1 receptors in prefrontal cortical regions (Lidow and Goldman-Rakic, 1994), and increased glial density in specific cortical layers (Selemon et al., 1999) without loss of neurons. In monkeys, long-term exposure to haloperidol did not result in neuronal loss. However, alterations of dendritic and spine-associated proteins were detected in various frontal cortical areas, but not in the primary visual cortex (Lidow et al., 2001). This was interpreted as evidence of collapse of dendritic structure in regions rich in dopamine receptors secondary to dopamine blockade. Because NAA is located in the neuronal soma as well as its processes, a reduction in the density of dendrites could theoretically result in lowering of NAA. Several limitations of the present study should be considered when interpreting our results. First, animals were exposed to drugs for only 6 weeks. In this time frame, a metabolic effect is apparent in schizophrenia patients (Holcomb et al., 1996; Miller et al., 2001) and a variety of microstructural changes have been demonstrated in rodents (Harrison, 1999). However, the NAA reductions in patients treated with antipsychotic agents have been described after 6 –12 months of treatment (Bustillo et al., 2002; Williamson et al., 2001). Therefore, it is possible that longer exposure in animals will result in similar NAA reductions. Second, the brain regions studied were rather large and a putative drug effect on NAA may be limited to more discrete areas. However, we specifically selected regions in which NAA reductions have been repeatedly described in chronically treated patients. Third, we studied only two antipsychotic drugs albeit ones very different in terms of neurological side effects. Clozapine and haloperidol are the most representative agents of the so-called atypical and typical class of antipsychotics, respectively. They
offer the additional advantage of being the two antipsychotic drugs for which blood levels have been linked to clinical effects (Palao et al., 1994; VanderZwaag et al., 1996), allowing a more meaningful comparison between exposure in humans and animals than if based only on dosage administered. Fourth, the animals studied were healthy and not representative of the fundamental neurobiological substrate that underlies schizophrenia, raising the possibility of an interaction between the disease and the therapeutic or deleterious effects of the medications. Finally, our design was cross-sectional and a longitudinal study may be more powerful to detect within subject changes. Future animal studies with repeated in vivo 1 H-MRS measures over several months would address this limitation. In summary, we found no evidence in rodents that exposure to dosages of clozapine or haloperidol consistent with clinical practice result in NAA reductions in several brain regions. These results suggest that the preliminary findings describing NAA reductions early in the course of schizophrenia (Williamson et al., 2001; Bustillo et al., 2002) may not be entirely accounted for by treatment with antipsychotic medications.
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Treatment of rats with antipsychotic drugs: lack of an effect on brain N-acetyl aspartate levels Juan Bustillo a,b,1,2, Christina Wolff a, Adan Myers-y-Gutierrez c, Todd S. Dettmer b,3, Tom B. Cooper d,e, Andrea Allan b, John Lauriello a,1, C. Fernando Valenzuela b,*,4 a
b
Department of Psychiatry, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA Department of Neurosciences, University of New Mexico Health Sciences Center, Basic Medical Sciences Building, Room 145, Albuquerque, NM 87131, USA c Mental Illness Neuroscience Discovery Institute, USA d Nathan Kline Institute, USA e College of P&S Columbia University, USA Received 6 June 2002; received in revised form 2 December 2002; accepted 12 December 2002
Abstract Background: Proton magnetic resonance spectroscopy (1H-MRS) studies of schizophrenia suggest an effect of the disease or of antipsychotic medications on brain N-acetyl aspartate (NAA), a marker of neuronal viability. We studied in the rat the effect of antipsychotic drugs on NAA in several brain regions where NAA reductions have been reported in chronically medicated patients with schizophrenia. Methods: Three groups of nine rats each were treated with haloperidol (6 mg/kg/day), clozapine (70 mg/kg/day) and vehicle for 6 weeks and were sacrificed. Concentrations of NAA were determined by high-performance liquid chromatography (HPLC) from the following brain regions: cortex, striatum, thalamus, hippocampus and cerebellum. Results: Mixed-factorial ANOVA of NAA concentrations revealed no significant effect of drug group [ F(2, 24) = 0.034; p = 0.966] or a group by brain region interaction [ F(8, 44) = 0.841; p = 0.572]. There was a significant main effect of region [ F(4, 21) = 6.104; p = 0.002] with higher NAA in the cortex. Conclusions: These results are consistent with the only other study of the effect of typical and atypical antipsychotic drugs on NAA in the rat brain. The well-documented lower NAA in chronically treated schizophrenia patients is probably not a simple effect of antipsychotic medications. D 2003 Elsevier Science B.V. All rights reserved. Keywords: N-acetyl aspartate; Clozapine; Haloperidol; Antipsychotic; Schizophrenia; HPLC
* Corresponding author. Tel.: +1-505-272-3128; fax: +1-505-272-8082. E-mail address: [email protected] (C.F. Valenzuela). 1 Financial support: State of New Mexico Antipsychotic Algorithm Project Consultation Grant from New Mexico Department of Health. 2 Financial support: Mental Illness Neuroscience Discovery Institute. 3 Financial support: UNM-Minority Biomedical Research Support Program funded by NIH Grant R25 GM 60201-2. 4 Financial support: NIH Grants MH63126 and AA12684. 0920-9964/$ - see front matter D 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0920-9964(02)00528-5
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1. Introduction Proton magnetic resonance spectroscopy ( 1HMRS) studies of schizophrenia have reported reduced N-acetyl aspartate (NAA) mainly in frontal and mesial temporal, but also in thalamic and cerebellar regions (see Rowland et al., 2001, for review). The predominant view regarding NAA is that it is produced in the neuronal mitochondria, found almost exclusively in the neuron and is more concentrated in gray than white matter (Tsai and Coyle, 1995; Moffet and Namdoori, 1995; Pfefferbaum et al., 1999). NAA may play a role in osmoregulation (Taylor et al., 1994), myelination (Bhakoo and Pearce, 2000), glial cell signaling (Baslow, 2000) and neuronal function (Daughtry et al., 2000), but its specific functions are far from clear. Once thought to be a relatively stable marker of neuronal density, NAA is now also considered a sensitive marker of neuronal viability and function. In a longitudinal study of Alzheimer’s dementia and healthy subjects, NAA became globally reduced in the demented group before cortical atrophy was apparent (Adalsteinsson et al., 2000). Finally, NAA is related to cognitive functioning. For example, a direct relationship between cognitive performance and NAA levels has been described in: systemic lupus erythematosus (Friedman et al., 1998), traumatic brain injury (Ross et al., 1998; Brooks et al., 1999), HIV encephalopathy (Lopez-Villegas et al., 1997), temporal lobe epilepsy (Martin et al., 1999; Pauli et al., 2000; Kikuchi et al., 2001), schizophrenia (Bertolino et al., 2000; Bustillo et al., 2001; Braus et al., 2002) and in healthy volunteers (Jung et al., 1999). The majority of schizophrenia studies that have documented NAA reductions involved patients chronically treated with antipsychotic medications. The few studies assessing antipsychotic-naı¨ve patients have been inconclusive, perhaps due to methodological differences (Rowland et al., 2001). We have presented preliminary longitudinal data in schizophrenia patients with history of minimal previous treatment that documents reductions in frontal NAA during the first year of treatment with antipsychotic medications (Bustillo et al., 2002). Antipsychotic drugs exert both their therapeutic and deleterious extrapyramidal side effects by blocking dopamine D2 receptors and have been shown to lead to both structural and metabolic changes in the brain (Chakos et al., 1995; Keshavan
et al., 1994; Holcomb et al., 1996). Hence, it is possible that NAA reductions in schizophrenia are the result of antipsychotic medications, but separating the impact of the disease from the treatment is problematic in clinical populations. One study that assessed the effect of 1 week exposure to antipsychotic agents on NAA measures in rat brain found negative results (Lindquist et al., 2000). In the current study, we investigated the effects of a more prolonged exposure (6 weeks) to two antipsychotic medications, haloperidol and clozapine, on NAA levels on various brain regions in rats.
2. Methods All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of New Mexico (UNM) Health Sciences Center and were performed according to the guidelines of the National Institute of Health. Two-monthold male Sprague –Dawley rats weighing about 200 g each were purchased from Harlan (Indianapolis, IN), housed in pairs and allowed to acclimatize for 2 weeks before the start of the study. Rats were housed at the UNM animal facility under a 12:12 light/dark cycle (lights on at 7 am) and received Purina rat chow and tap water ad libitum. After a training period of about a week, rats were administered by gastric intubation 70 mg/kg/day of clozapine (n = 9; kindly provided by Anna-Maria Sutter, Novartis Pharma, Basel, Switzerland) and 6 mg/kg/day of haloperidol (n = 9; Sigma, St. Louis, MO). These dosages are significantly higher than those used in other studies in rats. However, pilot studies in our laboratory confirmed their usefulness in achieving blood levels comparable to those considered therapeutic in humans (Palao et al., 1994; VanderZwaag et al., 1996). Dosages were given twice a day (9 am and 5 pm) for 39 – 40 days. The drugs were dissolved in 1 M ascorbic acid as 23 and 1.8 mg/ml stock solutions for clozapine and haloperidol, respectively. Control rats (n = 9) received equivalent amounts of this vehicle. At the end of the treatment, rats belonging to the control, haloperidol and clozapine groups weighed 374 F 7, 344 F 13 and 351 F 9 g, respectively. These values were found not to be statistically significantly different by ANOVA ( p>0.09).
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Rats were euthanized by decapitation with a guillotine under halothane anesthesia 4 – 6 h after the morning gavages. Trunk blood was collected in heparinized tubes and centrifuged to obtain plasma to assay for drug levels. Clozapine was quantified as described by Simpson and Cooper (1978) with two modifications: norclozapine was measured using a capillary column and a nitrogen detector was substituted for the electron capture detector originally described. This latter modification gives clean chromatography with excellent stability over time. Interassay coefficient of variation (CV) is 4.5% for clozapine and 6.2% for norclozapine at 300 ng/ml. Haloperidol was determined in plasma using a gas chromatography procedure as described by Bianchetti and Moselli (1978) with two modifications: the use of chlorohaloperidol as an internal standard and the simultaneous quantitation of reduced haloperidol. The interassay CVs for haloperidol and reduced haloperidol were both < 6%. Brains were rapidly removed and regions of interest (cortex, striatum, thalamus, hippocampus and cerebellum, all bilateral) were dissected out on ice and placed in plastic tubes that had been precooled on dry ice. Samples were stored at 80 jC until ready to use. Samples were prepared essentially as described by Florian et al. (1996). Twenty milligrams of thawed tissue from each region was homogenized by sonication in 400 Al of 12% perchloric acid and incubated on ice for 30 min. Samples were centrifuged at 3000 g for 10 min and the supernatant was transferred to a new tube. Supernatants were neutralized to pH 5– 6 with KOH and centrifuged again as described above. Supernatants were then stored at 20 jC. Concentrations of NAA were determined by highperformance liquid chromatography (HPLC) using a Partisil-10 SAX anion exchange column (Whatman, Clifton, NJ). A HPXL solvent delivery HPLC system (Rainin, Woburn, MA) controlled by a Macintosh IISi computer was used for these determinations. This system is equipped with a Dynamax absorbance detector Model UV-DII. The sampling interval was 0.5 s and the detector wavelength was 214 nm. The mobile phase was 0.1 M potassium phosphate monobasic and 0.025 M potassium chloride at pH 4.5. Buffer was passed through a 0.22-Am filter and degassed under vacuum and bath sonication for 30 min. The column was equilibrated with this buffer for
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40 min at a flow rate of 1.5 ml/min. NAA standard (Sigma) was diluted in this buffer. To analyze the samples from the brain regions of interest, an aliquot of the supernatants corresponding to 2 mg of wet tissue was injected in a total volume of 500 Al of the mobile phase buffer. Samples were injected manually. Area under the curve for the peaks of interest was determined with Dynamax HPLC Method Manager software (Rainin). Concentrations of NAA were determined by extrapolation into a standard curve measured on the same day as unknowns. The total run time for each sample was 9 – 10 min. NAA levels were analyzed with a 3 (group) by 5 (region) mixed-factorial ANOVA with repeated measures for region using SPSS software. Data are presented as mean F S.E.M. in all cases.
3. Results We first injected increasing amounts of NAA standard dissolved in mobile phase buffer. These standards eluted as single peaks with retention times ranging between 5.4 and 5.6 min (data not shown). These values are in agreement with previous reports (Koller et al., 1984). Quantification of these peaks revealed that the area under the curve increases linearly with respect to the NAA amount injected (data not shown). These values were used to generate standard curves on each of the different days the assay was performed. The concentration of NAA in the five
Fig. 1. Sample chromatogram obtained by injection of an amount of cortical extract from a clozapine-treated rat corresponding to 0.66 mg of wet tissue. The injection volume was 500 Al. The dotted lines represent the baseline used by the computer program to calculate peak area. The flow rate was 1.5 ml/min and sampling interval was 0.5 s. The arrow indicates the time of injection.
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regions of the rat brain was then quantified using the respective standard curve acquired on a given day. Fig. 1 illustrates a typical chromatogram obtained by injected extracts from the rat cortex. Similar chromatograms were obtained from other brain regions. As in the case of the standards, NAA eluted as peaks with retention times ranging between 5.4 and 5.6 min. The quantification of NAA levels in the cortex, striatum, thalamus, hippocampus and cerebellum from control, haloperidol and clozapine-treated rats is shown in Fig. 2. The mixed-factorial ANOVA revealed no significant group by region interaction [Wilk’s k = 0.734, F(8, 44) = 0.841, p = 0.572] and no main effect of group [ F(2, 24) = 0.034, p = 0.966]. These results do not support an effect of antipsychotic drug exposure on NAA levels in any of the regions studied. However, there was a significant main effect of region [Wilk’s k = 0.462, F(4, 21) = 6.104, p = 0.002] with follow-up comparisons revealing significantly higher NAA in cortex compared to striatum ( p = 0.01), thalamus ( p = 0.01) and hippocampus ( p = 0.01). These higher concentrations in cortex are consistent with previous rat studies using HPLC methods (Koller et al., 1984; Florian et al., 1996). The plasma concentrations of clozapine and norclozapine were 242.9 (S.E. = 51.1) and 980.2
Fig. 2. Distribution of NAA levels in the cortex (CTX), striatum (Stri), thalamus (Thal), hippocampus (Hipp) and cerebellum (Cer). Samples were injected as described in the legend for Fig. 1. NAA levels were measured in samples from rats belonging to the control (C), haloperidol (H) and clozapine (Cl) groups. Each bar represents the mean F S.E.M. NAA level obtained in each region (n = 9 separate rats per group).
(S.E. = 92.6) ng/ml, respectively. The concentration of haloperidol was 31.8 (S.E. = 4.1) ng/ml.
4. Discussion In rats exposed to clozapine or haloperidol, we found no differences in NAA concentrations in cortex, striatum, thalamus, hippocampus or cerebellum, compared to animals treated with vehicle. We are aware of only one previous study assessing the impact of antipsychotic drugs on NAA brain levels in animals. Lindquist et al. (2000) treated rats with intraperitoneal daily injections of olanzapine (1 mg/kg/day; n = 8), clozapine (10 mg/kg/day; n = 8), haloperidol (0.2 mg/ kg/day; n = 8) and vehicle (n = 8) for 1 week. Ratios of NAA to creatine (NAA/Cre) were calculated from spectra measured at 4.7 T with single-voxel 1H-MRS before, after 1 day and at the end of treatment. The voxel studied was 0.216 cm3 and located ‘‘. . . in front of the cerebellum, and within the volume defined by the left and right ventricle’’ (Lindquist et al., 2000). There were no differences in NAA/Cre between the four groups at the end of treatment. There was a small reduction in NAA/Cre between baseline and 1 week values only in the olanzapine-treated group (1.16 vs. 1.12; p < 0.04). However, this was not a predicted result, uncorrected for multiple comparisons and the authors interpreted the overall findings as not supportive of a medication effect on NAA/Cre ratios. In contrast to the shortage of animal studies, there are many reports using 1H-MRS to study NAA in schizophrenia populations treated with antipsychotic medications. Most 1H-MRS schizophrenia studies involve cross-sectional assessments of patients chronically treated with mainly typical antipsychotics (like haloperidol, potent blockers of dopamine D2 receptors more likely to induce extrapyramidal side effects— EPS). These studies report lower NAA or NAA/Cre in several brain structures like: mesial temporal lobe, which include the hippocampus (Nasrallah et al., 1994; Fukuzako et al., 1995; Maier et al., 1995; Bertolino et al., 1996; Yurgelun-Todd et al., 1996; Deicken et al., 1998; but not Buckley et al., 1994; or Heimberg et al., 1998); anterior cingulate cortex (Deicken et al., 1997; Thomas et al., 1998; Ende et al., 2000); dorsolateral prefrontal cortex (DLPFC; Bertolino et al., 1996; but not Kegeles et al., 2000); larger
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(8– 12 cm3) prefrontal locations (Buckley et al., 1994; Lim et al., 1998; Bustillo et al., 2001; but not Fukuzako et al., 1995; or Omori et al., 2000); thalamus (Deicken et al., 2000; Omori et al., 2000; but not Bertolino et al., 1996); and the cerebellum (Deicken et al., 2001; but not Eluri et al., 1998). Three cross-sectional studies specifically investigated differential effects between typical and atypical medications (drugs with a lower EPS profile, like clozapine). In two studies, schizophrenia patients treated with typical agents had lower frontal NAA than normal controls while patients receiving atypical medications had intermediate, but not significantly different levels (Ende et al., 2000; Bustillo et al., 2001). A recent report found higher anterior cingulate NAA in patients treated with atypicals (mostly clozapine) compared to a group receiving typical agents (Braus et al., 2001). Interestingly, these authors also found a direct relationship between length of time on atypical and NAA that was interpreted as a restorative effect of atypical drugs on the metabolite. The few studies that investigated NAA early in schizophrenia and before treatment have produced inconsistent results. Renshaw et al. (1995) and Cecil et al. (1999) found lower NAA/Cre in mesial temporal regions but Bartha et al. (1999) found normal NAA concentrations. Choe et al. (1994) and Cecil et al. (1999) reported lower frontal NAA/Cre, whereas Stanley et al. (1996), Bartha et al. (1997) and Bustillo et al. (2002) found frontal NAA concentrations similar to healthy controls. These contradicting results are probably due to methodological differences, like large voxels with varied tissue composition and use of metabolite ratios. Because NAA, Cho and Cre can vary independently in the human brain (Pfefferbaum et al., 1999), metabolite concentrations may be a more valid measure than ratios. Few longitudinal studies of schizophrenia have assessed NAA in the context of antipsychotic drug treatment. Choe et al. (1996) found no changes in left frontal NAA/Cre during naturalistic treatment with typical and atypical agents. Bertolino et al. (2001), in a retrospective study, reported higher NAA/Cre in the DLPFC in patients while treated with antipsychotic medication as compared to when they were medication-free. We found reductions in left frontal NAA in patients with minimal prior lifetime antipsychotic exposure (less than 3 weeks)
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during the first year of randomized treatment with either haloperidol or quetiapine (Bustillo et al., 2002). Williamson et al. (2001) studied 10 antipsychotic-naı¨ve patients before and after treatment with atypical antipsychotic drugs. They report normal NAA in the anterior cingulate and thalamus before medications, with subsequent reductions within 1 year of treatment in both regions. Again, inconsistent findings in these longitudinal studies likely reflect methodological differences, but ours (Bustillo et al., 2002) and the preliminary results of Williamson et al. (2001) suggest frontal NAA reductions early in schizophrenia in the context of initial antipsychotic exposure and regardless of the use of atypical agents. Results from the present study do not support a simple medication effect. Progression of the disease or an interaction between disease and antipsychotic medications are alternative explanations. Antipsychotic drugs clearly have metabolic, macroand microstructural brain effects that go beyond their well-described receptor blocking properties. The best documented macrostructural effect in persons with schizophrenia is the enlargement of striatal volumes with typical agents, which reverses when subjects are switched to treatment with the atypical drug clozapine (Keshavan et al., 1994; Chakos et al., 1995). Several reports of first-episode schizophrenia patients longitudinally followed with magnetic resonance imaging (MRI) demonstrate subtle global brain volume reductions over the first few years of treatment (DeLisi et al., 1997; Gur et al., 1998; Giedd et al., 1999; Lieberman et al., 2001). Because antipsychotic medications can cause neurotoxic effects (e.g. tardive dyskinesia), their potential impact on these structural findings has not been excluded. However, the preponderance of evidence does not support antipsychotic-induced neuronal death (Baldessarini et al., 1997; Selemon et al., 1999; Lidow et al., 2001). Lower frontal glucose metabolism and cerebral blood flow have been found in positron emission tomography (PET) studies of schizophrenia patients treated with antipsychotic medications compared to a drug-free state (Holcomb et al., 1996; Miller et al., 2001). Also, a single dose of haloperidol resulted in a similar reduction in cortical metabolism in healthy volunteers (Bartlett et al., 1994). This hypometabolic frontal effect appears to be more severe with typical than with atypical antipsychotics (Miller et al., 2001),
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but it is not established if it relates to the therapeutic and/or the deleterious effects of the medications. A variety of microstructural effects have been described in studies of rodents exposed to antipsychotics drugs (Nielsen and Lyon, 1978; Benes et al., 1985; Roberts et al., 1995; Meshul et al., 1996; Kelley et al., 1997). These mainly involve an increase in the proportion of symmetric synapses in the striatum (see Harrison, 1999, for review). In nonhuman primates, prolonged exposure to both typical and atypical agents results in up-regulation of dopamine D2 and down-regulation of D1 receptors in prefrontal cortical regions (Lidow and Goldman-Rakic, 1994), and increased glial density in specific cortical layers (Selemon et al., 1999) without loss of neurons. In monkeys, long-term exposure to haloperidol did not result in neuronal loss. However, alterations of dendritic and spine-associated proteins were detected in various frontal cortical areas, but not in the primary visual cortex (Lidow et al., 2001). This was interpreted as evidence of collapse of dendritic structure in regions rich in dopamine receptors secondary to dopamine blockade. Because NAA is located in the neuronal soma as well as its processes, a reduction in the density of dendrites could theoretically result in lowering of NAA. Several limitations of the present study should be considered when interpreting our results. First, animals were exposed to drugs for only 6 weeks. In this time frame, a metabolic effect is apparent in schizophrenia patients (Holcomb et al., 1996; Miller et al., 2001) and a variety of microstructural changes have been demonstrated in rodents (Harrison, 1999). However, the NAA reductions in patients treated with antipsychotic agents have been described after 6 –12 months of treatment (Bustillo et al., 2002; Williamson et al., 2001). Therefore, it is possible that longer exposure in animals will result in similar NAA reductions. Second, the brain regions studied were rather large and a putative drug effect on NAA may be limited to more discrete areas. However, we specifically selected regions in which NAA reductions have been repeatedly described in chronically treated patients. Third, we studied only two antipsychotic drugs albeit ones very different in terms of neurological side effects. Clozapine and haloperidol are the most representative agents of the so-called atypical and typical class of antipsychotics, respectively. They
offer the additional advantage of being the two antipsychotic drugs for which blood levels have been linked to clinical effects (Palao et al., 1994; VanderZwaag et al., 1996), allowing a more meaningful comparison between exposure in humans and animals than if based only on dosage administered. Fourth, the animals studied were healthy and not representative of the fundamental neurobiological substrate that underlies schizophrenia, raising the possibility of an interaction between the disease and the therapeutic or deleterious effects of the medications. Finally, our design was cross-sectional and a longitudinal study may be more powerful to detect within subject changes. Future animal studies with repeated in vivo 1 H-MRS measures over several months would address this limitation. In summary, we found no evidence in rodents that exposure to dosages of clozapine or haloperidol consistent with clinical practice result in NAA reductions in several brain regions. These results suggest that the preliminary findings describing NAA reductions early in the course of schizophrenia (Williamson et al., 2001; Bustillo et al., 2002) may not be entirely accounted for by treatment with antipsychotic medications.
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