- Email: [email protected]
Deficits of [3H]d -aspartate binding to glutamate uptake sites in striatal and accumbens tissue in patients with schizophrenia
Neuroscience Letters 232 (1997) 13–16
Deficits of [3H]d-aspartate binding to glutamate uptake sites in striatal and accumbens tissue in patients with schizophrenia M. Iraide Aparicio-Legarza, Andrew J. Cutts, Ben Davis, Gavin P. Reynolds* Department of Biomedical Science, The University of Sheffield, Sheffield S10 2TN, UK Received 10 June 1997; received in revised form 24 July 1997; accepted 24 July 1997
Abstract The hypothesis involving glutamate in the neuropathology of schizophrenia has attracted great interest. Several studies report dysfunctions in glutamatergic systems, including alterations in kainate and N-methyl-d-aspartate (NMDA) receptors in various areas, as well as changes in the number of glutamate uptake sites. We have studied this further using [3H]d-aspartate binding to glutamate uptake sites as a measure of the integrity of presynaptic glutamate systems in several areas (caudate nucleus, putamen, nucleus accumbens, frontal cortex and temporal cortex) of brain tissue taken at autopsy from schizophrenic patients and controls. A significant decrease in the number of glutamate uptake sites was apparent in caudate nucleus, putamen and nucleus accumbens in the schizophrenia group, indicating an impaired glutamatergic innervation of these subcortical regions. However, no significant changes were found in the two cortical regions studied. 1997 Elsevier Science Ireland Ltd. Keywords: Schizophrenia; Glutamate uptake sites; [3H]d-aspartate binding; Postmortem human tissue; Antipsychotic drugs
Glutamate is the most abundant amino acid in brain, where it plays an important role, not only in the synthesis of peptides and proteins, but as a well-established major excitatory neurotransmitter in the central nervous system (CNS). It has been suggested that reduced glutamate neurotransmission may be involved in the pathophysiology of schizophrenia. This hypothesis is based on several observations. Phencyclidine (PCP) is a drug which can induce a schizophrenia-like psychosis including both positive and negative symptoms [17] and exerts its action by blocking the ion channel linked to the N-methyl-d-aspartate (NMDA) glutamate receptor subtype [11]. This is supported by the observation that other non-competitive NMDA antagonists such as MK-801 and ketamine elicit PCP-like psychoses, whereas PCP derivatives that block dopamine (DA) reuptake but do not bind to the NMDA receptor fail to reproduce the PCP-induced effects [14]. Furthermore, it may be that there is a reduced glutamate release in the brain of schizophrenics. Although the reported decrease of glutamate concentration in cerebrospinal fluid of schizophrenic patients * Corresponding author. Tel.: +44 114 2224662; fax: +44 114 2765413; email: [email protected]
[13] has not been supported by further studies [20], it has been observed that there is a significantly reduced glutamate release induced by veratridine, kainate and NMDA in synaptosomal preparations from brains of schizophrenic patients [21]. A further line of evidence relates to the close relationship between dopamine and glutamate systems [2]; dopamine has long been implicated in schizophrenia since antipsychotic drugs are considered to exert their action via dopamine D2 receptor antagonism. Finally, numerous recent reports show alterations in glutamate receptor binding in postmortem assays [10], with [3H]kainate binding being increased in frontal cortex [6,19], MK-801 binding in-creased in putamen [15] and in temporal cortex [22], whereas [3H]AMPA binding shows no changes [8,12,16]. Such increases in the number of glutamate receptors have been interpreted as compensatory and due to a glutamatergic hypofunction [6,15]. This is supported by experiments by Ulas et al. [23] demonstrating that an increase in the number of NMDA and kainate receptors in ipsilateral hippocampus occurs after unilateral lesions of the entorhinal cortex in the rat. However, it is important to assess the function of the presynaptic glutamatergic system in schizophrenia using more specific and direct markers. Several
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0563-6
14
M.I. Aparicio-Legarza et al. / Neuroscience Letters 232 (1997) 13–16
studies using saturable binding of [3H]d-aspartate have been carried out to determine glutamate uptake sites, an indirect measurement of glutamatergic innervation. One such study observed increased [3H]d-aspartate binding in frontal cortex accompanied by decreased binding in left temporal cortex [6]. However a more recent report [5] used a centrifugation method to separate membrane-bound [3H]d-aspartate in the determination of glutamate uptake sites, and it was found that the results from previous studies appeared seriously to underestimate levels of glutamate uptake sites. The aim of this study was to determine presynaptic glutamatergic systems in frontal (BA11) and temporal (BA38) cortex and striatal regions from schizophrenic patients, and to determine the possible influence of chronic antipsychotic drug treatment on these findings. Human brain tissue taken at autopsy from schizophrenic patients and control subjects free of neurological disease, was stored at −70°C until used. The research diagnostic criteria (RDC) for the diagnosis of schizophrenia were applied to the cases studied. Patients with a hospital diagnosis of schizophrenia but not meeting the RDC, or without adequate case note documentation, were excluded from the study. This resulted in a experimental series of 14 brains from schizophrenic patients (seven males, seven females, aged 66.2 ± 6.4 years, postmortem delay 20.6 ± 9.3 h) all of which had previously received chronic treatment with neuroleptic drugs, and 13 control cases (11 males, two females, aged 66.7 ± 7.2 years, postmortem delay 26.5 ± 15.4 h). Tissue from each area for every case was not available, although within the series of measurements for each brain area the control group was matched approximately for age, sex and interval from death to freezing (postmortem delay). Samples were taken randomly from the left or right hemisphere, and in the case of temporal cortex (BA38) from both sides of the brain when tissue availability permitted. In this case a mean of the two values was used in the overall analysis. The method of Cutts and Reynolds [5] was used for the determination of glutamate uptake sites; this incorporated a centrifugation assay to maximise the yield of binding sites. The method gave non-specific binding values that were consistently below 10% of the total. The assay involved incubation in quadruplicate of 140 ml homogenate (10 mg tissue/ml), prepared in assay buffer (50 mM Tris-acetate, 300 mM NaCl, pH 7.4) following several centrifugation washes [5], with 100 nM [3H]d-aspartate (Amersham) in a total volume of 200 ml. Addition of unla-
belled d-aspartate allowed seven concentrations of ligand to be obtained in the range 100–6100 nM. Duplicate samples with 500 mM l-glutamate present defined non-specific binding. After 90 min at 0°C, incubation was stopped by centrifugation at 12 000 g for 1 min, the supernatant was aspirated and the pellet rapidly and superficially washed with 1 ml ice-cold buffer. Bound radioligand in the pellet at the tip of the centrifuge tube was determined by liquid scintillation counting. Binding site density and affinity were determined by Scatchard analysis using a computer package (Prism, GraphPad). Experiments were carried out to evaluate the influence of antipsychotic medication on the number of glutamate uptake sites. Male Wistar rats weighing 180–200 g at the beginning of the drug administration were injected intraperitoneally with 2.5 mg/kg clozapine in a HCl-saline solution, 1.5 mg/kg haloperidol in glacial acetic-saline solution, and a saline vehicle once daily for 21 days. Animals were sacrificed 24 h after the last dose, whereupon brain tissue was dissected and stored at −70°C until used. [3H]d-aspartate binding was found to be unchanged in the frontal and temporal cortex of the schizophrenic subjects (Table 1). However, these cases demonstrated a significant deficit below control values in the putamen, caudate nucleus and nucleus accumbens (Table 1). No significant differences in density of [3H]d-aspartate binding sites were observed between the animals administered with vehicle and either antipsychotic drug (Table 2). As previously reported [22] there was a significant negative correlation between age and number of glutamate uptake sites in some of the areas studied, this effect being more apparent in the control group. The Kd values were not different between the schizophrenic and control groups within any brain region (values ranged from 1.1 to 2.2 mM). Statistical analysis showed that other variables such as sex, and/or interval from death to freezing do not contribute to the significant changes. The decrease in saturable [3H]d-aspartate binding in the striatal and accumbens tissues in schizophrenia is certainly consistent with a deficit in the density of glutamate uptake sites, reflecting in turn the density of glutamatergic innervation in these regions. Although it is well established that glial cells also show high-affinity uptake of glutamate which does not appear to be functionally different from the uptake system in nerve terminals [1], other evidence supports the interpretation that these results primarily reflect effects on glutamatergic neuronal innervation. Thus, in
Table 1 Glutamate uptake site density in postmortem brain tissue
Schizophrenics Controls
Caudate (n)
Putamen (n)
N. accumbens (n)
BA11 (n)
BA38 (n)
24.6 ± 14.2** (10) 43.5 ± 15.6 (8)
17.8 ± 5.2*** (13) 33.1 ± 10.2 (8)
25.1 ± 7.5* (14) 41.4 ± 25.8 (12)
58.3 ± 23.6 (12) 60.0 ± 19.4 (12)
40.5 ± 14.1 (13) 48.6 ± 21.0 (10)
Data are Bmax values of saturable [3H]d-aspartate binding expressed as nmol/g tissue. Values are means ± SD. *P , 0.05, **P = 0.01,***P , 0.001 by Student’s t-test.
M.I. Aparicio-Legarza et al. / Neuroscience Letters 232 (1997) 13–16 Table 2 Rat striatal glutamate uptake site density following chronic antipsychotic drug administration
[3]
Striatum Clozapine-treated (n = 8) Haloperidol-treated (n = 8) Controls (n = 8)
53.7 ± 12.0 55.8 ± 13.6 58.7 ± 25.4
Data are Bmax values of saturable [3H]d-aspartate binding expressed as nmol/g tissue. Values are means ± SD.
[4]
[5]
[6]
comparison with glia, nerve terminals have a higher density of uptake sites and account for a higher proportion of the total tissue uptake [9]. Glial uptake is also more sensitive to homogenisisation [18], an integral part of the tissue preparation process. Furthermore, several groups report decreases in [3H]d-aspartate binding in postmortem brain tissue from disorders associated with glutamatergic loss including Alzheimer’s disease [4], Huntington’s disease [3] and cases of leucotomy [5], all situations where a gliosis is expected. This provides strong evidence that saturable [3H]d-aspartate binding defines neuronal glutamate uptake sites and thus we conclude that the method employed here provides a valuable measure of the integrity of glutamatergic neuronal terminals. Abnormalities of frontal cortical function have been implicated in schizophrenia, particularly in relation to the negative symptoms that are less responsive to classical antipsychotic treatment. Neuroanatomical and neurochemical correlates of this frontal dysfunction have been reported. Nevertheless, contrary to the previous study of Deakin et al. [6] we were unable to find an increase of [3H]d-aspartate binding in the orbito-frontal cortex of schizophrenics, and so these results do not support the hypothesis of an increased glutamatergic innervation of this area. However the levels of binding seen with the centrifugation assay [4] are far greater than those of previous methods [3], which could well account for the discrepancy between these studies. The decreases in [3H]d-aspartate binding observed in caudate nucleus, nucleus accumbens and putamen from schizophrenic patients suggest deficits in cortico-striatal innervation in this disease [7]. One confounding factor in postmortem studies of neuropsychiatric diseases is the influence of chronic drug treatment. The experiments carried out with rat tissue following chronic administration of two antipsychotics demonstrate no apparent effect on the number of glutamate uptake sites thus it seems likely that the observed changes reflect the pathophysiology underlying the disease. [1] Arriza, J.L., Fairman, W.A., Wadiche, J.I., Murdoch, G.H., Kavanaugh, M.P. and Amara, S.G., Functional comparison of three glutamate transporter subtypes cloned from human cortex, J. Neurosci., 14 (1994) 5559–5569. [2] Carlsson, M. and Carlsson, A., Interactions between glutamatergic and monoaminergic systems within the basal ganglia-implications
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
15
for schizophrenia and Parkinson’s disease, Trends Neurosci., 13 (1990) 272–276. Cross, A.J., Slater, P. and Reynolds, G.P., Reduced high-affinity glutamate uptake sites in the brains of patients with Huntington’s disease, Neurosci. Lett., 67 (1986) 198–202. Cross, A.J., Slater, P., Simpson, M., Royston, C., Deakin, J.F.W., Perry, R.H. and Perry, E.K., Sodium-dependent d-[3H]aspartate binding in cerebral cortex in patients with Alzheimer’s and Parkinson’s diseases, Neurosci. Lett., 79 (1987) 213–217. Cutts, A.J. and Reynolds, G.P., d-Aspartate binding to the glutamate uptake site in human brain tissue-effects of leucotomy, J. Neural Transm., 94 (1993) 147–152. Deakin, J.F.W., Slater, P., Simpson, M.D.C., Gilchrist, A.C., Skon, W.J., Royston, M.C., Reynolds, G.P. and Cross, C.J., Frontal cortical and left temporal glutamatergic dysfunction in schizophrenia, J. Neurochem., 52 (1989) 1781–1786. Fonnum, F., Storm-Mathisen, J. and Divac, I., Biochemical evidence for glutamate as a neurotransmitter in the cortico-striatal and corticothalamic fibres of rat brain, Neuroscience, 6 (1981) 863–875. Freed, W., Dillon-Carter, O. and Kleinman, J.E., Properties of [3H]AMPA binding in postmortem human brain from psychotic subjects and controls: increases in caudate nucleus associated with suicide, Exp. Neurol., 121 (1993) 48–56. Gundersen, V., Danbolt, N.C., Othersen, O.P. and Storm-Mathisen, J., Demonstration of glutamate/aspartate uptake activity in nerve endings by use of antibodies recognizing exogenous d-aspartate, Neuroscience, 57 (1993) 97–111. Ishimaru, M., Kurumaji, A. and Toru, M., Increases in strychnine insensitive glycine binding sites in cerebral cortex of chronic schizophrenics: evidence for glutamate hypothesis, Biol. Psychiatry, 35 (1994) 84–95. Javitt, D.C. and Zukin, S.R., Recent advances in the phencyclidine model of schizophrenia, Am. J. Psychiatry, 148 (1991) 1301–1308. Kerwin, R., Patel, S. and Meldrum, B., Quantitative autoradiographic analysis of glutamate binding sites in the hippocampal formation in normal and schizophrenic brain post mortem, Neuroscience, 39 (1990) 25–32. Kim, J.S., Kornhuber, H.H., Schmid-Burgk, W. and Holzmuller, B., Low cerebrospinal fluid glutamate in schizophrenic patients and a new hypothesis on schizophrenia, Neurosci. Lett., 20 (1980) 379– 382. Koek, W., Colpaert, F.C., Woods, J.H. and Kamenka, J.M., The phencyclidine (PCP) analog N-[1(2-benzo[b]thiphenyl-cyclohexyl]piperidine shares cocaine-like but not other characteristic behavioural effects with PCP, ketamine and MK-801, J. Pharmacol. Exp. Ther., 250 (1989) 1019–1027. Kornhuber, J., Mack-Burkhardt, F., Riederer, P., Hebenstret, G.F., Reynolds, G.P., Andrews, H.B. and Beckmann, H., [3H]MK-801 binding sites in post mortem brain regions of schizophrenic patients, J. Neural Transm., 77 (1989) 231–236. Kurumaji, A., Ishimaru, M. and Toru, M., a-[3H]Amino-3-hydroxy5-methylisoxazole-4-propionic acid binding to human cerebral cortical membranes: minimal changes in postmortem brains of chronic schizophrenics, J. Neurochem., 59 (1992) 829–837. Luisada, P.V., The phencyclidine psychosis. In R.C. Petersen and R.C. Stillman (Eds.), Phencyclidine (PCP) abuse, NIDA Research Monograph 21, US Government Printing Office, Washington DC, 1978, pp. 241–253. Lund Karlsen, R., Neurotransmitters in the mammalian visual system. In F. Fonnum (Ed.), Amino Acids as Chemical Transmitters, Plenum Press, New York, 1978, pp. 241–256. Nishikawa, T., Takashima, M. and Toru, M., Increased [3H]kainic acid binding in the prefrontal cortex in schizophrenia, Neurosci. Lett., 40 (1983) 245–250. Perry, T.L., Normal cerebrospinal fluid and brain glutamate levels in schizophrenics do not support the hypothesis of glutamatergic neuronal dysfunction, Neurosci. Lett., 28 (1982) 81–85.
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[21] Sherman, A.D., Hegwood, T.S., Baruah, S. and Waziri, R., Deficient NMDA-mediated glutamate release from synaptosomes of schizophrenics, Biol. Psychiatry, 20 (1991) 1191–1198. [22] Slater, P., McConnell, S.W., Barson, A.J., Simpson, M.D.C. and Gilchrist, A., Age-related changes in binding to excitatory amino acid uptake site in temporal cortex of human brain, Brain Res., 65 (1992) 157–160.
[23] Ulas, J., Monaghan, D.T. and Cotman, C.W., Kainate receptors in the rat hippocampus: a distribution and time course of changes in response to unilateral lesions of the entorhinal cortex, J. Neurosci., 10 (1990) 2352–2362.
Deficits of [3H]d-aspartate binding to glutamate uptake sites in striatal and accumbens tissue in patients with schizophrenia M. Iraide Aparicio-Legarza, Andrew J. Cutts, Ben Davis, Gavin P. Reynolds* Department of Biomedical Science, The University of Sheffield, Sheffield S10 2TN, UK Received 10 June 1997; received in revised form 24 July 1997; accepted 24 July 1997
Abstract The hypothesis involving glutamate in the neuropathology of schizophrenia has attracted great interest. Several studies report dysfunctions in glutamatergic systems, including alterations in kainate and N-methyl-d-aspartate (NMDA) receptors in various areas, as well as changes in the number of glutamate uptake sites. We have studied this further using [3H]d-aspartate binding to glutamate uptake sites as a measure of the integrity of presynaptic glutamate systems in several areas (caudate nucleus, putamen, nucleus accumbens, frontal cortex and temporal cortex) of brain tissue taken at autopsy from schizophrenic patients and controls. A significant decrease in the number of glutamate uptake sites was apparent in caudate nucleus, putamen and nucleus accumbens in the schizophrenia group, indicating an impaired glutamatergic innervation of these subcortical regions. However, no significant changes were found in the two cortical regions studied. 1997 Elsevier Science Ireland Ltd. Keywords: Schizophrenia; Glutamate uptake sites; [3H]d-aspartate binding; Postmortem human tissue; Antipsychotic drugs
Glutamate is the most abundant amino acid in brain, where it plays an important role, not only in the synthesis of peptides and proteins, but as a well-established major excitatory neurotransmitter in the central nervous system (CNS). It has been suggested that reduced glutamate neurotransmission may be involved in the pathophysiology of schizophrenia. This hypothesis is based on several observations. Phencyclidine (PCP) is a drug which can induce a schizophrenia-like psychosis including both positive and negative symptoms [17] and exerts its action by blocking the ion channel linked to the N-methyl-d-aspartate (NMDA) glutamate receptor subtype [11]. This is supported by the observation that other non-competitive NMDA antagonists such as MK-801 and ketamine elicit PCP-like psychoses, whereas PCP derivatives that block dopamine (DA) reuptake but do not bind to the NMDA receptor fail to reproduce the PCP-induced effects [14]. Furthermore, it may be that there is a reduced glutamate release in the brain of schizophrenics. Although the reported decrease of glutamate concentration in cerebrospinal fluid of schizophrenic patients * Corresponding author. Tel.: +44 114 2224662; fax: +44 114 2765413; email: [email protected]
[13] has not been supported by further studies [20], it has been observed that there is a significantly reduced glutamate release induced by veratridine, kainate and NMDA in synaptosomal preparations from brains of schizophrenic patients [21]. A further line of evidence relates to the close relationship between dopamine and glutamate systems [2]; dopamine has long been implicated in schizophrenia since antipsychotic drugs are considered to exert their action via dopamine D2 receptor antagonism. Finally, numerous recent reports show alterations in glutamate receptor binding in postmortem assays [10], with [3H]kainate binding being increased in frontal cortex [6,19], MK-801 binding in-creased in putamen [15] and in temporal cortex [22], whereas [3H]AMPA binding shows no changes [8,12,16]. Such increases in the number of glutamate receptors have been interpreted as compensatory and due to a glutamatergic hypofunction [6,15]. This is supported by experiments by Ulas et al. [23] demonstrating that an increase in the number of NMDA and kainate receptors in ipsilateral hippocampus occurs after unilateral lesions of the entorhinal cortex in the rat. However, it is important to assess the function of the presynaptic glutamatergic system in schizophrenia using more specific and direct markers. Several
0304-3940/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940 (97 )0 0563-6
14
M.I. Aparicio-Legarza et al. / Neuroscience Letters 232 (1997) 13–16
studies using saturable binding of [3H]d-aspartate have been carried out to determine glutamate uptake sites, an indirect measurement of glutamatergic innervation. One such study observed increased [3H]d-aspartate binding in frontal cortex accompanied by decreased binding in left temporal cortex [6]. However a more recent report [5] used a centrifugation method to separate membrane-bound [3H]d-aspartate in the determination of glutamate uptake sites, and it was found that the results from previous studies appeared seriously to underestimate levels of glutamate uptake sites. The aim of this study was to determine presynaptic glutamatergic systems in frontal (BA11) and temporal (BA38) cortex and striatal regions from schizophrenic patients, and to determine the possible influence of chronic antipsychotic drug treatment on these findings. Human brain tissue taken at autopsy from schizophrenic patients and control subjects free of neurological disease, was stored at −70°C until used. The research diagnostic criteria (RDC) for the diagnosis of schizophrenia were applied to the cases studied. Patients with a hospital diagnosis of schizophrenia but not meeting the RDC, or without adequate case note documentation, were excluded from the study. This resulted in a experimental series of 14 brains from schizophrenic patients (seven males, seven females, aged 66.2 ± 6.4 years, postmortem delay 20.6 ± 9.3 h) all of which had previously received chronic treatment with neuroleptic drugs, and 13 control cases (11 males, two females, aged 66.7 ± 7.2 years, postmortem delay 26.5 ± 15.4 h). Tissue from each area for every case was not available, although within the series of measurements for each brain area the control group was matched approximately for age, sex and interval from death to freezing (postmortem delay). Samples were taken randomly from the left or right hemisphere, and in the case of temporal cortex (BA38) from both sides of the brain when tissue availability permitted. In this case a mean of the two values was used in the overall analysis. The method of Cutts and Reynolds [5] was used for the determination of glutamate uptake sites; this incorporated a centrifugation assay to maximise the yield of binding sites. The method gave non-specific binding values that were consistently below 10% of the total. The assay involved incubation in quadruplicate of 140 ml homogenate (10 mg tissue/ml), prepared in assay buffer (50 mM Tris-acetate, 300 mM NaCl, pH 7.4) following several centrifugation washes [5], with 100 nM [3H]d-aspartate (Amersham) in a total volume of 200 ml. Addition of unla-
belled d-aspartate allowed seven concentrations of ligand to be obtained in the range 100–6100 nM. Duplicate samples with 500 mM l-glutamate present defined non-specific binding. After 90 min at 0°C, incubation was stopped by centrifugation at 12 000 g for 1 min, the supernatant was aspirated and the pellet rapidly and superficially washed with 1 ml ice-cold buffer. Bound radioligand in the pellet at the tip of the centrifuge tube was determined by liquid scintillation counting. Binding site density and affinity were determined by Scatchard analysis using a computer package (Prism, GraphPad). Experiments were carried out to evaluate the influence of antipsychotic medication on the number of glutamate uptake sites. Male Wistar rats weighing 180–200 g at the beginning of the drug administration were injected intraperitoneally with 2.5 mg/kg clozapine in a HCl-saline solution, 1.5 mg/kg haloperidol in glacial acetic-saline solution, and a saline vehicle once daily for 21 days. Animals were sacrificed 24 h after the last dose, whereupon brain tissue was dissected and stored at −70°C until used. [3H]d-aspartate binding was found to be unchanged in the frontal and temporal cortex of the schizophrenic subjects (Table 1). However, these cases demonstrated a significant deficit below control values in the putamen, caudate nucleus and nucleus accumbens (Table 1). No significant differences in density of [3H]d-aspartate binding sites were observed between the animals administered with vehicle and either antipsychotic drug (Table 2). As previously reported [22] there was a significant negative correlation between age and number of glutamate uptake sites in some of the areas studied, this effect being more apparent in the control group. The Kd values were not different between the schizophrenic and control groups within any brain region (values ranged from 1.1 to 2.2 mM). Statistical analysis showed that other variables such as sex, and/or interval from death to freezing do not contribute to the significant changes. The decrease in saturable [3H]d-aspartate binding in the striatal and accumbens tissues in schizophrenia is certainly consistent with a deficit in the density of glutamate uptake sites, reflecting in turn the density of glutamatergic innervation in these regions. Although it is well established that glial cells also show high-affinity uptake of glutamate which does not appear to be functionally different from the uptake system in nerve terminals [1], other evidence supports the interpretation that these results primarily reflect effects on glutamatergic neuronal innervation. Thus, in
Table 1 Glutamate uptake site density in postmortem brain tissue
Schizophrenics Controls
Caudate (n)
Putamen (n)
N. accumbens (n)
BA11 (n)
BA38 (n)
24.6 ± 14.2** (10) 43.5 ± 15.6 (8)
17.8 ± 5.2*** (13) 33.1 ± 10.2 (8)
25.1 ± 7.5* (14) 41.4 ± 25.8 (12)
58.3 ± 23.6 (12) 60.0 ± 19.4 (12)
40.5 ± 14.1 (13) 48.6 ± 21.0 (10)
Data are Bmax values of saturable [3H]d-aspartate binding expressed as nmol/g tissue. Values are means ± SD. *P , 0.05, **P = 0.01,***P , 0.001 by Student’s t-test.
M.I. Aparicio-Legarza et al. / Neuroscience Letters 232 (1997) 13–16 Table 2 Rat striatal glutamate uptake site density following chronic antipsychotic drug administration
[3]
Striatum Clozapine-treated (n = 8) Haloperidol-treated (n = 8) Controls (n = 8)
53.7 ± 12.0 55.8 ± 13.6 58.7 ± 25.4
Data are Bmax values of saturable [3H]d-aspartate binding expressed as nmol/g tissue. Values are means ± SD.
[4]
[5]
[6]
comparison with glia, nerve terminals have a higher density of uptake sites and account for a higher proportion of the total tissue uptake [9]. Glial uptake is also more sensitive to homogenisisation [18], an integral part of the tissue preparation process. Furthermore, several groups report decreases in [3H]d-aspartate binding in postmortem brain tissue from disorders associated with glutamatergic loss including Alzheimer’s disease [4], Huntington’s disease [3] and cases of leucotomy [5], all situations where a gliosis is expected. This provides strong evidence that saturable [3H]d-aspartate binding defines neuronal glutamate uptake sites and thus we conclude that the method employed here provides a valuable measure of the integrity of glutamatergic neuronal terminals. Abnormalities of frontal cortical function have been implicated in schizophrenia, particularly in relation to the negative symptoms that are less responsive to classical antipsychotic treatment. Neuroanatomical and neurochemical correlates of this frontal dysfunction have been reported. Nevertheless, contrary to the previous study of Deakin et al. [6] we were unable to find an increase of [3H]d-aspartate binding in the orbito-frontal cortex of schizophrenics, and so these results do not support the hypothesis of an increased glutamatergic innervation of this area. However the levels of binding seen with the centrifugation assay [4] are far greater than those of previous methods [3], which could well account for the discrepancy between these studies. The decreases in [3H]d-aspartate binding observed in caudate nucleus, nucleus accumbens and putamen from schizophrenic patients suggest deficits in cortico-striatal innervation in this disease [7]. One confounding factor in postmortem studies of neuropsychiatric diseases is the influence of chronic drug treatment. The experiments carried out with rat tissue following chronic administration of two antipsychotics demonstrate no apparent effect on the number of glutamate uptake sites thus it seems likely that the observed changes reflect the pathophysiology underlying the disease. [1] Arriza, J.L., Fairman, W.A., Wadiche, J.I., Murdoch, G.H., Kavanaugh, M.P. and Amara, S.G., Functional comparison of three glutamate transporter subtypes cloned from human cortex, J. Neurosci., 14 (1994) 5559–5569. [2] Carlsson, M. and Carlsson, A., Interactions between glutamatergic and monoaminergic systems within the basal ganglia-implications
[7]
[8]
[9]
[10]
[11] [12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
15
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