[1] 11w Not the Frontal Cortex or Caudate Nucleus from Subjects with Schizophrenia

[1] 131w Paroxetine binding to particulate membrane from tissue, obtained at autopsy, from the hippocampus, frontal cortex, and caudate nucleus from subjects who had or had not had schizophrenia was measured . The density of [3H]paroxetine binding to membranes from subjects who had or had not had schizophrenia did not differ. Similarly, the affinity of [H]paroxetine binding in the frontal cortex and caudate nucleus was not different . By contrast, the affinity of [H]paroxetine binding to hippocampal membrane from subjects who had schizophrenia was significantly lower than the affinity of binding for the nonschizophrenic subjects (0.40 ± 0 .06 vs. 0.26 ± 0 .02 ; p < 0.05) . As [3H]paroxetine binds to the serotonin transporter, these data suggest that the serotonin transporter is altered in the hippocampus in subjects with schizophrenia .

[1] 23w with typical neuroleptic drugs before death, none of them had been treated with antidepressants, and there was no history of major depression .

[2] 217w [ 3H]Paroxetine binding to particulate membrane In all cases the Scatchard plot of [3H]paroxetine binding to particulate membranes was linear, suggesting the presence of a single binding site. Furthermore, there was no difference in either the affinity (KD) or density (Bm.,x) of [3H]paroxetine binding to particulate membranes from the frontal cortex or caudate nucleus from subjects who had schizophrenia and subjects who had not had schizophrenia (Fig. 1 and Table 2) . Similarly, there was no difference between the density of [ 3H]paroxetine binding to particulate membrane from the hippocampus of subjects who had or those who did not have schizophrenia . By contrast, there was a significant decrease in the affinity (increased KD) of binding of [ 3 H] paroxetine to particulate membrane from the hippocampus of subjects with schizophrenia compared with that for the nonschizophrenic subjects (p < 0.05; Fig . 1 and Table 2) . Further analysis of these data showed that the decreased affinity of [3H]paroxetine binding was mostly attributable to a change in the subjects who had been schizophrenic and had committed suicide (p < 0.025 ; Fig . 1 and Table 2) . By contrast, the affinity of [3H]paroxetine binding tended to be lower for the schizophrenic subjects who had not committed suicide, but this difference did not reach significance .

[3] 31w There were no significant correlations between the KD or the Bmax for ['H]paroxetine binding to hippocampal, caudate nucleus, or frontal cortical tissue and age of the donor or the PMI .

[1] 83w This study has shown that the affinity, but not the density, of [3H]paroxetine binding is altered in the hippocampus but not frontal cortex or caudate nucleus of subjects with schizophrenia . These results differ from a previous study that showed a decreased density of [ 3H]paroxetine binding in the frontal but not the occipital pole from subjects with schizophrenia (Laruelle et al., 1993) . However, both studies suggest that there are changes in [3H]paroxetine binding in brain tissue from subjects with schizophrenia .

[2] 119w In addition to showing that the affinity of [ 3H]paroxetine binding is altered in the hippocampus from subjects who had been schizophrenic, this study has shown that the change in affinity of binding is most apparent in subjects who had been schizophrenic and had committed suicide. This observation is significant as changes in serotonergic function have been suggested to play a role in precipitating suicidal behavior (Mann et al ., 1989) . Furthermore, the incidence of suicide is thought to increase greatly in subjects with depressive illness . Significantly, like two prior studies (Lawrence et al., 1990;Hrdina et al ., 1993) schizophrenia and either did or did not commit suicide, this observation can only be regarded as preliminary .

[3] 31w A much more extensive study of [ 3H]paroxetine binding to hippocampal tissue from subjects with schizophrenia who had or had not committed suicide would be required to confirm this hypothesis .

[4] 146w Several studies have been published reporting the distribution of serotonin transporters in the human J. Neurochein., Vol. 64, No. 3, 1995 B. DEAN ET AL. brain and have shown a lower number of transporters in the frontal cortex compared with the hippocampus and caudate nucleus (Laruelle et al ., 1988;Lawrence et al., 1990;Plenge et al., 1990) . The data presented in this study also show a lower density of serotonin transporter in the frontal cortex compared with the hippocampus and caudate nucleus (Table 2) . Significantly, both the density and the affinity of [3H]-paroxetine binding to the serotonin transporter for the nonschizophrenic subjects are in close agreement with those reported in the published study using tissue from subjects with schizophrenia (Laruelle et al ., 1993) . This suggests the differing results obtained in the two studies are not likely to be due to methodological considerations .

[5] 94w No correlation was found between the affinity or density of [ 3 H]paroxetine binding and donor age or the PMl for the tissue . Hence, as in a previous study (Andersson et al ., 1992), these data suggest that the serotonin transporter is a stable molecule that is suitable for study in postmortem tissue . Therefore, the fact that the schizophrenic individuals who had not committed suicide were significantly older than the other subjects should not have influenced the overall findings because there was no relationship between age and [ 3 H]paroxetine binding .

[6] 131w All subjects from whom tissue was collected at autopsy who were diagnosed as schizophrenic had been treated with neuroleptic drugs . It therefore remains possible that the change in [ 3 H]paroxetine binding observed was drug related rather than a pathophysiological effect . Unfortunately, there are no studies on the effects of neuroleptic drugs on the serotonin transporter in animals that could help to support or disprove this hypothesis . However, we have shown that the addition of the neuroleptic drug, haloperidol, at 10 -'M, does not affect [ 3 H]paroxetine binding . This suggests that if changes in ['H]paroxetine binding were caused by neuroleptic drugs, this is not likely to be due to residual neuroleptic drugs that could be present in the brain interfering with the ['H]paroxetine binding assay .

[7] 17w However, further studies of neuroleptic drug treatment on ['H]paroxetine binding in the rat would be warranted .

[8] 204w Significantly, the findings from this study differ from those reported previously (Laruelle et al ., 1993) in that a change in affinity rather than in density of [ 3 H ]paroxetine binding was found . However, the two studies measured [ 3 H]paroxetine binding in different brain regions, and thus it could be possible that there are both regionally and functionally different changes in ['H]paroxetine binding in the schizophrenic brain . These studies, plus those showing changes in serotonin 5-HT 2 receptors in Brodmann's area 9 (Bennett et al ., 1979 ;Mita et al ., 1986 ;Arora and Meltzer, 1991) of the frontal cortex but not in other Brodmann's areas associated with the frontal cortex (Whitaker et al ., 1981 ;Reynolds et al ., 1983), suggest that regional changes in serotonin neurotransmission occur in the schizophrenic brain . Thus, it will be important to determine if the differing changes in serotonergic receptors and transporters always occur concurrently in tissue from subjects with schizophrenia . If that were the case, this could provide a rationale for why some individuals may respond to atypical rather than typical neuroleptic drugs, as the atypical neuroleptic drugs are more active on serotonergic neurons (Matsubara et al ., 1993) .

[9] 126w In summary, [ 3 H ] paroxetine has been shown to bind with high affinity and specificity to the serotonin transporter (Laruelle et al ., 1988 ;Backstrom et al ., 1989 ;Plenge et al ., 1990) . The changes in [ 3 H]paroxetine binding to brain tissue from subjects with schizophrenia, reported in this and a previous study (Laruelle et al ., 1993), suggest a change in the serotonin transporter in the brains of subjects with schizophrenia . Such changes in the serotonin transporter suggest that changes in serotonergic neurotransmission occur in the schizophrenic brain . Hence, these findings seem to support the hypothesis that, at least in some individuals, changes in serotonergic neurotransmission are involved in the pathogenesis of schizophrenia (Kahn and Davidson, 1993) .

[1] 74w With ethics committee approval, brain tissue was collected at autopsy from subjects stated to have been or not to have been schizophrenic . The subjects who were not schizophrenic were selected so that overall, the schizophrenic and nonschizophrenic subjects were of a similar age and of the same sex ratio . The diagnoses of schizophrenia were confirmed, by a senior psychiatrist, using DSM-111-R criteria (American Psychiatric Association, 1987), following an extensive case history review.

[2] 37w Tissue was collected, at autopsy, from the hippocampus, caudate nucleus, and frontal cortex (Brodmann's area 9) of the left hemisphere of the brain . Once collected, the tissue was rapidly frozen to -70°C and stored until required.

[3] 129w Tissue was collected from 14 (12 male and two female) subjects with schizophrenia, seven (all male) of whom had committed suicide, and 14 (12 male and two female) subjects with no known history of mental illness, seven (six male and one female) of whom had committed suicide (Table 1) . There was no statistical difference between the mean age of the subjects who had a history of schizophrenia and those who did not (Table 2) . By contrast, the subjects who had been schizophrenic but had not committed suicide were significantly older than subjects who had been schizophrenic and committed suicide (p < 0.01) and the subjects who had not been schizophrenic but who did (p < 0.05) or did not (p < 0.01) commit suicide (Table 2) .

[4] 27w There was no difference between the time of death and tissue collection [postmortem intervals (PMI)] for the tissue from the schizophrenic and nonschizophrenic subjects (Table 2) .

[5] 23w Although all the subjects with schizophrenia from whom tissue was obtained at autopsy had been treated 1. Neurochem., Vol. 64, No. 3, 1995

[1] 28w confirm that the serotonin transporter is altered in the schizophrenic brain . Thus, [3H]paroxetine binding to brain tissue, obtained at autopsy, from subjects with schizophrenia was measured .

[2] 340w Tissue blocks were removed from the -70°C freezer and placed in 5 volumes (vol/wt) of ice-cold Tris buffer (50 mM Tris, 120 mM NaCl, and 5 mM KCl, pH 7.4) and chopped . The chopped tissue was homogenized using three 15-s bursts of a Janke and Kunkel Ultra Turrax t25 tissue homogenizer and centrifuged at 30,000 g for 10 min at 4°C, and the supernatant was discarded . The pellet of particulate membrane was suspended in ice-cold Tris buffer, homogenized, and centrifuged at 30,000 g for 10 min at 4°C twice, and then the washed membrane was suspended in Tris buffer. The particulate membrane protein concentration was then measured using the Bio-Rad kit method based on the method of Bradford (1976) . Finally, the particulate membrane was diluted to give a final concentration of 2.0 mg of protein/ml . The binding of [;H]paroxetine to particulate membrane, from hippocampus, cortex, or caudate nucleus, at a final concentration of 0.2 mg of protein/ml, was measured in the presence of a range of concentrations of [ 3H]paroxetine (0.2-2 .0 nM) in the absence or presence of 10 -5 M citalopram in Tris buffer. After incubating with the particulate membrane for 2 h at room temperature, the bound [3H]paroxetine was separated from free, after addition of 3 ml of ice-cold Tris buffer, by filtration over GF/C filters on a Brandel cell harvester . Following three washes with 5 ml of 0.9% NaCl, the filter paper containing the membrane-bound [ 3H ] paroxetine was transferred into 5 ml of Beckman Readiprotein liquid scintillation cocktail, and the radioactivity on each filter was counted in a Canberra-Packard model 1500 liquid scintillation counter. Specific binding of [ ;H] paroxetine to particulate membrane was taken as the radioactivity bound to membrane without citalopram minus the radioactivity bound in the presence of citalopram. The KD and B,ax for [ aH]paroxetine binding were calculated from Scatchard plots of these data using the Ligand computer program . Statistical analysis was carried out using the ANOVA test in the Sigmastat computer program .

[3] 46w All the schizophrenic individuals from whom tissue was obtained had been treated with neuroleptic drugs. Thus, to determine the possible effect of neuroleptic drugs, [3H]paroxetine binding to hippocampul and caudate membranes was measured as described above in the absence or presence of 10 -'M haloperidol .

[4] 61w ['H]paroxetine binding to particulate membranes from the hippocampus and caudate from a single individual was similar in the absence (KD = 0.26 and 0.34 nM and B ,ax = 315 and 420 fmol/mg of protein, respectively) or presence (KD = 0.25 and 0.37 n M and Bmax = 310 and 390 fmol/mg of protein, respectively) of 10 -' M haloperidol .