[3H]Raclopride Binding to Brain Tissue from Subjects with Schizophrenia: Methodological Aspects

doi:10.1016/S0028-3908(97)00067-1

Neuropharmacology

Volume 36, Issue 6, June 1997, Pages 779-786

https://doi.org/10.1016/S0028-3908(97)00067-1 Get rights and content

Abstract

The binding of [³H]raclopride to particulate membrane and frozen sections (with quantitative autoradiography) from the caudate-putamen, obtained at autopsy from schizophrenic and non-schizophrenic subjects, was measured. The affinity of [³H]raclopride to particulate membrane was significantly decreased in the schizophrenic compared to non-schizophrenic subjects. The density of [³H]raclopride binding to tissue from subjects with schizophrenia was increased, unchanged or decreased depending on the methodology used. Finally, there was an age-dependent decrease in [³H]raclopride binding in the frozen sections from the caudate-putamen of the non-schizophrenic subjects. This age-dependent decrease was not apparent using particulate membrane from schizophrenic or non-schizophrenic subjects or tissue sections from the schizophrenic subjects. We conclude that the binding of [³H]raclopride is dependent on methodology and therefore data from in vitro and in vivo studies using this drug should be interpreted with caution. © 1997 Elsevier Science Ltd.

Section snippets

Materials

[³H]Raclopride was obtained from Du Pont (Australia) Ltd, Sydney, Australia. (+)-Butaclamol and S-(−)-sulpiride were obtained from Research Biochemicals International (Natick, MA, U.S.A.) whilst 5′-guanylyl-imidodiphosphate was from Sigma Aldrich Pty Ltd (Castle Hill, NSW, Australia). All other chemicals were obtained from Ajax Chemicals (Auburn, NSW, Australia).

Tissue collection

After gaining ethical approval, caudate-putamen was obtained at autopsy from the left hemisphere of 15 subjects with an initial

RESULTS

As would be expected, there was no difference in mean age between the schizophrenic subjects and non-schizophrenic subjects from whom tissue was obtained and used in the study of particulate membrane binding (n = 10 per group) or just for autoradiography (n = 15 per group) (Table 1). In addition, there was no relationships between the dose of neuroleptic drugs received by the schizophrenic subjects prior to death and any of the [³H]raclopride binding parameters. Finally, there was no

DISCUSSION

Depending on methodology, we have shown the density of [³H]raclopride binding sites in caudate-putamen from schizophrenic subjects to be decreased, not changed or increased. As tissue used in all methods was from the same individuals, it would seem most likely that the differing results are due to the minor changes in methodological conditions. The density of [³H]raclopride binding in the caudate-putamen from schizophrenic subjects has previously been reported to be increased (Sumiyoshi et al.,

Acknowledgements

The authors wish to thank The NH and MRC Schizophrenia Research Unit, The Woods Family Research Trust and The Rebecca L. Cooper Medical Research Foundation for supporting this research. The authors also wish to thank Professor Nicholas Keks and Ms. Christine Hill for carrying out case history reviews and diagnostic confirmation.

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Using this approach we are unable to determine whether the change represents fluctuations in mGlu2, mGlu3, or both. Moreover, using postmortem tissue we are unable to dissociate the irrevocable confound of suicidality upon binding levels.
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We have shown a decrease in cortical serotonin_2A receptors using tissue sections, but not with washed membranes, from the same cohort of subjects. These discrepant findings led us to determine if we could obtain similar results using samples from the same tissue block. Our studies used single-point saturation analyses to estimate the total number of [³H]ketanserin binding sites in tissue sections, crude homogenate, membrane-enriched and cytosol-enriched tissue samples from Brodmann's area 9. There were significant decreases in the levels of [³H]ketanserin binding using tissue sections (mean ± SD: 38 ± 16 vs. 56 ± 16 fmol/mg ETE; p = 0.008) and crude tissue homogenates (131 ± 53 vs. 168 ± 38 fmol/mg protein; p < 0.05) from subjects with schizophrenia compared to that in controls. By contrast, there was no significant difference in radioligand binding to the membrane-enriched (155 ± 95 vs. 145 ± 48 fmol/mg protein; p = 0.72) or cytosol-enriched (8.6 ± 14 vs. 7.5 ± 10 mol/mg protein; p = 0.85) tissue fraction. Significantly, adding 10^{− 5} M risperidone or chlorpromazine, as surrogates for residual antipsychotic drugs in the CNS, to crude homogenate from control subjects did not alter [³H]ketanserin binding. Our data therefore is consistent with the hypothesis that apparent decreases in serotonin_2A receptors in schizophrenia are due to altered levels of a regulatory factor(s) that modulates the binding of ligands to the serotonin_2A receptor and that separating the membrane and cytosol removes this regulatory control.
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Because chronic neuroleptic administration upregulates D2 receptor density (Burt et al., 1977), it is likely that these postmortem findings are related to prior neuroleptic exposure rather than to the disease process per se. In light of these very consistent results with [3H]spiperone, it is interesting to note that the striatal binding of [3H]raclopride has been reported to increase in many studies (Dean et al., 1997; Marzella et al., 1997; Ruiz et al., 1992; Sumiyoshi et al., 1995), but normal in several others (Knable et al., 1994; Lahti et al., 1996; Seeman et al., 1993), even in patients exposed to neuroleptic drugs prior to death. This observation suggests that the increase in [3H]raclopride binding is of lower magnitude than the one of [3H]spiperone binding.
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[3H]Raclopride Binding to Brain Tissue from Subjects with Schizophrenia: Methodological Aspects

Abstract

Section snippets

Materials

Tissue collection

RESULTS

DISCUSSION

Acknowledgements

European Journal of Pharmacology

Biology of Psychiatry

Biochemistry and Pharmacology

European Journal of Pharmacology

European Journal of Pharmacology

Brain Research

European Journal of Pharmacology

D2 dopamine receptors in neuroleptic-naive schizophrenic patients: A positron emission tomography study with [11C]raclopride

Archives of General Psychiatry

Variability in D2-dopamine receptor density and affinity: A PET study with [11C]raclopride binding

Synapse

Gpp(NH)p stimulates [3H]raclopride binding to homogenates from human putamen and accumbens

Neuroscience Letters

[³H]Raclopride Binding to Brain Tissue from Subjects with Schizophrenia: Methodological Aspects

D₂ dopamine receptors in neuroleptic-naive schizophrenic patients: A positron emission tomography study with [¹¹C]raclopride

Variability in D₂-dopamine receptor density and affinity: A PET study with [¹¹C]raclopride binding

Gpp(NH)p stimulates [³H]raclopride binding to homogenates from human putamen and accumbens