E L S E V I E R Schizophrenia Research 21 (1996) 133-139

SCHIZOPHRENIA RESEARCH

Decreased frontal cortical serotonin2A receptors in schizophrenia

Brian Dean a,b,,, Wendy Hayes a,c a The Rebecca L. Cooper Research Laboratories, The Mental Health Research Institute of Victoria, Locked Bag 11,

Parkville, Victoria 3052, Australia b The NH&MRC Schizophrenia Research Unit, Parkville, Victoria, Australia

c The Department of Medicinal Chemistry, The Victoria College of Pharmacy, Monash University, Parkville, Victoria, Australia

Received 15 December 1995; accepted 1 April 1996

Abstract

5HT2A receptors were measured in the frontal cortex from schizophrenic and non-schizophrenic subjects. There was a decrease in the density of 5HT2A receptors in Brodmann's areas 8, 9 and 10 from the schizophrenic subjects. In addition, there was an age-dependent decrease in the density of 5HT2A receptors in Brodmann's areas 9 from the non-schizophrenic subjects, which was absent in the schizophrenic subjects. Available evidence does not suggest that the change in 5HT2A receptors in the schizophrenic subjects was a result of drug treatment before death. These data may indicate that decreased 5HTEA receptors in the frontal cortex are involved in the pathology of schizophrenia.

1. In~oducfion

The serotonin2A (5HT2A) receptor is thought to be an important therapeutic site for anti-psychotic drugs (Ashton, 1992; Janssen et al., 1988; Leysen et al., 1978) and could, therefore, be involved in the genesis of a psychotic episode. Significantly, the study of tissue obtained at autopsy has shown there to be a decrease in the density of 5HT2A receptors in Brodmann's areas 9 from the frontal cortex of subjects with schizophrenia (Arora and Meltzer, 1991; Bennett et al., 1979; Hashimoto et al., 1993; Laruelle et al., 1993). By contrast, studies of other areas of the frontal cortex did not show a change in the 5HT2A receptor in schizo-

* Corresponding author. Tel.: (61-3) 9388-1633; Fax: (61-3) 938-75061; e-mail: Bdean@papyrus.mhri.edu.au

0920-9964/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved PII S0920-9964(96) 00034-5

phrenia (Reynolds et al., 1983; Whitaker et al., 1981). These data suggest there are regionally specific changes in 5HT2A receptors in the frontal cortex of subjects with schizophrenia. Because of this, we have measured the density of 5HT2A receptors in Brodmann's areas 8, 9 and 10 of the frontal cortex from schizophrenic and non-schizo- phrenic subjects.

2. Methods

2.1. Tissue collection

Tissue from Brodmann's areas 8, 9 and 10 of the frontal cortex from the left hemisphere was obtained at coronial autopsy from subjects with a provisional diagnosis of schizophrenia. This tissue

134 Brian Dean, Wendy Hayes/Schizophrenia Research 21 (1996) 133-139

was rapidly frozen to -70°C and stored at this temperature until required. Tissue was then obtained from subjects with no history of schizo- phrenia who were matched for sex and closely matched for age to the subjects with a confirmed diagnosis of schizophrenia. Because of this collec- tion process, there was no difference in the mean age of the schizophrenic and non-schizophrenic subjects (Table l).

2.2. Postmortem interval

In most cases where death did not involve suicide, tissue in this study was collected from subjects whose death was witnessed. In that situa- tion, the PMI is the time from death to autopsy. Where death was not witnessed, tissue was only taken from individuals who had been seen alive up to 5 h before being found dead. In those cases, the PMI is the interval that was half way between the donor being found dead and being last seen alive. In all cases, the cadavers were refrigerated within 5 h of being found dead. In this study, the mean postmortem interval (PMI) for the tissue from the non-schizophrenic subjects was slightly less (P=0.05) than it was for the schizophrenic subjects (Table 1).

room temperature with 10 nM [3H]ketanserin in 170 mM Tris buffer (pH 7.7). Non-specific binding of radioligand to tissue was taken as the binding of 10 nM [3H]ketanserin to the other two sections in the presence of 10 -6 M spiperone. After the incubation all sections were washed twice for 15 rain in ice cold Tris buffer and then rinsed in ice cold distilled water. The washed sections were air dried and stored desiccated, under vacuum for 12h and then apposed to Amersham 3H hyperfilm® for 14 days. The hyperfilm was devel- oped and the density of the resulting images ana- lysed using the MCID image analysis system and compared to Amersham [3H]microscales® for quantification.

The concentration of [3H ] ketanserin used in this study was significantly higher than the K a (1 .8+l .2nM: mean_+SEM) for [3H]ketanserin binding to the human 5HT2, receptor (Dean et al., 1995). The density (Bma,) of 5HT2, receptors was taken as the binding of [3H]ketanserin without spiperone minus the binding of the radioactive drug in the presence of spiperone. Thus, this study used a single point estimation of receptor density under conditions of receptor saturation.

2.5. Statistics

2.3. Diagnosis

The provisional diagnosis of schizophrenia, obtained from preliminary reports to the Coroner's Office, was confirmed by a senior psychologist and senior psychiatrist after an extensive case history review (Hill et al., 1996). In this study all diagnoses were confirmed using the DSM-III-R criteria (American Psychiatric Association, 1987).

2.4. Measurement of 5HTea receptors

The density of 5HT2A receptors was measured in tissue from Brodmann's areas 8, 9 and 10 using [3H]ketanserin binding and autoradiography essentially as described previously (Fishette et al., 1987). Blocks of tissue were brought to -20°C in a cryostat and five 20-gm sections cut and thaw mounted onto gelatin coated slides. Three sections from each subject were incubated for one hour at

Statistical significance was measured using the Mann-Whitney test in the Sigrnastat ® computer program. Pearson product-moment correlation coefficients were calculated using an assumed straight line model.

3. Results

3.1. The measurement of 5HT2a receptors

The 5HTzA receptors were shown to be concen- trated in a number of cortical layers in Brodmann's areas 8, 9 and 10 but the resolution of the auto- radiographs did not allow the separate analysis of the receptors in each layer. Thus, the data pre- sented are the summed measure of the receptors in each of the three brain regions from the 40 individuals from whom tissue was obtained. There was no significant variation in the density of

Brian Dean, Wendy Hayes/Schizophrenia Research 21 (1996) 133-139 135

5HT2A receptors between the three brain regions within the frontal cortex from each subject (Table 1) (schizophrenic: B8 vs. B9, P=0.47; B8 vs. B10, P---0.53; and B9 vs. B10, P=0.97; non- schizophrenic: B8 vs. B9, P=0.53; B8 vs. B10, P = 0.31; and B9 vs. B10, P=0.11). By contrast, compared to the same regions from the non- schizophrenic subjects, the density of 5HT2A recep- tors was significantly decreased in Brodmann's areas 8, 9 and 10 from the frontal cortex of subjects with schizophrenia (Tables 1 and 2).

There was no difference in the density of 5HT2A receptors in Brodmann's areas 8, 9 or l0 from subjects who had or had not committed suicide (Table 2). For the schizophrenic subjects there was no relationship between the density of 5HT2A receptors and age in Brodmann's areas 8 (r= -0.18; P=0.43), 9 (r= -0.16; P=0.48) or 10 ( r - - -0 .27; P=0.24). By contrast, in the non- schizophrenic subjects, there was a significant inverse relationship between the density of 5HT2A receptors in Brodmann's area 9 with the age of the donor (r= -0.62; P<0.005). This rela- tionship was almost statistically significant in Brodmann's area 8 ( r = - 0 . 4 2 ; P=0.06) but was absent in Brodmann's area 10 ( r = - 0 . 2 0 ; P = 0.39). There was no relationship between the PMI for the tissue and the density of 5HT2A receptors in Brodmann's areas 8 ( r = - 0 . 0 6 or 0.3), 9 (r-- 0.02 or 0.18) or 10 (r=0.10 or 0.09) for the schizophrenic and non-schizophrenic subjects, respectively.

All individuals with schizophrenia from whom tissue was obtained for this study had been treated with neuroleptic drugs (Table 1). However, there was no difference in the density of 5HT2A receptors in the tissue of subjects treated with differing neuroleptic drugs (i.e., haloperidol vs. chlopromaz- ine vs. fluphenazine vs. other; Tables 1 and 2). Furthermore, there was no relationship between the last recorded dose of neuroleptic drug and the density of 5HT2A receptors in Brodmann's areas 8 ( r = - 0 . 1 2 ) , 9 ( r = - 0 . 2 ) or l0 ( r = - 0 . 1 2 ) (Table 1 ). There was no difference in 5HT2A recep- tor density in tissue from schizophrenic and non- schizophrenic individuals who had or had not been treated with benztropine, antidepressant drugs or benzodiazepines (Table 2).

4. Discussion

This study has shown the density of 5HT2A receptors to be decreased in Brodmann's areas 8, 9 and 10 from schizophrenic compared to non- schizophrenic subjects. This finding agrees with most other studies of 5HT2A receptors in Brodmann's area 9 from subjects with schizophre- nia (Arora and Meltzer, 1991; Bennett et al., 1979; Hashimoto et al., 1993; Laruelle et al., 1993). There is one study, also using autoradiography, that did not show a change in the density of 5HT2A receptors in Brodmann's area 9 from sub- jects with schizophrenia (Joyce et al., 1993). Significantly, that study used [3H]LSD, a less specific radioligand for the 5HT2A receptors (Leysen et al., 1982), and therefore the differing results obtained using autoradiography could be due to the use of different radioligands used to measure the density of 5HT2 receptors in human brain.

This study has shown that the density of 5HT2A receptors in Brodmann's area 9 from non-schizophrenic subjects decreases with age. Furthermore, there was a trend toward a negative correlation between the density of 5HT2A receptors and donor age for Brodmann's area 8 from the non-schizophrenic subjects. The finding of an age- dependent decrease in 5HT2A receptors in the frontal cortex from subjects with no history of mental illness agrees with other studies (Bennett et al., 1979; Cheetham et al., 1988; Gross-Isseroff et al., 1990; Laruelle et al., 1993; Lowther et al., 1994). Significantly, an in vivo study of 5HT2A receptors using positron emission tomography has also shown a decreasing density of 5HT2A receptors with age (Iyo and Yamasaki, 1993). However, this is the first study to suggest the loss of 5HT2A receptors in subjects with no history of mental illness is localised to specific regions of the frontal cortex. It is also important to note that the density of the 5HT2A receptors are decreased in schizo- phrenic individuals of all ages. This suggests low densities of 5HT2A receptors are always present in the brain of subjects who develop schizophrenia. These data would therefore favour the argument that schizophrenia involves altered neurodevelop-

136 Brian Dean, Wendy Hayes~Schizophrenia Research 21 (1996) 133-139

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mental, rather than neurodegenerative, processes (Murray, 1994).

Unlike two previous studies (Reynolds et al., 1983; Whitaker et al., 1981), this study has shown 5HTzA receptors to be decreased in Brodmann's areas 8 and 10 from subjects with schizophrenia. In one of these studies (Whitaker et al., 1981), the 5HT2A receptors were measured on pooled particulate membrane from Brodmann's areas 4, 10 and 11, and it is not possible to determine if the density of 5HT2A receptors was altered in any single region. In the other study, which used [3H]ketanserin binding and only tissue from Brodmann's area 10 (Reynolds et al., 1983), it is difficult to explain why a change in 5HTzA recep- tors in schizophrenia was not detected. However, in our first study of 5HTzA receptors using particu- late membrane we failed to measure any differences in the density of 5HT2A receptors in Brodmann's area 9 from subjects with schizophrenia (Dean et al., 1995). After that study we suggested we had taken tissue from a region of Brodmann's area 9 in which 5HT2A receptors were not changed. This hypothesis is supported by our latest finding of decreased 5HTzA receptor density using autoradi- ography to examine larger and deeper areas of the frontal cortex. A similar hypothesis could therefore be extended to explain the differing results using tissue from Brodmann's area 10 in an autoradio- graphic verus particulate membrane study.

It has been reported that 5HT2A receptors are increased in the brain tissue of subjects who have committed suicide (Arango et al., 1990; Hrdina et al., 1993; Mann et al., 1989). By contrast, in this study, there was no difference in the density of 5HT2A receptors in subjects who had committed suicide. Thus, the data in this study would be more in line with other studies that were unable to show a change in 5HT2A receptors in subjects who had committed suicide (Arranz et al., 1994; Cheetham et al., 1988; Lowther et al., 1994).

The subjects from whom tissue was obtained for this study had been treated with neuroleptic drugs, some of which have been shown to decrease the levels of 5HT2A receptors in rat brain (Andree et al., 1986). In such studies rats treated with chlopromazine, but not haloperidol, showed a 60% decrease in 5HTEA receptor density. This study failed to show the change in the density of

138 Brian Dean, Wendy Hayes~Schizophrenia Research 21 (1996) 133-139

Table 2 The density (mean±SEM) of 5HT2A receptors in schizophrenic and non-schizophrenic subjects, who had or had not committed suicide and subjects treated with different drugs

Subjects Brodmann's area

8 9 10

Receptor density* p Receptor density* p Receptor density* p

Schizophrenic 45 ± 3.4 Non-schizophrenic 60 ± 2.2 Suicide 54 +_ 3.5 Not suicide 52 ± 3.2 Neuroleptie drug treatment Haloperidol 47 ± 7.1 Chlorpromazine 35 ± 6.6 Fluphenazine 47 ± 7.1 Other 62 ± 15 Other drug treatments Benztropine 44 ± 6.1 No benztropine 45 ± 5.3 Antidepressants 38 ± 4.8 No antidepressants 47 ± 3.9 Benzodiazepines 47 ± 14 No benzodiazepines 45 ± 3.1

<0.001 42±3.2 63±2.4

NS 54±4.6 51±3.0

40±6.9 31±5.1 40±7.6 55±9.8

<0.001

NS

41±3.6 56±2.6 49±4.4 48±3.2

39±8.0 31±9.1 39±7.4 54±7.3

NS 41±7.5 NS 42±6.4 NS 42±3.1 41±4.7

NS 37±3.7 NS 33±5.6 NS 43±3.7 42±4.1

NS 42±13 NS 40±13 NS 42±2.7 42±3.6

<0.005

NS

NS, not significant; *fmol/mg TE.

5HT2A receptors to be limited to tissue from schizophrenic subjects treated with chlopromazine. Thus, if the study of neuroleptic drugs in the rat is valid for studies in humans, then the change in 5HT2A receptors in schizophrenia is not likely to be a drug-induced effect.

Antidepressant drugs have been reported to up-regulate 5HT2A receptors (Hrdina and Vu, 1993). This study produced no evidence to show a change in 5HT2A receptors in the schizophrenic subjects treated with antidepressants. This would argue against the change in 5HT2A receptors in schizophrenia being an affect of antidepressant drug treatment. Similarly, benztropine had been used to treat a number of schizophrenic individuals from whom tissue was obtained for this study. Again, there was no evidence that the change in 5HT2A receptors was limited to individuals treated with benztropine.

This study has shown the 5HT2A receptor to be decreased in the frontal cortex from subjects with schizophrenia. Significantly, LSD has been shown to induce schizophrenic-like psychoses in non- schizophrenic individuals (Ashton, 1992) and to

decrease the density of 5HT2A receptors in treated rats (Buckholtz et al., 1990). Taken together, these two findings could indicate that a decreased density of 5HT2A receptors could precipitate a psychosis. If this were the case, then the decreased density of 5HT2A receptors in subjects with schizophrenia would be consistent with a change that could induce a psychotic episode. Furthermore, Brodmann's areas 8, 9 and l0 form part of the frontal cortex that is believed to control higher cognitive function and suppress extraneous influ- ences that could lead to inappropriate actions (Fuster, 1989). Changes in these functions are apparent in many subjects with schizophrenia. Perhaps, changes in 5HTEA receptors could be linked to such higher functional abnormalities because they have altered the functioning of critical areas of the frontal cortex in subjects with schizophrenia.

Acknowledgment

The authors would like to thank Dr. K. Opeskin, Mr. G. Pavey, Ms. P. O'Brien and Ms.

Brian Dean, Wendy Hayes~Schizophrenia Research 21 (1996) 133-139 139

J. Leditschke for assistance in the collection of tissue. We also thank Ms. C. Hill and Professor N. Keks for carrying out case history reviews and confirming the diagnoses. This research was sup- ported in part by a grants in aid from the Stanley Foundation Research Awards Program, the Woods Family Research Project and the Rebecca L. Cooper Medical Research Foundation.

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