Benzodiazepine receptors in the post-mortem brain of suicide victims and schizophrenic subjects

Psychiatry Research 71 Ž1997. 137]149

Benzodiazepine receptors in the post-mortem brain of suicide victims and schizophrenic subjects Ghanshyam N. Pandey a,U , Robert R. Conley b , Subhash C. Pandey a , Sudhir Goel a , Rosalinda C. Roberts b , Carol A. Tammingab, Dennis Chute c , John Smialek c a

The Psychiatric Institute, Uni®ersity of Illinois, Chicago, IL 60612, USA b Maryland Psychiatric Research Center, Baltimore, MD, USA c Department of Pathology, Uni®ersity of Maryland, School of Medicine, Baltimore, MD, USA Received 8 December 1996; revised 14 May 1997; accepted 2 June 1997

Abstract To examine the role of benzodiazepine ŽBZ. receptors in suicide and schizophrenia, we determined BZ receptors in post-mortem brain ŽBrodmann’s area 10. obtained from suicide victims, schizophrenic patients, and control subjects using w 3 HxRO15-1788 as the radioligand. The maximum number of binding sites Ž Bmax . of BZ receptors in the cortex of suicide victims was significantly higher compared with controls, but this increase was mainly due to those suicide victims who died by violent means and whose Bmax was significantly higher than of those who died by non-violent means or control subjects. In schizophrenic patients, Bmax was not significantly different from that of control subjects. When the schizophrenic subjects were separated into two groups, those on neuroleptics and those off neuroleptics for at least 12 months, however, the mean Bmax of BZ receptors in the prefrontal cortex in post-mortem brain obtained from schizophrenic patients on neuroleptics was significantly lower than Bmax in drug-free schizophrenic patients or normal controls. There were no significant differences among groups in values of the apparent dissociation constant Ž K D . of w 3 HxRO15-1788 binding. These results suggest that BZ receptors are up-regulated in the cortex of suicide victims, specifically those who used violent means, and that neuroleptic treatment may result in decreased central BZ receptor binding in the cortex of schizophrenic patients. Thus, the method of suicide and previous exposure to neuroleptics should be considered in the interpretation of data on BZ receptors. Q 1997 Elsevier Science Ireland Ltd. Keywords: Benzodiazepine receptors; Schizophrenia; Neuroleptic drugs; Suicide

U

Corresponding author, The Psychiatric Institute, 1601 West Taylor Street, Chicago, IL 60612, USA. Tel.: q1 312 4338352; fax: q1 312 4338360. 0165-1781r97r$17.00 Q 1997 Elsevier Science Ireland Ltd. All rights reserved. PII S0165-1781Ž97. 00060-7

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1. Introduction Several studies suggest that benzodiazepine ŽBZ. receptors may be altered in the post-mortem brain of suicide victims and schizophrenic patients. The BZ receptors in the brain mediate the central pharmacological action of the benzodiazepines ŽHaefely et al., 1985; Martin, 1987.. BZ receptors are thought to be located proximate to and are functionally coupled to both recognition sites for gamma-aminobutyric acid ŽGABA. and for chloride ions, and they are one of the regulatory sites of the benzodiazepinerGABA receptor-chloride ion complex ŽOlsen, 1981; Haefely et al., 1993.. The benzodiazepine-GABA receptor protein is functionally linked to a chloride ionophore, which probably is the effector component of the receptor complex and regulates the transmembrane flux of chloride ions through the occupation of allosteric recognition sites on this complex ŽGuidotti et al., 1979; Martin, 1987; Haefely, 1989.. Based on molecular cloning studies, GABA A receptors are composed of five subunits, a , b , g , d , and r , and each subunit has several isoforms, each encoded by multiple genes ŽOlsen and Tobin, 1990; Burt and Kamatchi, 1991; Costa and Guidotti, 1996.. Pharmacologically, there are two types of BZ receptors, BZ 1 and BZ 2 , which are classified according to their sensitivity to Cl218,872, a partial agonist. BZ 1 has a higher affinity and BZ 2 has a lower affinity for Cl218,872. Type II receptors are abundant in the hippocampus, the striatum, and the spinal cord. The co-expression of a 1 , b 1 , and g 1 receptor subunits displays the characteristics of the type I receptor, whereas a 2,3, or 5 , b 1 , and g 2 subunits display the characteristics of the type II receptor ŽYakushizi et al., 1993.. Benzodiazepine receptors may be altered during anxiety and stress ŽMedina et al., 1983; Norman and Burrows, 1986; Weizman et al., 1987., and treatment with benzodiazepines, antidepressants, or neuroleptics can cause changes in the density of BZ receptors ŽGuidotti et al., 1979; Kimber et al., 1987; Gavish et al., 1988; Todd et al., 1995.. In the last decade, several reports have ap-

peared suggesting possible alterations in GABAmediated neural pathways in schizophrenia ŽGerner et al., 1984; Van Kammen et al., 1989.. Recently, w 3 Hxmuscimol binding to GABA A receptors in the post-mortem cingulate cortex was reported to be increased in schizophrenia ŽHanada et al., 1987; Benes et al., 1992.. Since BZ receptors are part of the GABA A receptor, it is quite possible that altered GABA A receptors in the post-mortem brain of schizophrenic patients are also associated with changes in BZ receptors. Benes et al. Ž1992., who found increased GABA A receptor binding in the post-mortem brain of schizophrenics, however, did not observe any changes in BZ receptors in the post-mortem brain of schizophrenics ŽVincent et al., 1995.. The reasons for this dissociation between GABA A and BZ receptors are unclear. On the other hand, Squires et al. Ž 1993 . found decreased w 3 Hxflunitrazepam binding in several areas of the post-mortem brain of schizophrenics. BZ receptors have also been shown to be altered in the post-mortem brain of suicide victims by some researchers ŽCheetham et al., 1988., but not by others ŽManchon et al., 1987; Stocks et al., 1990; Rochet et al., 1992.. Since many schizophrenics die by committing suicide and since BZ receptors have been shown to be altered in the post-mortem brain of suicide victims, the role of BZ receptors in suicide and schizophrenia warrants further investigation. We have therefore studied central BZ receptor recognition sites in the post-mortem brain of schizophrenic patients, suicide victims Žwith or without schizophrenia., and normal controls. Since most, but not all of the schizophrenic brain samples came from patients who were treated chronically with neuroleptics, we also examined the effect of chronic neuroleptic treatment on BZ receptors. 2. Methods 2.1. Subjects and brain sample collection Benzodiazepine receptors were determined in the prefrontal cortex ŽBrodmann’s area 10. in brain tissue obtained from 13 schizophrenic

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patients Žeight suicides, five non-suicides., 13 suicide victims without schizophrenia, and 15 normal control subjects. Toxicology data indicated that out of 13 non-schizophrenic suicide victims, six had a history of mood disorders, three had a history of alcohol abuse, two showed polysubstance abuse, and two had no known history of mental illness. Among the 13 schizophrenic subjects, eight were on neuroleptics alone or neuroleptics with other antidepressants, five were free of neuroleptics Žone was on lithium, one was taking verapamil off and on, and three had no psychoactive drugs.. Toxicology data were based on blood and urine samples and are presented in Table 1 together with demographic data and clinical characteristics of subjects. Post-mortem brain samples from normal control subjects matched for age and post-mortem interval were used for comparison purposes. Normal control subjects were free of any history of psychiatric disorders and died of causes other than suicide. Brain samples came from the Maryland Brain Collection Program, a collaboration between the Maryland Psychiatric Research Center and the Maryland Medical Examiner’s Office in Baltimore. Brain tissue was removed as soon after death as possible and immediately chilled in ice. Subsequent to collection and dissection, tissue was stored at y808C until analyzed. 2.2. Diagnostic methodology All subjects in the study were diagnosed in the following manner; at least one family member was interviewed after giving informed consent. These interviews were based on the Diagnostic Evaluation After Death ŽDEAD. ŽSalzman et al., 1983. and the Schedule for Clinical Interviews for the DSM-III-R ŽSCID. ŽSpitzer et al., 1992.. In cases in which there was a prior history of mental health treatment and in all cases of suicide, family members were requested to give permission for clinical records to be obtained from prior mental health treatment providers. An attempt was made to collect all available records on each case, and then the appropriate data were abstracted from the records and collected using the DEAD. Two senior psychiatrists made independent DSM-III-

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R-based diagnoses using all the gathered available data. These diagnoses were compared, and all discrepancies were resolved through a consensus conference. Thus, all DSM-III-R diagnoses represent a consensus diagnosis of two psychiatrists. Similarly, all normal controls were verified as being free of mental illness by the same diagnostic process. Data on suicide cases were collected using the Circumstances of Suicide form during the same interview process. Cases were included as suicides only if the manner of death was considered to be suicide by the medical examiner. 2.3. Determination of [ 3 H]RO15-1788 binding to central benzodiazepine receptors in the brain The binding of w 3 HxRO15-1788 Žan imidazobenzodiazepine., which is a selective, high-affinity competitive antagonist at central BZ receptors ŽHunkeler et al., 1981. to rat and human brain, has been characterized by several investigators ŽKopp et al., 1990; Mohler and Richards, 1981; Maloteaux et al., 1988.. It has also been shown that it binds with high affinity and is temperature dependent. Changing the temperature has no effect on Bmax , but K D increases as the temperature increases ŽKopp et al., 1990.. In-vitro binding is not influenced by the presence of GABA ŽMohler and Richards, 1981.. Central BZ receptors were determined in the frontal cortex using w 3 HxRO15-1788 as the radioligand by a slight modification of a procedure described earlier by Maloteaux et al. Ž1988. and Kopp et al. Ž1990. as follows. The tissue was homogenized in 10 ml of 0.32 M sucrose using a polytron Žsetting 7 for 30 s. and then centrifuged at 1000 = g for 10 min to remove nuclei and cell debris. The resulting supernatant was spun at 49 000 = g for 15 min, and the pellet thus obtained was suspended in 10 ml of tris buffer Ž50 mM, pH 7.4.. The suspension was again centrifuged at 49 000 = g for 15 min, and the resulting pellet was washed one more time with tris buffer. The pellet thus obtained was suspended in an incubation buffer Ž50 mM tris, pH 7.4., and the binding was carried out in duplicate by incubating an aliquot Ž200 m l. of membrane sus-

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Table 1 Clinical, demographic and toxicological characteristics of subjects Comparison groups

Age Žyears.

Sex

PMI Žh.

Normal controls 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Mean "S.D.

45 21 31 61 32 35 19 42 37 37 31 46 41 36 24 35.8 "10.6

M M M M F M M F M M M M M F M

15 14 17 18 14 26 6 9 9 5 9 10 19 17 15 13.5 "5.59

Suicide Victims 1 2

37 21

M M

10 11

3 4 5 6

27 44 24 36

M F M F

7 8

24 43

9 10 11

34 42 41

12 13 Mean "S.D.

39 40 34.76 "8.05

Schizophrenicrsuicide victims 1 29 2 3 4 5 6 7 8 Mean "S.D.

42 41 20 54 20 40 39 35.6 "11.7

Cause of death

Toxicology

MI, ASCVD Cardiac myopathy ASCVD ASCVD GSW homicide Drug intoxication Smoke inhalation ASCVD ASCVD Card. arrest GSW MVA Liver cirrhosis ASCVD Drowning

Codeine-morphine ETOH None detected None detected None detected None detected None detected None detected Morphine-cocaine None detected Lidocaine None detected None detected None detected None detected

CO Proloxin

MD MD

24 13 22 13

CO intoxication Drug overdose Cardiac arrythmia GSW Drug overdose GSW GSW

MD Alc. abuse MDr None MD

M M

9 11

GSW Drug overdose

M M F

16 12 16

GSW Drowning Drug overdose

M M

17 25 14.5 "4.5

None detected Nortriptyline Atropine Acetominophen, diphenhydramin Butalbital Propoxyphene, acetaminophen ETOH None detected ETOH, AMI, DMI, nortriptyline Chlorodifluromethane ETOH

F

8

M F F M M M M

12 3 11 12 24 17 19 14.7 "5.52

Aspyyxia GSW

Stab wound Hanging Drowningrjumping Overdose Jump from height Jump from height Drowning Smoke inhalation

CPZ, morphine, doxepin Haldol, Prozac Lithium Haldol None detected Prolixin Stelazine Verapamil

Diagnosis

Alcohol abuse Polysubstance abuse ETOH abuse None MD Substance abuse MD

Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia

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Table 1 Ž Continued. Schizophrenics 1 2 3 4 5 Mean "S.D.

28 39 42 47 22 35.6 "10.3

M M M F M

18 18 11 19 8 14.8 "4.96

ASCVD MNS ASCVD ASCVD Unknown

None detected Haldol, Prozac Haldol, Prozac None detected Thioridazine

Schizophrenia Schizophrenia Schizophrenia Schizophrenia Schizophrenia

MI, myocardial infarction; ASCVD, atherosclerotic cardiovascular disease; GSW, gunshot wound; ETOH, alcohol; AMI, amitriptyline; DMI, desipramine; CPZ, chlorpromazine; MD, mood disorders; MNS, malignant neuroleptic syndrome.

pension with w 3 HxRO15-1788 at six concentrations Ž0.4]5 nM. with or without 1 m M diazepam in a total volume of 0.5 ml. The suspension was incubated at 48C for 60 min and was then separated by filtration through a Whatman ŽGFrB. filter using a Brandel Harvester. The filters were then placed in vials with scintillation cocktail and the radioactivity was counted in a liquid scintillation counter. Specific binding was defined as the difference between the total binding and the binding observed in the presence of 1 m M diazepam, and it ranged from 95 to 82% depending upon the concentration of the ligand. The maximum number of binding sites Ž Bmax . and the apparent dissociation constant Ž K D . were calculated by Scatchard analysis using the EBDA program ŽMcPherson, 1985., and the protein content was determined by the method of Lowry et al. Ž1951.. 2.4. Statistical analysis A one-way analysis of variance ŽANOVA. was used to analyze the data obtained for the different groups. In the presence of a significant f statistic, a post-hoc comparison between the groups was performed using Tukey’s studentized range ŽHSD. test. All values are the mean " S.D. 3. Results 3.1. Normal controls ®s. suicides The binding of w 3 HxRO15-1788 to human cortical membranes was saturable, and the Scatchard

analysis indicated a single class of high-affinity binding sites, with Bmax of 719 fmolrmg protein and K D of 0.82 nM in a brain sample from a normal control subject as shown in Fig. 1. The saturation isotherm and the Scatchard analysis in the frontal cortex of this normal control subject are shown in Fig. 1. Demographic, toxicological, and clinical characteristics of the tissue donors as well as w 3 HxRO15-1788 binding characteristics in the different groups are shown in Table 2. Both age and the post-mortem interval were matched and are similar in each diagnostic group, and there were no significant differences between groups. We compared Bmax of w 3 HxRO15-1788 binding in the frontal cortex of suicide victims without schizophrenia with that of normal control subjects and observed that Bmax was significantly higher Ž P) 0.01. in the non-schizophrenic suicide victims. There were no significant differences in K D between the two groups ŽFig. 2.. Among the suicidal patients, two were taking antidepressants Žnortriptyline, amitriptyline. at the time of death. The mean Bmax of the 11 non-schizophrenic suicidal patients free of antidepressant treatment Ž1269 " 146 fmolrmg protein. was still significantly higher compared to controls Ž Ps 0.01.. The mean Bmax of the two suicide subjects taking antidepressants was 760 fmolrmg protein. There were no schizophrenics who were on antidepressants only; however, there were four schizophrenics who were on Haldol and antidepressants Žthree were on Haldol and Prozac., and their mean Bmax was 420 " 121. This decrease is probably related to treatment with neuroleptics as discussed later.

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Fig. 1. Saturation isotherm of w 3 HxRO15-1788 binding to central BZ receptors in the human frontal cortex. Membranes were incubated with different concentrations of ligand in the presence and absence of 1 m M diazepam. The inset shows a Scatchard plot of the binding data, which are the means of duplicate determinations of a single experiment. BrF, bound over free w 3 HxRO15-1788 Žfmolrmg proteinrnM.; Bound, w 3 HxRO15-1788 specifically bound Žfmolrmg protein.. For this particular experiment, binding indices are K D s 0.82 nM; Bmax s 719 fmolrmg protein; r s 0.98.

3.2. Violent ®s. non-®iolent Since it has been reported that the method of committing suicide Žviolent vs. non-violent. may affect 5HT1A receptors ŽMatsubara et al., 1991., we compared Bmax and K D values of BZ receptor binding in suicide subjects who died by violent versus non-violent means. The violent suicide victims were defined as those who died by such methods as gunshot wound, stabbing, hanging, or jumping off a bridge, and non-violent suicide victims were those who died of an overdose of drugs or CO poisoning. As shown in Fig. 2, we found that mean Bmax Žfmolrmg protein. of w 3 HxRO151788 binding in six violent suicide victims Ž1581 " 341. was significantly higher than that found in the seven non-violent suicide victims Ž Bmax s 855 " 301. or normal control subjects. There was no significant difference in Bmax between non-violent suicide victims and control subjects. It thus ap-

pears that the up-regulation of Bmax in suicide victims is related to and restricted to violent suicide. There were no significant differences in K D among any of these subgroups. 3.3. Effects of diagnosis and toxicology on benzodiazepine receptors To examine if the observed increase in BZ receptors in the brain of non-schizophrenic suicide victims was related to some form of mental illness or to the presence of alcohol or other drugs, we grouped the suicidal subjects by diagnosis. Out of 13 suicidal subjects, six had a history of mood disorder, three had a history of alcohol abuse, two had a history of polysubstance abuse, and three had no known history of any mental illness. The mean Bmax in the suicidal subjects with a history of a mental or mood disorder was 1159 " 299 fmolrmg protein, n s 5; in those with

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Fig. 2. Bargraph and scattergram of Bma x of w 3 HxRO15-1788 binding in various diagnostic groups and subgroups.

a history of alcoholism, the mean Bmax was 1425 " 722 fmolrmg protein, n s 3; in those with polysubstance abuse, the mean Bmax was 840 " 30 fmolrmg protein, n s 2; and subjects with no such history showed a mean Bmax of 1440 " 488 fmolrmg protein, n s 2. Although the number of subjects in each group was too small for a meaningful analysis, subjects who had a history of alcoholism appeared to have higher Bmax . Similarly, we analyzed the data in terms of drug toxicology. The mean Bmax in the suicidal subjects with alcohol toxicity was 1482 fmolrmg protein, whereas in those who did not have alcohol toxicity the mean Bmax was 1061 fmolrmg protein, n s 9. On the other hand, those with certain drug toxicities Že.g. antidepressants . had a Bmax of 1082 fmolrmg protein, n s 6, compared with 1283 fmolrmg protein, n s 7, for those with no such drug toxicity, so there was no significant

difference between the two. There were only three subjects with neither drug toxicity nor presence of alcohol, and their mean Bmax was similar to those with drug toxicity. 3.4. Schizophrenic patients ®s. normal controls: effect of treatment with neuroleptics We also compared Bmax of w 3 HxRO15-1788 binding in the frontal cortex of schizophrenic patients and of normal control subjects and observed that the mean Bmax in schizophrenic patients was not significantly different from that of normal control subjects as shown in Table 2 and Fig. 3. To examine the effects of neuroleptic treatment on w 3 HxRO15-1788 binding, we compared Bmax and K D values of the neuroleptictreated and the neuroleptic-free schizophrenics. The schizophrenic population consisted of five

144

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Fig. 3. Effect of neuroleptic treatment on Bma x of w 3 HxRO15-1788 binding to central BZ receptors in the frontal cortex of schizophrenics. U Significantly different from the group of neuroleptics or normal controls Ž P - 0.05..

patients who were free of neuroleptic treatment for at least 12 months prior to death as well as eight patients who were taking neuroleptics at the time of death. Out of five neuroleptic-free

schizophrenic patients, three were free of any medication, one was taking verapamil off and on, and one was on lithium. Bmax in the schizophrenic patients who were on neuroleptics Ž n s 8; Bmax s

Table 2 Benzodiazepine receptors in the post-mortem brain of schizophrenic patients, suicide victims, and normal controls Diagnostic groups

w3 Hx-RO15-1788 binding Bma x Žfmolrmg protein. mean " S.D.

Normal controls Ž n s 15. Suicides without schizophrenia Ž n s 13. Violent suicides Ž n s 6. Non-violent suicides Ž n s 7.

787 q 319 U

K D ŽnM. mean " S.D. 0.70" 0.26

1190 q 451 1581 " 341U 855 " 301

0.86" 0.17 1.00" 0.05 0.72" 0.12

Total schizophrenics Ž n s 13. Suicidal schizophrenics Ž n s 8. Non-suicidal schizophrenics Ž n s 5.

578 " 212 599 " 209 546 " 238

0.53" 0.07 0.53" 0.09 0.52" 0.01

Neuroleptic-treated schizophrenics Ž n s 8.

498 " 216U

0.53" 0.08

Neuroleptic-free schizophrenics Ž n s 5.

715 " 126

0.52" 0.06

U

Significantly different from control subjects.

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498 " 76 fmolrmg protein. was significantly lower Ž F s 4.48; df s 1,16; P- 0.05. when compared with Bmax of schizophrenic patients who were neuroleptic-free Ž n s 5; Bmax s 713 " 57 fmolrmg protein. or with Bmax of normal control subjects Ž n s 15; Bmax s 787 " 82 fmolrmg protein.. Bmax in neuroleptic-free schizophrenics was not significantly different from that of normal controls. 3.5. Schizophrenics who died by suicide Since we observed a significant increase in BZ receptors in suicide, we examined the role of BZ receptors in the schizophrenics who died of suicide. Eight schizophrenic patients died by suicide, and five schizophrenic patients died of other causes. We compared Bmax of BZ receptor binding sites in schizophrenic suicides with Bmax of non-suicide schizophrenic patients and normal control subjects. The mean Bmax for schizophrenic suicides Ž n s 8; Bmax s 599 " 69 fmolrmg protein. was not significantly different from that of non-suicide schizophrenics Ž n s 5, Bmax s 547 " 106 fmolrmg protein. or of normal controls. Since it has been observed that neuroleptic treatment causes a decrease in benzodiazepine receptors, we separated the schizophrenic suicides into two groups on- and off-neuroleptics. The number of neuroleptic-free suicides Ž n s 3. was too small, however, for a meaningful comparison. Since we observed an increase in BZ receptors in violent suicide, we divided the schizophrenic suicides into violent and non-violent groups. The mean Bmax Ž576 " 105. in the five violent schizophrenic suicides was not significantly different from the three non-violent suicides Ž641 " 96. or the normal controls. 4. Discussion In the present investigation, we examined the role of central BZ receptors in suicide and in schizophrenia. An important finding of the investigation is that BZ receptors are increased in the frontal cortex of suicide victims as compared with normal control subjects. This increase may be related to the method of suicide, since we observed that it is significantly higher in violent

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compared with non-violent suicide victims. Although we did not observe any significant differences in BZ receptors between schizophrenic patients and normal controls, we observed a significant decrease in BZ receptors in the prefrontal cortex obtained from those schizophrenic patients who were on neuroleptic treatment compared with the patients who were off neuroleptics or normal control subjects. This result suggests that neuroleptic treatment causes down-regulation of BZ receptors in the brain of schizophrenic victims. The role of BZ receptors in suicide has been studied by several investigators. Cheetham et al. Ž1988. studied BZ receptors in frontal and temporal cortices obtained from depressed suicide victims. Although they observed an increase in BZ receptors in the frontal cortex, no changes were found in the temporal cortex when compared with control subjects. Stocks et al. Ž1990., who investigated BZ receptors in the amygdala and the hippocampus, and Manchon et al. Ž1987. and Rochet et al. Ž1992., who studied BZ receptors in the hippocampus, observed no differences in BZ receptors in the hippocampus or the amygdala between suicide victims and control subjects. Rochet et al. Ž1992., who studied a small number of subjects Ž n s 6., observed a significant change in K D but not in Bmax . Taken together, there is only one study of BZ receptors in the frontal cortex comparable to our study, that of Cheetham et al. Ž1988., and it found an increase in BZ receptors, similar to our study. We observed that the increase in BZ receptors is restricted to the cortex of only those suicidal patients who died by violent means, but BZ receptors are not increased in suicidal subjects who died by non-violent means. It is not clear at this point if the increase in BZ receptors observed in violent suicide victims is related to levels of anxiety or stress, or to other features that might distinguish them from non-violent suicides. Thus, the biological factors associated with violent and non-violent suicide may be different. We also observed that Bmax of schizophrenic suicides who died by violent means was not significantly different from Bmax of those schizophrenic suicides who died by non-violent means, or from normal

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controls. It is not clear whether this lack of difference between violent and non-violent schizophrenic suicides is related to exposure to the neuroleptics, to an increase in BZ receptors in violent suicides, or is restricted to non-schizophrenic suicide. The increase in BZ receptors we observed in non-schizophrenic suicide subjects does not appear to be related to previous treatment with antidepressant drugs, since it was still significantly higher in the 11 patients free of antidepressant medication compared to control subjects. Furthermore, the mean Bmax in the two subjects taking antidepressants Ž760 fmolrmg protein. was similar to the mean Bmax of the normal controls. Four schizophrenics were on antidepressants and neuroleptics and had a mean Bmax of 420 " 121 fmolrmg protein, which was lower than controls, however, this may be due to treatment with neuroleptics rather than antidepressants, or may be related to the combined effect of neuroleptics and antidepressants. Inconsistent results have been obtained in animal studies. The effect of repeated administration of antidepressants on BZ receptors has been studied by several investigators, but they have reported inconsistent results: whereas Suranyi-Cadotte et al. Ž1984, 1990. reported a significant decrease in BZ receptors in the rat brain after chronic treatment, Giardino et al. Ž1993. found an increase in BZ receptors after treatment with sertraline. On the other hand, Kimber et al. Ž1987. and Todd et al. Ž1995. found no change in BZ receptors in the rat brain after treatment with several different antidepressants. Our observation of no change in BZ receptors in the frontal cortex of schizophrenic patients is similar to the results of Reynolds and Stroud Ž1993., who observed no changes in BZ receptors in the hippocampus of schizophrenic patients. Recently, Vincent et al. Ž1995., who studied BZ receptors in the cingulate cortex of schizophrenic patients, observed no difference in the number of BZ receptor sites between schizophrenic patients and control subjects. Kiuchi et al. Ž1989. observed an increase in BZ receptors in the post-mortem brain of schizophrenic patients; however, the study was performed at one concentration of w 3 Hxflunitrazepam using the autoradiographic

technique. It is also not clear if the patients were on medication or were drug-free at the time of the study. However, Squires et al. Ž1993. observed decreased w 3 Hxflunitrazepam in certain cortical areas of schizophrenic patients compared to controls, using autoradiographic techniques. Taken together, it appears that BZ receptor studies in the post-mortem brain of schizophrenic patients have provided inconsistent results. It is important to note the dissociation between the GABA A and the benzodiazepine receptors in the post-mortem brain of schizophrenia as reported by the Benes group ŽBenes et al., 1992; Vincent et al., 1995.. Several schizophrenic patients in our study died by committing suicide. Since we observed an increase in BZ receptors in the non-schizophrenic suicide population, we also examined if BZ receptors in psychotic suicides were similar to those of non-psychotic suicides. We found no significant differences in Bmax or K D between normal controls and schizophrenic suicides, however. There may be two explanations for why we did not observe any alteration in BZ receptors in the schizophrenic suicides. Since five of the eight suicidal schizophrenics were on neuroleptics and since we observed that neuroleptic treatment decreased w 3 HxRO15-1788 binding, it is quite possible that we observed no differences in BZ receptors between normal controls and psychotic suicides because of the latter’s neuroleptic exposure. The number of drug-free schizophrenic suicides was too small Ž n s 3. for any meaningful statistics. It is also possible that psychotic suicides may differ from non-psychotic suicides, and this needs further investigation. Although we did not observe any changes in BZ receptors in the post-mortem brain of schizophrenic subjects, it became apparent that treatment with neuroleptics had an effect on the Bmax of binding sites in the schizophrenic group when they were separated into those who were neuroleptic-free Žfor at least 12 months. and those who were treated with neuroleptics until their death. Bmax of the BZ receptors was significantly lower in neuroleptic-treated schizophrenics, compared with drug-free schizophrenics and normal control subjects. The effect of neuroleptic treatment on BZ re-

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ceptors in the rat brain has beenstudied by Gavish et al. Ž1988.. Although they observed an upregulation of peripheral benzodiazepine receptors in the cerebral cortex, the heart, and the kidney after treatment with haloperidol or chlorpromazine, no significant change was observed in central BZ receptors in the cortex. These results thus suggest that neuroleptic treatment does not cause changes in the number of central BZ receptors. Our results that neuroleptic treatment may decrease BZ receptors in the post-mortem brain differ from those observed in rats by Gavish et al. Ž1988.. Several factors might account for this difference, including species differences, duration of treatment, and chronicity of illness. Another issue is the significance of these observations. Although it is generally believed that neuroleptics may exert their therapeutic effects primarily through their interaction with the dopaminergic system ŽBurt et al., 1977; Liskowsky and Potter, 1987., it is possible that they may also interact with other neurotransmitters or modulators and may thus alter more than one distinct CNS function. Whether changes in BZ receptors caused by neuroleptics are of any clinical consequence is not clear. It is quite possible that alterations in BZ receptors may have an indirect influence on the dopaminergic system, since it has been shown that the GABA system might modulate the dopaminergic system ŽGarbutt and Van Kammen, 1983.. It has been shown that BZ receptors are part of a benzodiazepinerGABA receptor-chloride ion channel complex ŽTallman et al., 1978; Haefely, 1989.. Therefore, changes in BZ receptors may have an indirect influence on GABA function in schizophrenic patients. GABA is believed to be associated with schizophrenic illness, as suggested by studies of GABA levels in the CSF of schizophrenic patients ŽGerner et al., 1984; Van Kammen et al., 1989.. This suggestion is also supported by the observation that neuroleptic treatment may cause changes in the GABA system. Zimmer et al. Ž1981. studied GABA levels in the CSF of 13 schizophrenic patients before and during treatment with neuroleptics. They observed that the levels of GABA in the CSF of schizophrenic patients during neuroleptic treatment were sig-

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nificantly higher as compared with levels during the drug-free period. It is unclear if changes in BZ receptors are related to such changes in CSF GABA levels, but this does suggest that the benzodiazepine-GABA system may be altered by neuroleptic treatment. In conclusion, our studies indicate that BZ receptors are up-regulated in the prefrontal cortex of suicide victims, specifically those who used violent means, and that previous exposure to neuroleptics may decrease central BZ receptors in the cortex of schizophrenic patients. Future studies should take into account patient histories of treatment with neuroleptics in the interpretation of BZ receptor studies regarding either psychiatric illness or suicidal behavior. Further studies with larger numbers of drug-free schizophrenics and schizophrenic suicides need to be carried out to delineate the relationship of BZ receptors to schizophrenia and suicide. Acknowledgements This work was supported by a grant RO1 MH 48153 to Dr. G.N. Pandey, and the brain collection was supported in part by the Grant MH 40279. We thank Ms. Rosemary Schmitt and Terri U’Prichard for their assistance in collection of psychological and demographic data. References Benes, F.M., Vincent, S.L., Alsterberg, G., Bird, E., Giovanni, G., 1992. Increased GABA A receptor binding in superficial layers of cingulate cortex in schizophrenics. Journal of Neuroscience 12, 924]929. Burt, D.R., Kamatchi, G.L., 1991. GABA A receptor subtypes from pharmacology to molecular biology. FASEB Journal 5, 2916]2923. Burt, D.R., Creese, I., Snyder, S.H., 1977. Antischizophrenic drugs: chronic treatment elevates dopamine receptor binding in brain. Science 196, 326]328. Cheetham, S.C., Crompton, M.R., Katona, C.L.E., Parker, S.J., Horton, R.W., 1988. Brain GABA A rbenzodiazepine binding sites and glutamic acid decarboxylase activity in depressed suicide victims. Brain Research 460, 114]123. Costa, E., Guidotti, A., 1996. Benzodiazepines on trial: a research strategy for their rehabilitation. Trends in Pharmacological Sciences 17, 192]200. Garbutt, J.C., Van Kammen, D.P., 1983. The interaction between GABA and dopamine: implications for schizophrenia. Schizophrenia Bulletin 9, 336]353.

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