unaltered number of brain serotonin uptake sites in suicide victims

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http://jop.sagepub.com/Journal of Psychopharmacology

http://jop.sagepub.com/content/6/4/509The online version of this article can be found at:

DOI: 10.1177/026988119200600406

1992 6: 509J PsychopharmacolAnna Andersson, Anders Eriksson and Jan Marcusson

Unaltered number of brain serotonin uptake sites in suicide victims

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Unaltered number of brain serotonin uptake sites in suicide victims

AnnaAndersson1,Anders Eriksson2 and Jan Marcusson31Department of Internal Medicine,2Department of Forensic Medicine, University of Ume, Ume, Swedenand3Department of Geriatric Medicine, University of Linkoping, Linkoping, Sweden

The 5-HT uptake sites were studied with[3H ] paroxetine as radioligand in frontal cortex, cingulate cortex and

hypothalamus from a control group (n=22) and a group ofsuicide victims (n = 19). The binding was also analysedwith regard to the method ofsuicide (violent or non-violent) and history of depressive symptoms. The apparentdissociation constant (Kd) was the same, 0.07-0.10 nM, and did not differ between the two groups studied. Themaximum number of binding sites(Bmax) for the controls were frontal cortex 11221, cingulate gyrus 22792and hypothalamus 699240 fmol/mg protein. TheBmax values for the suicide group were not different fromthose ofthe control group. When the binding parameters were analysed according to the method of suicide (violentor non-violent) there were no differences in comparison to the control group or between these two suicide groups.

Similarly, suicides with and without history of depression did not differ in[3H ] paroxetine binding and were notdifferent from the control group. The control and suicide groups were not different with respect to age and post-

mortem storage time. Considering 5-HT uptake sites as indirect markers for 5-HT terminals, these data suggest thatthe 5-HT terminal system is intact in the neocortex, the limbic system and in the hypothalamus in suicide victims.

Key words: human brain; suicide; 5-HT uptake site; [3H] paroxetine

Introduction

The antidepressant action of imipramine and other tricyclicagents has been suggested to be at least partly due to theinhibition of the re-uptake of 5-HT into the nerveterminals (Carlsson et al., 1969; Lapin and Oxenkrug,1969). The binding sites for these antidepressant agentshave been extensively studied by the use of several radio-

ligands such as [ 3H ] imipramine, [ 3H ] norzimeldine,[ 3H ] paroxetine and [ 3H ] citalopram (for a review, seeMarcusson and Ross, 1990). The 5-HT uptake sites havebeen studied in suicide victims on post-mortem human

brain tissue using [ 3H ] imipramine, but the data are notconsistent. The number of cortical and hippocampal[ 3H ] imipramine binding sites were reduced in suicide(Stanley, Virgilio and Gershon, 1982) and depression(Perry et al., 1983), whereas unaltered numbers of siteswere reported by others (Owen et al., 1986;Arora and

Meltzer, 1989).An increase in [ 3H] imipramine binding insuicide brains has also been reported (Gross-Isserhoff,Israeli and Biegon, 1989). These studies were performedwith [ 3H] imipramine which has been shown to be hetero-

genous in nature and to contain binding not related to the5-HT uptake site (Marcusson et al., 1985; Marcusson,Backstrom and Ross, 1986; Backstrom and Marcusson,1987). Therefore, the above-mentioned studies on [ 3H ] -imipramine binding and suicide may be difficult to evaluate

regarding changes in the density of 5-HT uptake sites.Since, however, these first studies on [ 3H] imipramine

binding and suicide have made a clear impact on

psychiatric neuropharmacology research, we felt it was

necessary to repeat the experiments with a technique which

selectively labels the 5-HT uptake site.Amore selective

radioligand for the 5-HT-uptake site is [ 3H ] paroxetine,which has been used in post-mortem human brain tissue

(Laruelle, Vanisberg and Maloteaux, 1988; Bdckstr6m,Bergstrom and Marcusson, 1989; Plenge, Mellerup and

Laursen, 1990;Andersson, Sundman and Marcusson,1992). During the progress of our study, Lawrence et al.

(1990) reported on [ 3H ] paroxetine binding in 10 brain

regions of depressed suicide victims and controls. They didnot find any differences in binding between the twogroups.

We chose to study three different brain regions with

respect to brain function and affective mechanisms. The

frontal cortex is a reference region (Stanley, Virgilio andGershon, 1982). The cingulate cortex is part of the limbic

system which is known to be involved in affective mech-anisms and the hypothalamus is well known in endocrinefunctions which may be altered in depressive conditions.

Materials and methods

Materials

[ 3H ] Paroxetine (22.3 Ci/mmol) was obtained fromNew England Nuclear, Boston, MA, USA. Citalopramhydrobromide was a gift from H. Lundbeck and Co.AS,

Journal of Psychopharmacology 6(4) (1992) 509-513 ~1992 BntishAssociation for Psychopharmacology

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Table 1 Demographic data and post-mortem time (meanvalues + SD)

n = number of cases. There were no significant differences in age andpost-mortem time between suicide and control.

Copenhagen, Denmark.All other reagents were standard

laboratory reagents of analytical grade wherever possible.

Brain samples

Post-mortem human brains from 41 subjects, (31 males

and 10 females), covering an age spectrum between 16and 75 years, were collected at autopsy. The collected

brains consisted of a control group with 22 subjects anda suicide group with 19 subjects.

The suicide victims were grouped according to themethod of suicide. Nine of the suicide victims used violentmethods such as hanging (n = 8) and shooting (n =1),whereas five died from drug intoxication and five fromcarbon monoxide poisoning. Seven of the suicide victimshad a history of depressive symptoms before death. Thisinformation was obtained in these patients case books

from a general practitioner or a psychiatrist. There were&dquo;~ no records of depressive illness in the other 12 cases.

The causes of death for the controls were ischemic heart

disease (IHD), pulmonary disease, injuries from car

accidents, accidental drowning and suffocation. Drugmedication prior to death was noted in four of thecontrols and in nine of the suicide victims, but

psychoactive drugs were only taken by the five individualsthat committed suicide by overdoses of the same drugsand by no one in the control group. Three of these fiveindividuals had taken tricyclic antidepressants incombination with benzodiazepines, one with benzo-

diazepines alone and one with benzodiazepines and

analgesics (paracetamol, dextropopoxifen). The brainsshowed no signs of macroscopical pathological changes.The time between death and autopsy ranged between 19and 72 h (see Table 1). The brains were placed on an

ice-tray immediately after removal from the skulwhereafter dissection was performed on unfrozen tissu

Samples were taken from the frontal cortex, the cingulagyrus and the hypothalamus and were then stored in ai

tight plastic vials at - 80C until use.

Tissue preparation

The brain tissue was homogenized in 20 ml of ice-cobuffer (50mM Tris-HCI, 120mM NaCI, 5 mM KCIpH 7.4) using a Kinematica Polytron homogenize(Lfzem Switzerland) at setting 6 for 7 s.After centrifugtion (48 000 g, 10 min, 4C) the pellet was homogenizein the Tris-buffer and, following centrifugation (48 000

10 min, 4C), resuspended in the buffer to a fintissue concentration of 40-90 lg of protein/m( ~ 2 mg wet wt/ml) in the binding assay.

[ 3H ] Paroxetine binding

The homogenates were incubated for 60 min at 25 C wi

[3H]paroxetine in the presence or absence of 10 citalopram in a total volume of 1600 ~1.After the additiof 6 ml of ice-cold buffer, the homogenates were rapidfiltered through Whatman GF/C filters using a 2channel cell harvester (Brandel, Gaithersburg, MDUSA). Finally the filters were washed with three 6-mrinses of ice-cold buffer. The radioactivity trapped by tfilters was determined by liquid scintillation spectroscopSix concentrations of [ 3H ] paroxetine ranging from 0.to 0.5 nM were used. Specific binding was defined as th

displaced by 10 itM citalopram. Protein concentrationwere estimated by the method of Markwell et al. (1978

Data analysis and statistics

Values for binding maximum (8~9 and apparent affini(Kd) were calculated from Scatchard plots where t

regression lines were determined by least-squares regressianalyses. When presented as mean values, the Bm~ valuare arithmetic + SD and Kd geometric (range) valueStudents t-test (two-tailed) was used for the overa

comparison between the control and suicide groups

Table 2 [ 3H ] Paroxetine binding sites in controls and suicide victims

The maximum number of binding sites, Bmax (fmol/mg protein) and apparent binding affinity, Kd (nM) are

presented. The data represent mean values + SD and mean (range) values. n = number of individuals. Two-tailedStudents t-test was used for testing mtergroup differences and the p-values (suicide versus control) are given.



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Figure 1 [3H] ] Paroxetine binding in a typical experiment on human frontal cortex of a control and suicide sample.Six concentrations of [H ] paroxetine ranging from 0.01 to 0.5 nM were used. Specific bmdmg was defined asthat displaced by 10 gM citalopram. In the upper section total, non-specific and specific binding are illustrated,in the lower section there are Scatchard plots of the specific bmding. Indicated are 2~ values (fmol/mg protein),Kd values (nM) and the correlation coefficient (r) .

When testing differences between subgroups of suicides,three and four group models including controls were usedand the intergroup differences were checked by analysisof variance (single factor factorialANOVA) usingthe computer program Statview Graphics . If theoverall F-test was not significant, subgroup differences

were neglected. The level of significance was definedas p < 0.05.

Results

[ 3H ] Paroxetine binding was clearly saturable with Kd of0.07-0.10 nM in the regions studied (Table 2, Fig. 1). The

binding capacity (Bmax) was around 100 fmol/mg proteinin the frontal cortex, 2-fold higher in the cingulate gyrusand 6-fold higher in the hypothalamus. There were no

differences in either Bmax or Kd between the two groupsin any of the brain regions studied (Table 2).The binding parameters were also analysed according

to the method of suicide. There were no differences

in either Bmax or Kd between any of the groupstested (Table 3). Also the suicide victims with a

history of depressive symptoms were analysed separately.They did not show any differences in either Bmax or Kdin comparison with control or with non-depressivesuicides.

Five of the suicide victims were on psychoactivetreatment prior to death. The [ 3H ] paroxetine bindingfor this group was not different from the other suicides

or controls (p>0.05 single factorANOVAfor both

Bmax and Kd in all three brain regions). There were nodifferences in age or post-mortem storage time between

the control and suicide groups (Table 1).



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Table 3 [ 3H ] Paroxetine binding sites in controls and suicide victims

The maximum number of binding sites, B&dquo;,ax (fmol/mg protein) and apparent binding affinity, Kd (nM) are presented. The data represent mevalues SD and mean (range) values. n = number of individuals. Differences between controls and subgroups of suicides, were checked by analyof variance (single factor factorialANOVA). If the overall F-test was not significant, subgroup differences were neglected. Indicated are

p-values for the F-test

DiscussionThe association of inhibition of 5-HT uptake and anti-

depressent effect has led to numerous radioligand bindingstudies on the 5-HT transporter both in brain and platelets(for a review, see Langer et al., 1987). Since the reports ofdecreased numbers of ( 3H ] imipramine binding sites in thecortices of suicide victims (Stanley, Vergitio and Gershon,1982; Stanley, Mann and Gershon, 1983) the interest inthe role of the 5-HT synapse in depressive disorders hasbeen even more increased. Even though these data wereconfirmed by Perry et al. (1983), later studies did notdetect any changes in ~ax (Owen et al., 1986;Arora and

Meltzer, 1989). Since [ 3H ] paroxetine has a several-fold

higher proportion of specific binding and a 100-fold higheraffinity (Backstrom, Bergstr6m and Marcusson, 1989;Marcusson et al., 1988a) in comparison with [ 3H ] imipra-mine, it was of interest to repeat the suicide-control studyon the 5-HT uptake site using this improved technique.

During the progress of tissue collection for the presentstudy, Lawrence et al. (1990) published a thorough studyof [ 3H ] paroxetine binding in suicide victims, demons-trating no differences in binding parameters betweenviolent and non-violent suicide victims. The findings in our

study confirm the data on [ 3H ] paroxetine binding

reported by Lawrence et al. (1990). Thus, there were nodifferences in either Bm~ or Kd between suicide victims andcontrols nor between violent and non-violent suicides or

between depressive and non-depressive suicides. In additionto frontal cortex, we chose to study cingulate cortex and

hypothalamus, as examples of brain regions with limbicand neuroendocrine brain functions. The choice of the three

functionally different brain regions did not result in anydifferences between the regions in the suicide-control data.

Seven of the suicide victims had records of depressivesymptoms before death. We found no alterations in

~maxbetween

depressiveand

non-depressivesuicides n

between depressive suicides and controls. These resulare in line with those of Owen et al. (1986) and Lawrenet al. (1990), who reported that suicide victims with

history of depression did not represent a subgroup wialtered binding values.The binding values were also compared between tho

victims who had received psychoactive drugs and thowho had not. It appeared also that this parameter dnot influence the binding.

In conclusion [3H]paroxetine binding to the 5-H

uptake sites in the frontal cortex, the cingulate cortex anthe hypothalamus is not altered in suicide victims. N

differences in binding were noted between violent annon-violent methods of suicide, even though suicivictims dying from carbon monoxide poisoning had low

Bmax values. Considering that [ 3H ] paroxetine bindilabels the neuronal 5-HT uptake sites and reflects 5-Hterminal density (Bdckstr6m, Bergstr6m and Marcusso1989), the 5-HT uptake sites can be regarded as structurmarkers for 5-HT terminals. Therefore, it is conceivab

that 5-HT terminals are intact in suicide victims. Stabiliof the 5-HT uptake sites has also been indicated by the fathat the 5-HT uptake sites are not up- or down-regulatafter antidepressant therapy (Marcusson, Backstrom an

Ross, 1988b). Considering the data reported by Lawrenet al. (1990) it appears that the previous findings ofdecreased number of [ 3H ] imipramine binding sitessuicide should be questioned.

Acknowledgements

J. M. was supported by the Swedish Medical ResearCouncil (B92-12X-08648-04B). This study was founded bgrants from the Stohne Foundation, the Swedish Socie



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for Medicine, Centrala Frsksdjursnmnden, GamlaTjanarinnor and the County Councils of Northern Sweden.

Address for correspondence

Dr Jan Marcusson

Departmentof Geriatric Medicine

University of LinkbpingVallaragen 6S-582 46 Link6pingSweden

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unaltered number of brain serotonin uptake sites in suicide victims

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