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imidazoline receptors in suicide victims: a postmortem brain autoradiographic analysis
European Neuropsychopharmacology 10 (2000) 265–271 www.elsevier.com / locate / euroneuro
Unaltered a 2 -noradrenergic / imidazoline receptors in suicide victims: a postmortem brain autoradiographic analysis a, b a a Ruth Gross-Isseroff D.Sc. *, Abraham Weizman M.D. , Sheila J. Fieldust B.A. , Malka Israeli B.A. , a,c Anat Biegon Ph.D. a
b
Department of Neurobiology, The Weizman Institute of Science, 76100 Rehovot, Israel The Geha Psychiatric Hospital, Felsenstein Medical Research Center, Beilinson Medical Center, Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel c Pharmos Ltd., Kiryat Weizmann, Rehovot, Israel Received 13 January 1999; accepted 29 February 2000
Abstract In vitro quantitative autoradiography of a 2 -adrenergic / imidazoline receptors, using [ 125 I]iodoclonidine as a ligand, was performed on 24 human brains postmortem. Twelve brains were obtained from suicide victims and 12 from matched controls. We found no significant, region-dependent alterations in the density of a 2 -adrenergic receptors in brains of suicide victims as compared to matched controls. We also report age-dependent reductions in binding in the prefrontal cortex and hippocampus, as well as significant recent alcohol ingestion-dependent reductions in binding in the prefrontal cortex. Sex and time from death to autopsy did not affect iodoclonidine binding in our sample. 2000 Elsevier Science B.V. All rights reserved. Keywords: a 2 -Adrenergic receptors; Autoradiography; Human brain; Suicide; Alcohol; Aging
1. Introduction The central noradrenergic system has been implicated in the pathophysiology of a variety of psychopathologies (e.g., Crow et al., 1984; Shimohama et al., 1986). It has been of particular interest in the context of depression, and especially in studies using the brain of suicide victims as a model of depression, which were inspired by the catecholaminergic hypothesis of affective disorders (Schildkraut, 1965). The a 2 -adrenergic receptor was extensively studied. Six reports described elevated binding to these receptors in suicide brains (Ferrier et al., 1986; Meana and GarciaSevilla, 1987; De Paermentier et al., 1991a; Meana et al., 1992; Gonzales et al., 1994; Ordway et al., 1994). Another report has found a decrease in binding to these receptors (Crow et al., 1984) while three others failed to demonstrate *Corresponding author. Department of Molecular Genetics, Ullmann Building, The Weizman Institute of Science, 76100 Rehovot, Israel. E-mail address: [email protected] (R. Gross-Isseroff)
significant differences between the binding of these receptors in suicide brains as compared to controls (Arango et al., 1993; Sastre and Garcia-Sevilla, 1993; Callado et al., 1998). More specifically, Ferrier et al. (1986) studied single samples from prefrontal cortex in a total of 15 brains obtained from a variety of mood disorder patients none of whom died by committing suicide, and 11 of whom had a history of antidepressant medication up to the time of death. Meana and Garcia-Sevilla (1987) studied five violent suicide victims who had a diagnosis of major depressive disorder, two of whom had a history of antidepressant treatment at the time of death. De Paermentier et al. (1991a) studied about 15 brains of drug-free depressed suicide victims and reported an elevation of 24% in receptor density in temporal cortex. The same group also showed a lack of effect of antidepressants on binding in prefrontal cortex, which may strengthen the results of the previously quoted groups De Paermentier et al. (1991b). Meana et al. (1992) studied samples of 41 drugfree violent suicide victims diagnosed as a mixture of mood disorders and reported no significant differences in
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clonidine binding but elevated UK14304 binding in prefrontal cortex of a group of 19 suicides and cerebellar cortex in a group of 10 suicides. Their experiments seem to indicate a presynaptic supersensitivity of the high affinity state of the a 2A receptor in suicide. This study was further expanded (Gonzales et al., 1994) and its neuroanatomical resolution refined by the use of quantitative autoradiography using UK14304 as a ligand in 17 suicide brains of various diagnostic groups. Molecular layer of CA1 and outer layers of prefrontal cortex showed significant elevations in binding. Ordway et al. (1994) found autoradiographically higher binding to the a 2A receptor in the locus coereleus, where this receptor is an autoreceptor, of a group of 10 suicide victims. Arango et al. (1993) studied in autoradiographic detail the clonidine binding in prefrontal cortex of 12 suicide victims, and though they found trends of reduction they failed to reach statistical significance. Crow et al. (1984), using rauwloscine as a ligand found a decrease in binding in seven to eight suicide brains judged to be depressed by clinical postmortem diagnostic evaluation. Thus, the total number of brains for which the data are presented in detail is about 60, for 40 of which increased densities of a 2 receptors were reported while for the other 20 reduced densities of these receptors were observed. One of the controversies in the literature concerns adequacy of using phentolamine to define nonspecific binding in the presence of clonidine and other imidazolinelike substances, as phentolamine binds to imidazoline binding sites and these are therefore added to the a 2 high affinity specific binding. One solution to this controversy is that both clonidine type substances and phentolamine pick up a maximum of 5% of the imidazoline binding sites in the human brain and most of the effects reported about suicide brains are much larger than that, even when they fail to reach statistical significance. Another solution was recently suggested which claims that a 2 receptors are unaltered in brains of suicide victims but imidazoline binding sites are decreased in these brains (Sastre and Garcia-Sevilla, 1997). Another controversy in the literature stems from the exact type of diagnosis of the patients studied. As we and others (for a review, see Gross-Isseroff et al., 1998) have previously stated suicide appears to be a type of behaviour that cuts across diagnostic borders and is possibly a bad model for studying mood disorders. Finally, some of the controversy in the existing literature may have stemmed from lack of uniformity in neuroanatomical definitions between the different reports. The present experiment is part of a larger project, portions of which have been previously published, in which as many monoaminergic receptors and binding sites as possible were mapped autoradiographically in a pool of suicide and control brains on consecutive coronal sections taken at the level of the prefrontal pole (similar to the sections employed by Arango et al., 1993) and at the level of the
frontoparietal transition (cf. Gross-Isseroff et al., 1988, 1990a,b,c). As none of the studies in the field were guided by specific hypotheses as to the nature of the brain regional neurochemical defect in suicide, as such a hypothesis does not exist, we thought it would be worthwhile to examine as large a sample of neuroanatomical regions as possible. Such purely exploratory phenomenological studies may lead in the future to the creation of refutable neuroanatomical hypotheses, especially since the forebrain adrenergic innervation in the primate brain has begun to be unravelled (Morrison et al., 1982; Zecevic and Verney, 1995).
2. Experimental procedures
2.1. Subjects Brains of 24 neuropathology-free subjects were obtained at autopsy from the New York City (NY) medical examiner and from the Institute of Forensic Medicine at Jaffa (Israel). Subjects with a known history of schizophrenia, alcoholism or drug abuse, according to their medical records, were excluded. Twelve subjects were suicide victims and 12 matched controls. Matching of subjects was done for sex, age, postmortem delay and, whenever possible, cause of death. For example, a jump from height was matched to a fall from height. Mean age (6S.D.) of the subjects was 42.67 (622.76) years, ranging from 15 to 81 years in the control group and 42.25 (621.63), range 14–72 in the suicide group. Postmortem delay (time from death to autopsy) ranged from 8 to 48 h with a mean (6S.D.) of 24.58 (612.7) in the control group and 5–45 h with a mean (6S.D.) of 23.08 (611.2) in the suicide group. Each group included seven males and five females. Suicide was committed by jumping from heights (three cases), hanging (three cases), suffocation (two cases), gunshot (one case) and unknown means (three cases). Although the means of suicide was unknown in these cases it was clear that they committed suicide from suicide letters they have left. Cause of death was most probably from overdose of agents undetectable in routine toxicological examination. Cause of death, in the control group, included multiple trauma (six cases), burns (one case), gastrointestinal bleeding (one case), heart failure (two cases), drowning (one case), and food poisoning (one case). Multiple trauma deaths were due to accidents. According to the available medical records the controls were free of mental disorders, including alcoholism. Toxicological tests were performed at autopsy, screening for alcohol, barbiturates, analgetics, phenothiazines and salicylates. To the best of our knowledge, based on toxicology as well as medical records, the subjects were free of drugs which might interfere with the adrenergic system. Presence of tricyclic antidepressants was determined in brain tissue by radioreceptor assay. In four subjects alcohol was detected in blood tests at autopsy. Levels of alcohol were 14, 21, 34
R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271
and 71 mg% (female suicide aged 72 years, male control aged 61 years, male control aged 52 years and male control aged 25 years, respectively). These and all other brains used in the study were otherwise drug free. Freezing storage time of the brains was 3 months to 2 years.
2.2. Preparation of sections Brains were removed from the cranium, cut into approximately 2.5-cm coronal blocks which were immediately frozen over dry ice and kept at 2708C. Coronal sections, 40-mm thick, cut on a Bright whole body cryotome at 2158C, were thaw mounted onto gelatin-coated glass slides and kept overnight at 2208C.
2.3. Autoradiography Slides were preincubated for 20 min, at 258C, in 170 mM Tris–HCl buffer, pH 7.6. They were then incubated for 60 min, at 258C with 0.5 nM [ 125 I]iodoclonidine (New England Nuclear, Boston, MA, specific activity 2200 Ci / mmol) in 170 mM Tris–HCl buffer, pH 7.4, containing 0.01% ascorbic acid and 10 mM MgCl 2 . Non-specific binding was determined in the presence of 100 mM phentolamine. A 10-min wash period in 170 mM Tris–HCl ice-cold buffer, pH 7.6, followed the incubation. These conditions label both a 2 -adrenergic receptors and imidazoline binding sites. Slides were then dipped in ice-cold water to remove buffer salts, dried on a 508C hot plate and apposed against tritium-sensitive film (Amersham [ 3 H]Hyperfilm) for 6 h. The film was then developed and fixed with Kodak developer and fixer. Sections were stained with cresyl violet for anatomical reference and several neuroanatomical texts (Barr, 1974; DeArmond et al., 1974; Carpenter, 1976; Nieuwenhuys et al., 1981) were used for identification of structures. All sections were obtained from the right cerebral hemisphere, at the level of the amygdala or hippocampus, head of the caudate nucleus and at the level of prefrontal cortex. Typically four consecutive sections from each brain and anatomical level were used, two for the determination of total binding and two for the determination of nonspecific binding. Experiments were run in a matched pair design so that each one included sections from a suicide brain and its matched control.
2.4. Quantitative analysis of the autoradiograms Autoradiograms were analyzed with the aid of an IBMPC based computerized image analysis system with a PC-vision digitizing board (Imaging Technology, Israel) and customized software. Standard curves for converting gray level readings into ligand concentration were obtained by exposing film to commercial (Amersham) 125 I standards for 6 h. Non-specific binding was subtracted from total binding, to obtain specific binding. Both total and non-
267
specific binding were measured in duplicate in each region, for each brain.
2.5. Homogenate binding assays Saturation binding experiments were performed using tritiated para-amino clonidine (NEN, specific activity, 58.2 Ci / mmol). We used a different ligand than the one used for autoradiography in order to increase validity. Brain samples, obtained from the superior frontal gyrus, were homogenized in 100 volumes of 170 mM Tris–HCl buffer, pH 6.0, and centrifuged for 20 min at 40 0003g. The procedure was repeated and the pellet resuspended in 10 ml / 100 mg original wet weight of incubation buffer: 170 mM Tris–HCl, 10 mM MgCl 2 and 0.01% ascorbic acid. Tissue homogenate samples (800 ml) were incubated with increasing concentrations of radioligand (0.2–12 nM final concentration, 100 ml) for 30 min at 258C. Non-specific binding was assessed in the presence of excess clonidine (100 mM final concentration, 100 ml). Total and nonspecific binding were assayed in triplicate for each concentration. Samples were filtered under vacuum through Whatman GF / B filters and washed in 335 ml ice-cold Tris–HCl buffer. Filters were counted in Packard liquid scintillation counter. The results of saturation experiments were subjected to Scatchard analysis to estimate Kd and Bmax .
2.6. Statistical analyses Statistical analyses were performed using SAS software. Two-tailed student t-tests for independent samples, twoway analyses of variance (age3group, age3sex) corrected for unbalanced designs and Pearson coefficients of correlation were used as appropriate. a was preset at P50.05.
3. Results
3.1. Autoradiography The distribution of iodoclonidine binding in the various neuroanatomical regions examined is shown in Table 1 for young and old subjects. The highest level of binding occurs in portions of the prefrontal cortex and the dentate gyrus. Less binding was found in other cortical areas and the hippocampus. Low binding was observed in the basal ganglia and thalamus, and almost no binding was found in the white matter. The binding in the cortex was heterogeneous as well as being characterized by a unique pattern of lamination. There were two visible bands: an outer dark band overlaying cortical layers I–III, and a lighter inner band overlaying cortical layers IV–VI. The effect of alcohol was tested by two-tailed t-tests, and significant reductions in iodoclonidine binding in brains taken from subjects in which alcohol was detected
R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271
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Table 1. Continued
Table 1 The dependence of a 2 receptor distribution on age a Region
Frontoparietal gyri: Cingulate Internal band External band Precentral Internal band External band Postcentral Internal band External band Insula Internal band External band Transverse temporal Internal band External band Superior temporal Internal band External band Middle temporal Internal band External band Inferior temporal Internal band External band Lateral occipitotemporal Internal band External band Parahippocampal Internal band External band Basal ganglia Caudate nucleus: Head Tail Putamen
Old (n)
r(P)
30.065.0 (10) 26.564.5 (10) 33.065.7 (10) 25.764.9 (9) 23.664.4 (9) 26.865.6 (9) 24.364.7 (9) 22.364.2 (9) 25.564.7 (9) 24.063.7 (9) 21.362.9 (9) 26.264.2 (9) 23.064.7 (8) 22.564.8 (8) 23.464.8 (8) 31.965.9 (8) 30.666.1 (9) 31.265.5 (9)
13.364.1*(10) 11.363.2*(10) 14.464.4*(10) 11.963.7 (9) 11.663.7 (9) 12.263.6 (9) 14.765.9 (9) 14.565.8 (9) 14.165.6 (9) 15.264.9 (9) 14.564.4 (9) 15.865.4 (9) 14.164.7 (9) 14.064.2 (9) 15.464.5 (9) 13.363.2*(10) 11.662.8*(10) 16.064.2*(10)
18.163.5 16.263.0 20.163.8 19.862.7 16.661.1 21.464.8 16.663.1 14.562.6 17.663.4 17.766.3 17.066.3 17.564.9 16.662.7 10.861.4 17.963.6 14.862.4 13.061.9 15.562.4 13.362.4 12.262.2 14.062.8 13.562.7 13.162.3 14.062.9
(5) (5) (5) (3) (3) (3) (6) (6) (6) (4) (4) (4) (5) (5) (5) (7) (7) (7) (6) (6) (6) (6) (6) (6)
12.762.3 12.463.2 12.862.5 9.663.2 8.263.0 10.463.0 8.762.3 8.062.3 8.962.3 14.261.1 13.760.9 14.261.4 11.261.4 10.861.6 10.961.7 10.261.4 9.361.3 10.761.6 9.661.8 9.561.7 9.361.9 10.561.3 10.361.3 10.561.1
(3) (3) (3) (3) (3) (3) (4) (4) (4) (3) (3) (3) (6) (6) (6) (5) (5) (5) (4) (4) (4) (6) (6) (6)
15.062.9 14.262.8 15.162.8 13.362.5 12.462.2 14.361.8
(7) (7) (7) (5) (5) (5)
10.961.8 11.062.0 10.961.8 10.962.1 10.761.8 11.662.1
(4) (4) (4) (5) (5) (5)
6.860.8 (10) 7.861.3 (6) 7.760.6 (10)
7.160.7 (10) 4.060.8*(5) 7.860.5 (8)
20.50(0.03) 20.53(0.02) 20.51(0.02) 20.49(0.04) 20.47(0.05) 20.49(0.04)
10.260.3 (3)
6.661.7 (5)
Old (n)
Hippocampus CA fields: Molecular layer Pyramidal layer
12.461.6 (6) 12.061.8 (6)
Dentate gyrus: Granular layer Molecular layer Hilus Subiculum
20.563.4 12.861.8 12.362.1 11.361.1
White matter
(7) (7) (6) (6)
6.060.9 (10)
r(P)
7.661.2*(8) 6.960.8*(8)
20.69(0.006) 20.66(0.01)
10.161.6*(8) 7.461.0*(8) 8.061.7 (8) 8.361.4 (8)
20.66(0.008) 20.64(0.01)
5.460.6 (10)
a
This table presents mean6S.E.M of specific [ 125 I]iodoclonidine binding in n brains, expressed as fmol / mg tissue. The sample was divided into two age groups at the median age of 35. The resulting young group had a mean (6S.D.) age of 21.1 (66.0) years, and the old group had a mean (6S.D.) age of 59.8 (612.4) years. *P,0.05 in a two-tailed t-test. r, Pearson coefficient of correlation between iodoclonidine binding and age. 20.62(0.004) 20.63(0.004) 20.53(0.02)
20.67(0.04) 20.64(0.05) 20.65(0.04)
at autopsy were observed in a number of regions (Table 2). The prefrontal cortex was most affected by alcohol, as well as the whole cingulate cortex. These four brains were discarded from further analyses of the data. The effect of age on iodoclonidine binding was tested by computing the coefficient of correlation between binding and age. These coefficients were mostly negative and in several regions highly significant (Table 1). The regions most affected by age were the prefrontal cortex and hippocampus. The tail of the caudate nucleus also exhibited remarkable decrease in binding with age. Since we found a remarkable age effect, the effect of suicide was tested by two-way analyses of variance (age3 Table 2 The dependence of a 2 receptors in the neocortex on alcohol a
20.65(0.03)
Region
No alcohol
Alcohol
P
Cortex Prefrontal gyri: Superior frontal Internal band External band Inferior frontal Internal band External band Orbital Internal band External band Cingulate Internal band External band
15.262.5 (20) 13.262.0 (20) 16.762.9 (20) 13.762.5 (18) 13.062.3 (18) 13.962.5 (18) 13.662.0 (18) 12.461.6 (18) 14.562.3 (18) 15.362.5 (19) 14.562.5 (19) 15.962.4 (19)
4.160.7 (4) 4.060.6 (4) 4.060.8 (4) 2.861.0 (4) 3.160.9 (4) 3.161.1 (4) 4.061.1 (4) 4.161.1 (4) 4.561.2 (4) 3.161.1 (4) 3.161.0 (4) 2.961.3 (4)
0.0004 0.0003 0.0004 0.0006 0.0007 0.0008 0.0006 0.03 0.001 0.0002 0.0004 0.0001
Frontoparietal level: Cingulate Internal band External band
16.162.4 (8) 14.862.2 (8) 17.462.8 (10)
6.562.2 (4) 5.661.5 (3) 6.362.2 (3)
0.03 0.02 0.03
a
Thalamus Medial nucleus
Mean6S.E.M. Young (n)
Mean6S.E.M. Young (n)
Cortex Prefrontal gyri: Superior frontal Internal band External band Middle frontal Internal band External band Inferior frontal Internal band External band Orbital Internal band External band Rectus Internal band External band Cingulate Internal band External band
Region
This table presents mean6S.E.M. of specific [ 125 I]iodoclonidine binding in n brains, expressed as fmol / mg tissue. P, probability of t, in a two-tailed t-test for independent samples.
R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271 Table 3 The effects of suicide and age on a 2 receptors in the human brain postmortem a Region
(Suicide / control) 3100
(Old / young) 3100
Cortex Prefrontal gyri: Superior frontal Internal band External band Middle frontal Internal band External band Inferior frontal Internal band External band Orbital Internal band External band Rectus Internal band External band Cingulate Internal band External band
131 129 145 118 107 135 114 113 122 101 91 106 101 101 108 127 135 124
44 43 44 46 49 46 69 65 55 63 68 60 61 62 66 42 38 51
Frontoparietal gyri: Cingulate Internal band External band Precentral Internal band External band Postcentral Internal band External band Transverse temporal Internal band External band Superior temporal Internal band External band Middle temporal Internal band External band Inferior temporal Internal band External band Lateral occipitotemporal Internal band External band Parahippocampal Internal band External band
65 65 71 52 50 51 61 63 59 83 95 82 91 95 93 87 88 84 104 105 106 76 79 78 92 101 91
70 77 64 48 49 49 52 55 51 67 100 61 69 72 69 72 78 66 78 79 75 73 77 72 82 86 81
Basal ganglia Caudate nucleus: Head Tail Putamen Claustrum
94 68 100 157
104 51 101
90 58 64 37
65
Thalamus Medial nucleus Posterior lateral Pulvinar Lateral geniculate body
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Table 3. Continued Region
Hippocampus CAI field Pyramidal layer CAIV field Molecular layer Pyramidal layer
(Suicide / control) 3100
(Old / young) 3100
69 96 86
61 58
Dentate gyrus: Granular layer Molecular layer Hilus Subiculum
74 66 82 101
49 58 65 73
White matter
108
90
a
This table presents the effects of suicide (left) and age (right) on iodoclonidine specific binding as percent of group means, where the young and old group were created as described in legend to Table 1. No significant differences between suicide and control were obtained in any of the examined brain regions.
group). Using these analyses no significant effects of suicide or interactions were found. The lack of statistical significance is probably due to the small number of brains in each age3group cell. As we have noticed non-significant differences between the suicide and control groups we have listed the suicide / control ratio in binding in Table 3. This presentation clarifies the magnitude of the results (left column), in comparison to the magnitude of the results for the age effect (right column). The difference between the two effects is most pronounced in the prefrontal cortex, where suicide induces elevations in binding while aging induces reductions. The effect of sex was tested in similar analyses of variance (age3sex). No effect of sex or interactions were found. The effect of postmortem delay was tested by computing the coefficient of correlation between the postmortem delay and specific binding. No effect of postmortem delay was found.
3.2. Homogenate binding In order to assess whether the observed differences in the autoradiographic result were due to changes in Kd or Bmax , we analyzed the superior frontal gyrus homogenate saturation binding experiments accordingly. The effect of recent alcohol ingestion was tested by a two-tailed t-test for independent samples. Bmax for the ‘alcohol present’ group (n53) was 22.45 fmol [ 3 H]p-amino clonidine / mg protein, whereas for the ‘alcohol absent’ group (n58) it was 42.97 fmol 3 H-labelled para-amino clonidine / mg protein. This difference is statistically significant (P5 0.03). No differences between the two groups were observed for Kd (‘alcohol present’ Kd 54.63 nM; ‘alcohol absent’ Kd 53.49 nM). The effect of age on Kd and Bmax
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R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271
was tested by Pearson coefficient of correlation in the alcohol-free group. The resulting correlation between age and Bmax was 20.35 and failed to reach statistical significance (P50.39) probably because of the small number of cases tested. No effect of age on Kd was found. The possible effects of group (control versus suicide) and sex on Kd and Bmax were tested by two-tailed t-tests for independent samples and no significant effects were found. The effect of postmortem delay on Kd and Bmax was tested by Pearson coefficient of correlation between the variables and no significant effects were found.
4. Discussion We have presented three major findings: brain a 2 -adrenergic receptors do not appear to be altered in suicide victims, age-related reductions in a 2 -adrenergic / imidazoline receptors in the human brain, and recent alcohol ingestion-related reductions in these receptors in the human brain. In addition we have presented the most detailed neuroanatomical distribution map of the density of these receptors to date. The age-related reduction in the density of these receptors is in contrast to a previous publication (Meana et al., 1989) and in agreement with others (Sastre and GarciaSevilla, 1993; Sastre and Garcia-Sevilla, 1994). The a 2 adrenergic receptor is but one of a series of monoaminergic receptors displaying such an age-related reduction in the human brain (Gross-Isseroff and Biegon, 1988; GrossIsseroff et al., 1990b; Dillon et al., 1991) and may be connected to age-related gene expression governed by other neurohumors such as steroid hormones (Jones et al., 1983; Jhnwar-Uniyal and Leibowitz, 1986; Karkanias and Etgen, 1995). The effect of alcohol presence on these receptors has not been reported before. There are a number of monoaminergic receptors affected by alcohol in the human brain, and the mechanism underlying this effect has yet to be elucidated (Gross-Isseroff and Biegon, 1988; Dillon et al., 1991). Due to the limited number of brains in this study, the effect of alcohol ingestion on a 2 -adrenergic / imidazoline receptors in the human brain warrants further investigation. In brains of suicide victims we noted a non-significant tendency toward elevated binding (roughly 20–45%) in most regions of prefrontal cortex, except regions of the base of the brain (orbital and rectus gyri). This is in agreement with others (Ferrier et al., 1986; Meana and Garcia-Sevilla, 1987; De Paermentier et al., 1991a; Gonzales et al., 1994; Callado et al., 1998). We have also noticed a trend toward reduced binding in all other cortical and subcortical regions represented in our sections. This is in agreement with Arango et al. (1993) and Crow et al. (1984). As none of the previous studies examined a complete pool of the neuroanatomical regions reported here, and most do not report results which fail to reach
statistical significance, there are discrepancies between the present report and previous ones. These may stem from either differences in ligand or neuroanatomical definitions. As we have previously stated, a trend meta-analysis of the pooled worldwide data should be interesting. It has to be borne in mind, though that the a 2 receptor is but one of a series of markers which can be monitored in suicide, and that the adrenergic system which it monitors is but one of a multiplicity of neurotransmitter systems which constantly interact with each other. The a 2 receptor has a regulatory function in norepinephrine and serotonin release and thus changes in its density may cause changes in neurotransmitter release. Changes in norepinephrine release may, in turn, affect other monoaminergic systems of neurotransmission. Indications of the existence of such interactions have been amply documented in animal studies (e.g., Fuchs et al., 1996; Gobert et al., 1998). a 2 -Adrenergic receptors are both pre- and post-synaptic in the central nervous system (for reviews, cf. Berlan et al., 1992; Bylund, 1992) and coexpressed on serotonergic as well as other neurons (for a review, cf. Mahoney and Seeley, 1990). The method used in the present experiment cannot differentiate between the pre- and post-synaptic sites and neither can it differentiate between the adrenergic versus other neurones (heteroreceptors). This limitation has to be borne in mind, and the present results treated with the appropriate caution. Further experimentation, possibly using in situ hybridization methods (e.g., Jones and Palacios, 1991) will probably shed further light on the possible role of a 2 receptors and heteroreceptors, in concert with others, in suicidal behaviour. Two possible caveats should be borne in mind when interpreting the results presented here. The first is the choice of ligands. Under the conditions of the present experiment both a 2 receptors and imidazoline binding sites were probably labeled. The second is the probable diagnostic non-uniformity of the brains obtained from suicide victims. Suicide victims suffer from a variety of axis I diagnoses such as: major depression, psychoactive substance use disorders, schizophrenia, bipolar disorder and other disorders or a combination of disorders. Some of the control group brains may have gone undiagnosed, suffering from axis I disorders which overlap the ones characterizing the suicide group. This may have masked any differences in a 2 binding between the groups. It would be of interest in the future to study brains of suicide victims with a known psychiatric history. An ideal study would include groups of suicide plus major depression, suicide plus schizophrenia, suicide without suicide and healthy controls. Such a design, where feasible, would facilitate the extraction of the ‘suicide effect’.
Acknowledgements This study was supported by a grant from the US–Israel Binational Science Foundation to Anat Biegon.
R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271
References Arango, V., Ernsberger, P., Sved, A.F., Mann, J.J., 1993. Quantitative autoradiography of a 1 - and a 2 -adrenergic receptors in the cerebral cortex of controls and suicide victims. Brain Res. 630, 271–282. Barr, M.L., 1974. In: The Human Nervous System. An Anatomical Viewpoint, Harper and Row, New York. Berlan, M., Montastruc, J.L., Lafontan, M., 1992. Pharmacological prospects for alpha 2 -adrenoceptor antagonist therapy. Trends Pharmacol. Sci. 13, 277–282. Bylund, D.B., 1992. Subtypes of a 1 - and a 2 -adrenergic receptors. FASEB J. 6, 832–839. Callado, L.F., Meana, J.J., Grijalba, B., Pazos, A., Sastre, M., GarciaSevilla, J.A., 1998. Selective increase of alpha2A-adrenoceptor agonist binding sites in brains of depressed suicide victims. J. Neurochem. 70, 1114–1123. Carpenter, M.B., 1976. In: Human Neuroanatomy, 7th Edition, Williams and Wilkins, Baltimore. Crow, T.J., Cross, A.J., Cooper, S.J., Deakin, J.F.W., Ferrier, I.N., Johnson, J.A., Joseph, M.H., Owen, F., Poulter, M., Lofthouse, R., Corsellis, J.A.N., Chambers, D.R., Blessed, G., Perry, E.K., Perry, R.H., Tomlinson, B.E., 1984. Neurotransmitter receptors and monoamine metabolites in the brains of patients with Alzheimer-type dementia and depression and suicides. Neuropharmacology 23, 1561– 1569. DeArmond, S.J., Fusco, M.M., Dewey, M.M., 1974. In: Structure of the Human Brain. A Photographic Atlas, Oxford University Press, New York. De Paermentier, F., Crompton, M.R., Katona, C.L.E., Horton, R.W., 1991a. a 2 -Adrenoceptor binding sites in cortical samples from depressed suicide victims. Br. J. Pharmacol. 102, 2. De Paermentier, F., Crompton, M.R., Katona, C.L.E., Horton, R.W., 1991b. Brain a 2 -adrenoceptor binding sites in antidepressant-treated suicides and controls. Br. J. Pharmacol. 104, 275. Dillon, K.A., Gross-Isseroff, R., Israeli, M., Biegon, A., 1991. Autoradiographic analysis of serotonin 5-HT 1A receptor binding in the human brain postmortem: effects of age and alcohol. Brain Res. 554, 56–64. Ferrier, I.N., McKeith, I.G., Cross, A.J., Perry, E.K., Candy, J.M., Perry, R.H., 1986. Postmortem neurochemical studies in depression. Ann. NY Acad. Sci. 487, 128–142. Fuchs, E., Kramer, M., Hermes, B., Netter, P., Hiemke, C., 1996. Psychosocial stress in the tree shrews: clomipramine counteracts behavioural and endocrine changes. Pharmacol. Biochem. Behav. 54, 219–228. Gobert, A., Rivet, J.M., Audinot, V., Newman-Tancredi, A., Cistarelli, L., Millan, M.J., 1998. Simultaneous quantification of serotonin, dopamine and noradrenaline levels in single frontal cortex dialysates of freely-moving rats reveals a complex pattern of reciprocal auto- and heteroreceptor-mediated control of release. Neuroscience 84, 413–429. Gonzales, A.M., Pasqual, J., Meana, J.J., Barturen, F., del Arco, C., Pazos, A., Garcia-Sevilla, J.A., 1994. Autoradiographic demonstration of increased a 2 -adrenoceptor agonist binding sites in the hippocampus and frontal cortex of depressed suicide victims. J. Neurochem. 63, 256–265. Gross-Isseroff, R., Biegon, A., 1988. Autoradiographic analysis of [ 3 H]imipramine binding in the human brain postmortem: Effects of age and alcohol. J. Neurochem. 51, 528–534. Gross-Isseroff, R., Israeli, M., Biegon, A., 1988. Autoradiographic analysis of [ 3 H]desmethylimipramine binding in the human brain postmortem. Brain Res. 456, 120–126. Gross-Isseroff, R., Dillon, K.A., Fieldust, S.J., Biegon, A., 1990a. Autoradiographic analysis of a 1 -noradrenergic receptors in the human brain postmortem: effect of suicide. Arch. Gen. Psychiatry 47, 1049– 1053.
271
Gross-Isseroff, R., Salama, D., Israeli, M., Biegon, A., 1990b. Autoradiographic analysis of age dependent changes in serotonin 5-HT 2 receptors of the human brain postmortem. Brain Res. 519, 223–227. Gross-Isseroff, R., Dillon, K.A., Israeli, M., Biegon, A., 1990c. Regionally selective increases in m opiate receptor density in brains of suicide victims. Brain Res. 530, 312–316. Gross-Isseroff, R., Biegon, A., Voet, H., Weizman, A., 1998. The suicide brain — a review of postmortem receptor / transporter binding studies. Neurosci. Biobehav. Rev. 22, 653–662. Jones, C.R., Palacios, J.M., 1991. Autoradiography of adrenoceptors in rat and human brain: alpha-adrenoceptor and idazoxan binding sites. Prog. Brain Res. 88, 271–291. Jones, S.B., Bylund, D.B., Rieser, C.A., Shekim, W.O., Nyer, J.A., Carr, G.W., 1983. a 2 -Adrenergic receptor binding in human platelets, alterations during the menstrual cycle. Clin. Pharmacol. Ther. 43, 90–96. Jhnwar-Uniyal, M., Leibowitz, S.F., 1986. Impact of circulating corticosterone on alpha 1 - and alpha 2 -noradrenergic receptors in discrete brain areas. Brain Res. 368, 404–408. Karkanias, G.B., Etgen, A.M., 1995. A thermodynamic analysis of estrogen regulation of alpha 2 -adrenoceptor binding. Brain Res. 593, 7–13. Mahoney, A., Seeley, H., 1990. Clonidine: old friend — new guises. Br. J. Hosp. Med. 44, 358–361. Meana, J.J., Garcia-Sevilla, J.A., 1987. Increased a 2 -adrenoceptor density in the frontal cortex of depressed suicide victims. J. Neural Transm. 70, 377–381. Meana, J.J., Barturen, F., Garcia-Sevilla, J.A., 1989. Characterization and regional distribution of a 2 -adrenoceptors in postmortem human brain using the full agonist [ 3 H]UK 14304. J. Neurochem. 52, 1210–1217. Meana, J.J., Barturen, F., Garcia-Sevilla, J.A., 1992. a 2 -Adrenoceptors in the brain of suicide victims: increased receptor density associated with major depression. Biol. Psychiatry 31, 471–490. Morrison, J.H., Foote, S.L., O’Connor, D., Bloom, F.E., 1982. Laminar, tangential and regional organization of the noradrenergic innervation of monkey cortex: dopamine-beta-hydroxylase immunohistochemistry. Brain Res. Bull. 9, 309–319. Nieuwenhuys, R., Voogd, J., van Huijzen, C., 1981. In: The Human Central Nervous System, 2nd Edition, Springer-Verlag, New York. Ordway, G.A., Widdowson, P.S., Smith, K.S., Halaris, A., 1994. Agonist binding to a 2 -adrenoceptors is elevated in the locus coeruleus from victims of suicide. J. Neurochem. 63, 617–624. Sastre, M., Garcia-Sevilla, J.A., 1993. Opposite age-dependent changes of alpha 2A -adrenoceptors and nonadrenoceptor [ 3 H]idazoxan binding sites (I2-imidazoline sites) in the human brain: strong correlation of I2 with monoamine oxidase-B sites. J. Neurochem. 61, 881–889. Sastre, M., Garcia-Sevilla, J.A., 1994. Density of alpha-2A adrenoceptors and Gi proteins in the human brain: ratio of high-affinity sites to antagonist sites and effect of age. J. Pharmacol. Exp. Ther. 269, 1062–1072. Sastre, M., Garcia-Sevilla, J.A., 1997. Densities of I2-imidazoline receptors, alpha2-adrenoceptors and monoamine oxidase B in brains of suicide victims. Neurochem. Int. 30, 63–72. Schildkraut, J.J., 1965. The catecholamine hypothesis of affective disorders: a review of supporting evidence. Am. J. Psychiatry 122, 509–522. Shimohama, S., Taniguchi, T., Fujiwara, M., Kameyama, M., 1986. Biochemical characterization of alpha-adrenergic receptors in human brain and changes in Alzheimer-type dementia. J. Neurochem. 47, 1294–1301. Zecevic, N., Verney, C., 1995. Development of the catecholamine neurons in human embryos and fetuses, with special emphasis on the innervation of the cerebral cortex. J. Comp. Neurol. 351, 509–535.
Unaltered a 2 -noradrenergic / imidazoline receptors in suicide victims: a postmortem brain autoradiographic analysis a, b a a Ruth Gross-Isseroff D.Sc. *, Abraham Weizman M.D. , Sheila J. Fieldust B.A. , Malka Israeli B.A. , a,c Anat Biegon Ph.D. a
b
Department of Neurobiology, The Weizman Institute of Science, 76100 Rehovot, Israel The Geha Psychiatric Hospital, Felsenstein Medical Research Center, Beilinson Medical Center, Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel c Pharmos Ltd., Kiryat Weizmann, Rehovot, Israel Received 13 January 1999; accepted 29 February 2000
Abstract In vitro quantitative autoradiography of a 2 -adrenergic / imidazoline receptors, using [ 125 I]iodoclonidine as a ligand, was performed on 24 human brains postmortem. Twelve brains were obtained from suicide victims and 12 from matched controls. We found no significant, region-dependent alterations in the density of a 2 -adrenergic receptors in brains of suicide victims as compared to matched controls. We also report age-dependent reductions in binding in the prefrontal cortex and hippocampus, as well as significant recent alcohol ingestion-dependent reductions in binding in the prefrontal cortex. Sex and time from death to autopsy did not affect iodoclonidine binding in our sample. 2000 Elsevier Science B.V. All rights reserved. Keywords: a 2 -Adrenergic receptors; Autoradiography; Human brain; Suicide; Alcohol; Aging
1. Introduction The central noradrenergic system has been implicated in the pathophysiology of a variety of psychopathologies (e.g., Crow et al., 1984; Shimohama et al., 1986). It has been of particular interest in the context of depression, and especially in studies using the brain of suicide victims as a model of depression, which were inspired by the catecholaminergic hypothesis of affective disorders (Schildkraut, 1965). The a 2 -adrenergic receptor was extensively studied. Six reports described elevated binding to these receptors in suicide brains (Ferrier et al., 1986; Meana and GarciaSevilla, 1987; De Paermentier et al., 1991a; Meana et al., 1992; Gonzales et al., 1994; Ordway et al., 1994). Another report has found a decrease in binding to these receptors (Crow et al., 1984) while three others failed to demonstrate *Corresponding author. Department of Molecular Genetics, Ullmann Building, The Weizman Institute of Science, 76100 Rehovot, Israel. E-mail address: [email protected] (R. Gross-Isseroff)
significant differences between the binding of these receptors in suicide brains as compared to controls (Arango et al., 1993; Sastre and Garcia-Sevilla, 1993; Callado et al., 1998). More specifically, Ferrier et al. (1986) studied single samples from prefrontal cortex in a total of 15 brains obtained from a variety of mood disorder patients none of whom died by committing suicide, and 11 of whom had a history of antidepressant medication up to the time of death. Meana and Garcia-Sevilla (1987) studied five violent suicide victims who had a diagnosis of major depressive disorder, two of whom had a history of antidepressant treatment at the time of death. De Paermentier et al. (1991a) studied about 15 brains of drug-free depressed suicide victims and reported an elevation of 24% in receptor density in temporal cortex. The same group also showed a lack of effect of antidepressants on binding in prefrontal cortex, which may strengthen the results of the previously quoted groups De Paermentier et al. (1991b). Meana et al. (1992) studied samples of 41 drugfree violent suicide victims diagnosed as a mixture of mood disorders and reported no significant differences in
0924-977X / 00 / $ – see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S0924-977X( 00 )00075-4
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R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271
clonidine binding but elevated UK14304 binding in prefrontal cortex of a group of 19 suicides and cerebellar cortex in a group of 10 suicides. Their experiments seem to indicate a presynaptic supersensitivity of the high affinity state of the a 2A receptor in suicide. This study was further expanded (Gonzales et al., 1994) and its neuroanatomical resolution refined by the use of quantitative autoradiography using UK14304 as a ligand in 17 suicide brains of various diagnostic groups. Molecular layer of CA1 and outer layers of prefrontal cortex showed significant elevations in binding. Ordway et al. (1994) found autoradiographically higher binding to the a 2A receptor in the locus coereleus, where this receptor is an autoreceptor, of a group of 10 suicide victims. Arango et al. (1993) studied in autoradiographic detail the clonidine binding in prefrontal cortex of 12 suicide victims, and though they found trends of reduction they failed to reach statistical significance. Crow et al. (1984), using rauwloscine as a ligand found a decrease in binding in seven to eight suicide brains judged to be depressed by clinical postmortem diagnostic evaluation. Thus, the total number of brains for which the data are presented in detail is about 60, for 40 of which increased densities of a 2 receptors were reported while for the other 20 reduced densities of these receptors were observed. One of the controversies in the literature concerns adequacy of using phentolamine to define nonspecific binding in the presence of clonidine and other imidazolinelike substances, as phentolamine binds to imidazoline binding sites and these are therefore added to the a 2 high affinity specific binding. One solution to this controversy is that both clonidine type substances and phentolamine pick up a maximum of 5% of the imidazoline binding sites in the human brain and most of the effects reported about suicide brains are much larger than that, even when they fail to reach statistical significance. Another solution was recently suggested which claims that a 2 receptors are unaltered in brains of suicide victims but imidazoline binding sites are decreased in these brains (Sastre and Garcia-Sevilla, 1997). Another controversy in the literature stems from the exact type of diagnosis of the patients studied. As we and others (for a review, see Gross-Isseroff et al., 1998) have previously stated suicide appears to be a type of behaviour that cuts across diagnostic borders and is possibly a bad model for studying mood disorders. Finally, some of the controversy in the existing literature may have stemmed from lack of uniformity in neuroanatomical definitions between the different reports. The present experiment is part of a larger project, portions of which have been previously published, in which as many monoaminergic receptors and binding sites as possible were mapped autoradiographically in a pool of suicide and control brains on consecutive coronal sections taken at the level of the prefrontal pole (similar to the sections employed by Arango et al., 1993) and at the level of the
frontoparietal transition (cf. Gross-Isseroff et al., 1988, 1990a,b,c). As none of the studies in the field were guided by specific hypotheses as to the nature of the brain regional neurochemical defect in suicide, as such a hypothesis does not exist, we thought it would be worthwhile to examine as large a sample of neuroanatomical regions as possible. Such purely exploratory phenomenological studies may lead in the future to the creation of refutable neuroanatomical hypotheses, especially since the forebrain adrenergic innervation in the primate brain has begun to be unravelled (Morrison et al., 1982; Zecevic and Verney, 1995).
2. Experimental procedures
2.1. Subjects Brains of 24 neuropathology-free subjects were obtained at autopsy from the New York City (NY) medical examiner and from the Institute of Forensic Medicine at Jaffa (Israel). Subjects with a known history of schizophrenia, alcoholism or drug abuse, according to their medical records, were excluded. Twelve subjects were suicide victims and 12 matched controls. Matching of subjects was done for sex, age, postmortem delay and, whenever possible, cause of death. For example, a jump from height was matched to a fall from height. Mean age (6S.D.) of the subjects was 42.67 (622.76) years, ranging from 15 to 81 years in the control group and 42.25 (621.63), range 14–72 in the suicide group. Postmortem delay (time from death to autopsy) ranged from 8 to 48 h with a mean (6S.D.) of 24.58 (612.7) in the control group and 5–45 h with a mean (6S.D.) of 23.08 (611.2) in the suicide group. Each group included seven males and five females. Suicide was committed by jumping from heights (three cases), hanging (three cases), suffocation (two cases), gunshot (one case) and unknown means (three cases). Although the means of suicide was unknown in these cases it was clear that they committed suicide from suicide letters they have left. Cause of death was most probably from overdose of agents undetectable in routine toxicological examination. Cause of death, in the control group, included multiple trauma (six cases), burns (one case), gastrointestinal bleeding (one case), heart failure (two cases), drowning (one case), and food poisoning (one case). Multiple trauma deaths were due to accidents. According to the available medical records the controls were free of mental disorders, including alcoholism. Toxicological tests were performed at autopsy, screening for alcohol, barbiturates, analgetics, phenothiazines and salicylates. To the best of our knowledge, based on toxicology as well as medical records, the subjects were free of drugs which might interfere with the adrenergic system. Presence of tricyclic antidepressants was determined in brain tissue by radioreceptor assay. In four subjects alcohol was detected in blood tests at autopsy. Levels of alcohol were 14, 21, 34
R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271
and 71 mg% (female suicide aged 72 years, male control aged 61 years, male control aged 52 years and male control aged 25 years, respectively). These and all other brains used in the study were otherwise drug free. Freezing storage time of the brains was 3 months to 2 years.
2.2. Preparation of sections Brains were removed from the cranium, cut into approximately 2.5-cm coronal blocks which were immediately frozen over dry ice and kept at 2708C. Coronal sections, 40-mm thick, cut on a Bright whole body cryotome at 2158C, were thaw mounted onto gelatin-coated glass slides and kept overnight at 2208C.
2.3. Autoradiography Slides were preincubated for 20 min, at 258C, in 170 mM Tris–HCl buffer, pH 7.6. They were then incubated for 60 min, at 258C with 0.5 nM [ 125 I]iodoclonidine (New England Nuclear, Boston, MA, specific activity 2200 Ci / mmol) in 170 mM Tris–HCl buffer, pH 7.4, containing 0.01% ascorbic acid and 10 mM MgCl 2 . Non-specific binding was determined in the presence of 100 mM phentolamine. A 10-min wash period in 170 mM Tris–HCl ice-cold buffer, pH 7.6, followed the incubation. These conditions label both a 2 -adrenergic receptors and imidazoline binding sites. Slides were then dipped in ice-cold water to remove buffer salts, dried on a 508C hot plate and apposed against tritium-sensitive film (Amersham [ 3 H]Hyperfilm) for 6 h. The film was then developed and fixed with Kodak developer and fixer. Sections were stained with cresyl violet for anatomical reference and several neuroanatomical texts (Barr, 1974; DeArmond et al., 1974; Carpenter, 1976; Nieuwenhuys et al., 1981) were used for identification of structures. All sections were obtained from the right cerebral hemisphere, at the level of the amygdala or hippocampus, head of the caudate nucleus and at the level of prefrontal cortex. Typically four consecutive sections from each brain and anatomical level were used, two for the determination of total binding and two for the determination of nonspecific binding. Experiments were run in a matched pair design so that each one included sections from a suicide brain and its matched control.
2.4. Quantitative analysis of the autoradiograms Autoradiograms were analyzed with the aid of an IBMPC based computerized image analysis system with a PC-vision digitizing board (Imaging Technology, Israel) and customized software. Standard curves for converting gray level readings into ligand concentration were obtained by exposing film to commercial (Amersham) 125 I standards for 6 h. Non-specific binding was subtracted from total binding, to obtain specific binding. Both total and non-
267
specific binding were measured in duplicate in each region, for each brain.
2.5. Homogenate binding assays Saturation binding experiments were performed using tritiated para-amino clonidine (NEN, specific activity, 58.2 Ci / mmol). We used a different ligand than the one used for autoradiography in order to increase validity. Brain samples, obtained from the superior frontal gyrus, were homogenized in 100 volumes of 170 mM Tris–HCl buffer, pH 6.0, and centrifuged for 20 min at 40 0003g. The procedure was repeated and the pellet resuspended in 10 ml / 100 mg original wet weight of incubation buffer: 170 mM Tris–HCl, 10 mM MgCl 2 and 0.01% ascorbic acid. Tissue homogenate samples (800 ml) were incubated with increasing concentrations of radioligand (0.2–12 nM final concentration, 100 ml) for 30 min at 258C. Non-specific binding was assessed in the presence of excess clonidine (100 mM final concentration, 100 ml). Total and nonspecific binding were assayed in triplicate for each concentration. Samples were filtered under vacuum through Whatman GF / B filters and washed in 335 ml ice-cold Tris–HCl buffer. Filters were counted in Packard liquid scintillation counter. The results of saturation experiments were subjected to Scatchard analysis to estimate Kd and Bmax .
2.6. Statistical analyses Statistical analyses were performed using SAS software. Two-tailed student t-tests for independent samples, twoway analyses of variance (age3group, age3sex) corrected for unbalanced designs and Pearson coefficients of correlation were used as appropriate. a was preset at P50.05.
3. Results
3.1. Autoradiography The distribution of iodoclonidine binding in the various neuroanatomical regions examined is shown in Table 1 for young and old subjects. The highest level of binding occurs in portions of the prefrontal cortex and the dentate gyrus. Less binding was found in other cortical areas and the hippocampus. Low binding was observed in the basal ganglia and thalamus, and almost no binding was found in the white matter. The binding in the cortex was heterogeneous as well as being characterized by a unique pattern of lamination. There were two visible bands: an outer dark band overlaying cortical layers I–III, and a lighter inner band overlaying cortical layers IV–VI. The effect of alcohol was tested by two-tailed t-tests, and significant reductions in iodoclonidine binding in brains taken from subjects in which alcohol was detected
R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271
268
Table 1. Continued
Table 1 The dependence of a 2 receptor distribution on age a Region
Frontoparietal gyri: Cingulate Internal band External band Precentral Internal band External band Postcentral Internal band External band Insula Internal band External band Transverse temporal Internal band External band Superior temporal Internal band External band Middle temporal Internal band External band Inferior temporal Internal band External band Lateral occipitotemporal Internal band External band Parahippocampal Internal band External band Basal ganglia Caudate nucleus: Head Tail Putamen
Old (n)
r(P)
30.065.0 (10) 26.564.5 (10) 33.065.7 (10) 25.764.9 (9) 23.664.4 (9) 26.865.6 (9) 24.364.7 (9) 22.364.2 (9) 25.564.7 (9) 24.063.7 (9) 21.362.9 (9) 26.264.2 (9) 23.064.7 (8) 22.564.8 (8) 23.464.8 (8) 31.965.9 (8) 30.666.1 (9) 31.265.5 (9)
13.364.1*(10) 11.363.2*(10) 14.464.4*(10) 11.963.7 (9) 11.663.7 (9) 12.263.6 (9) 14.765.9 (9) 14.565.8 (9) 14.165.6 (9) 15.264.9 (9) 14.564.4 (9) 15.865.4 (9) 14.164.7 (9) 14.064.2 (9) 15.464.5 (9) 13.363.2*(10) 11.662.8*(10) 16.064.2*(10)
18.163.5 16.263.0 20.163.8 19.862.7 16.661.1 21.464.8 16.663.1 14.562.6 17.663.4 17.766.3 17.066.3 17.564.9 16.662.7 10.861.4 17.963.6 14.862.4 13.061.9 15.562.4 13.362.4 12.262.2 14.062.8 13.562.7 13.162.3 14.062.9
(5) (5) (5) (3) (3) (3) (6) (6) (6) (4) (4) (4) (5) (5) (5) (7) (7) (7) (6) (6) (6) (6) (6) (6)
12.762.3 12.463.2 12.862.5 9.663.2 8.263.0 10.463.0 8.762.3 8.062.3 8.962.3 14.261.1 13.760.9 14.261.4 11.261.4 10.861.6 10.961.7 10.261.4 9.361.3 10.761.6 9.661.8 9.561.7 9.361.9 10.561.3 10.361.3 10.561.1
(3) (3) (3) (3) (3) (3) (4) (4) (4) (3) (3) (3) (6) (6) (6) (5) (5) (5) (4) (4) (4) (6) (6) (6)
15.062.9 14.262.8 15.162.8 13.362.5 12.462.2 14.361.8
(7) (7) (7) (5) (5) (5)
10.961.8 11.062.0 10.961.8 10.962.1 10.761.8 11.662.1
(4) (4) (4) (5) (5) (5)
6.860.8 (10) 7.861.3 (6) 7.760.6 (10)
7.160.7 (10) 4.060.8*(5) 7.860.5 (8)
20.50(0.03) 20.53(0.02) 20.51(0.02) 20.49(0.04) 20.47(0.05) 20.49(0.04)
10.260.3 (3)
6.661.7 (5)
Old (n)
Hippocampus CA fields: Molecular layer Pyramidal layer
12.461.6 (6) 12.061.8 (6)
Dentate gyrus: Granular layer Molecular layer Hilus Subiculum
20.563.4 12.861.8 12.362.1 11.361.1
White matter
(7) (7) (6) (6)
6.060.9 (10)
r(P)
7.661.2*(8) 6.960.8*(8)
20.69(0.006) 20.66(0.01)
10.161.6*(8) 7.461.0*(8) 8.061.7 (8) 8.361.4 (8)
20.66(0.008) 20.64(0.01)
5.460.6 (10)
a
This table presents mean6S.E.M of specific [ 125 I]iodoclonidine binding in n brains, expressed as fmol / mg tissue. The sample was divided into two age groups at the median age of 35. The resulting young group had a mean (6S.D.) age of 21.1 (66.0) years, and the old group had a mean (6S.D.) age of 59.8 (612.4) years. *P,0.05 in a two-tailed t-test. r, Pearson coefficient of correlation between iodoclonidine binding and age. 20.62(0.004) 20.63(0.004) 20.53(0.02)
20.67(0.04) 20.64(0.05) 20.65(0.04)
at autopsy were observed in a number of regions (Table 2). The prefrontal cortex was most affected by alcohol, as well as the whole cingulate cortex. These four brains were discarded from further analyses of the data. The effect of age on iodoclonidine binding was tested by computing the coefficient of correlation between binding and age. These coefficients were mostly negative and in several regions highly significant (Table 1). The regions most affected by age were the prefrontal cortex and hippocampus. The tail of the caudate nucleus also exhibited remarkable decrease in binding with age. Since we found a remarkable age effect, the effect of suicide was tested by two-way analyses of variance (age3 Table 2 The dependence of a 2 receptors in the neocortex on alcohol a
20.65(0.03)
Region
No alcohol
Alcohol
P
Cortex Prefrontal gyri: Superior frontal Internal band External band Inferior frontal Internal band External band Orbital Internal band External band Cingulate Internal band External band
15.262.5 (20) 13.262.0 (20) 16.762.9 (20) 13.762.5 (18) 13.062.3 (18) 13.962.5 (18) 13.662.0 (18) 12.461.6 (18) 14.562.3 (18) 15.362.5 (19) 14.562.5 (19) 15.962.4 (19)
4.160.7 (4) 4.060.6 (4) 4.060.8 (4) 2.861.0 (4) 3.160.9 (4) 3.161.1 (4) 4.061.1 (4) 4.161.1 (4) 4.561.2 (4) 3.161.1 (4) 3.161.0 (4) 2.961.3 (4)
0.0004 0.0003 0.0004 0.0006 0.0007 0.0008 0.0006 0.03 0.001 0.0002 0.0004 0.0001
Frontoparietal level: Cingulate Internal band External band
16.162.4 (8) 14.862.2 (8) 17.462.8 (10)
6.562.2 (4) 5.661.5 (3) 6.362.2 (3)
0.03 0.02 0.03
a
Thalamus Medial nucleus
Mean6S.E.M. Young (n)
Mean6S.E.M. Young (n)
Cortex Prefrontal gyri: Superior frontal Internal band External band Middle frontal Internal band External band Inferior frontal Internal band External band Orbital Internal band External band Rectus Internal band External band Cingulate Internal band External band
Region
This table presents mean6S.E.M. of specific [ 125 I]iodoclonidine binding in n brains, expressed as fmol / mg tissue. P, probability of t, in a two-tailed t-test for independent samples.
R. Gross-Isseroff et al. / European Neuropsychopharmacology 10 (2000) 265 – 271 Table 3 The effects of suicide and age on a 2 receptors in the human brain postmortem a Region
(Suicide / control) 3100
(Old / young) 3100
Cortex Prefrontal gyri: Superior frontal Internal band External band Middle frontal Internal band External band Inferior frontal Internal band External band Orbital Internal band External band Rectus Internal band External band Cingulate Internal band External band
131 129 145 118 107 135 114 113 122 101 91 106 101 101 108 127 135 124
44 43 44 46 49 46 69 65 55 63 68 60 61 62 66 42 38 51
Frontoparietal gyri: Cingulate Internal band External band Precentral Internal band External band Postcentral Internal band External band Transverse temporal Internal band External band Superior temporal Internal band External band Middle temporal Internal band External band Inferior temporal Internal band External band Lateral occipitotemporal Internal band External band Parahippocampal Internal band External band
65 65 71 52 50 51 61 63 59 83 95 82 91 95 93 87 88 84 104 105 106 76 79 78 92 101 91
70 77 64 48 49 49 52 55 51 67 100 61 69 72 69 72 78 66 78 79 75 73 77 72 82 86 81
Basal ganglia Caudate nucleus: Head Tail Putamen Claustrum
94 68 100 157
104 51 101
90 58 64 37
65
Thalamus Medial nucleus Posterior lateral Pulvinar Lateral geniculate body
269
Table 3. Continued Region
Hippocampus CAI field Pyramidal layer CAIV field Molecular layer Pyramidal layer
(Suicide / control) 3100
(Old / young) 3100
69 96 86
61 58
Dentate gyrus: Granular layer Molecular layer Hilus Subiculum
74 66 82 101
49 58 65 73
White matter
108
90
a
This table presents the effects of suicide (left) and age (right) on iodoclonidine specific binding as percent of group means, where the young and old group were created as described in legend to Table 1. No significant differences between suicide and control were obtained in any of the examined brain regions.
group). Using these analyses no significant effects of suicide or interactions were found. The lack of statistical significance is probably due to the small number of brains in each age3group cell. As we have noticed non-significant differences between the suicide and control groups we have listed the suicide / control ratio in binding in Table 3. This presentation clarifies the magnitude of the results (left column), in comparison to the magnitude of the results for the age effect (right column). The difference between the two effects is most pronounced in the prefrontal cortex, where suicide induces elevations in binding while aging induces reductions. The effect of sex was tested in similar analyses of variance (age3sex). No effect of sex or interactions were found. The effect of postmortem delay was tested by computing the coefficient of correlation between the postmortem delay and specific binding. No effect of postmortem delay was found.
3.2. Homogenate binding In order to assess whether the observed differences in the autoradiographic result were due to changes in Kd or Bmax , we analyzed the superior frontal gyrus homogenate saturation binding experiments accordingly. The effect of recent alcohol ingestion was tested by a two-tailed t-test for independent samples. Bmax for the ‘alcohol present’ group (n53) was 22.45 fmol [ 3 H]p-amino clonidine / mg protein, whereas for the ‘alcohol absent’ group (n58) it was 42.97 fmol 3 H-labelled para-amino clonidine / mg protein. This difference is statistically significant (P5 0.03). No differences between the two groups were observed for Kd (‘alcohol present’ Kd 54.63 nM; ‘alcohol absent’ Kd 53.49 nM). The effect of age on Kd and Bmax
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was tested by Pearson coefficient of correlation in the alcohol-free group. The resulting correlation between age and Bmax was 20.35 and failed to reach statistical significance (P50.39) probably because of the small number of cases tested. No effect of age on Kd was found. The possible effects of group (control versus suicide) and sex on Kd and Bmax were tested by two-tailed t-tests for independent samples and no significant effects were found. The effect of postmortem delay on Kd and Bmax was tested by Pearson coefficient of correlation between the variables and no significant effects were found.
4. Discussion We have presented three major findings: brain a 2 -adrenergic receptors do not appear to be altered in suicide victims, age-related reductions in a 2 -adrenergic / imidazoline receptors in the human brain, and recent alcohol ingestion-related reductions in these receptors in the human brain. In addition we have presented the most detailed neuroanatomical distribution map of the density of these receptors to date. The age-related reduction in the density of these receptors is in contrast to a previous publication (Meana et al., 1989) and in agreement with others (Sastre and GarciaSevilla, 1993; Sastre and Garcia-Sevilla, 1994). The a 2 adrenergic receptor is but one of a series of monoaminergic receptors displaying such an age-related reduction in the human brain (Gross-Isseroff and Biegon, 1988; GrossIsseroff et al., 1990b; Dillon et al., 1991) and may be connected to age-related gene expression governed by other neurohumors such as steroid hormones (Jones et al., 1983; Jhnwar-Uniyal and Leibowitz, 1986; Karkanias and Etgen, 1995). The effect of alcohol presence on these receptors has not been reported before. There are a number of monoaminergic receptors affected by alcohol in the human brain, and the mechanism underlying this effect has yet to be elucidated (Gross-Isseroff and Biegon, 1988; Dillon et al., 1991). Due to the limited number of brains in this study, the effect of alcohol ingestion on a 2 -adrenergic / imidazoline receptors in the human brain warrants further investigation. In brains of suicide victims we noted a non-significant tendency toward elevated binding (roughly 20–45%) in most regions of prefrontal cortex, except regions of the base of the brain (orbital and rectus gyri). This is in agreement with others (Ferrier et al., 1986; Meana and Garcia-Sevilla, 1987; De Paermentier et al., 1991a; Gonzales et al., 1994; Callado et al., 1998). We have also noticed a trend toward reduced binding in all other cortical and subcortical regions represented in our sections. This is in agreement with Arango et al. (1993) and Crow et al. (1984). As none of the previous studies examined a complete pool of the neuroanatomical regions reported here, and most do not report results which fail to reach
statistical significance, there are discrepancies between the present report and previous ones. These may stem from either differences in ligand or neuroanatomical definitions. As we have previously stated, a trend meta-analysis of the pooled worldwide data should be interesting. It has to be borne in mind, though that the a 2 receptor is but one of a series of markers which can be monitored in suicide, and that the adrenergic system which it monitors is but one of a multiplicity of neurotransmitter systems which constantly interact with each other. The a 2 receptor has a regulatory function in norepinephrine and serotonin release and thus changes in its density may cause changes in neurotransmitter release. Changes in norepinephrine release may, in turn, affect other monoaminergic systems of neurotransmission. Indications of the existence of such interactions have been amply documented in animal studies (e.g., Fuchs et al., 1996; Gobert et al., 1998). a 2 -Adrenergic receptors are both pre- and post-synaptic in the central nervous system (for reviews, cf. Berlan et al., 1992; Bylund, 1992) and coexpressed on serotonergic as well as other neurons (for a review, cf. Mahoney and Seeley, 1990). The method used in the present experiment cannot differentiate between the pre- and post-synaptic sites and neither can it differentiate between the adrenergic versus other neurones (heteroreceptors). This limitation has to be borne in mind, and the present results treated with the appropriate caution. Further experimentation, possibly using in situ hybridization methods (e.g., Jones and Palacios, 1991) will probably shed further light on the possible role of a 2 receptors and heteroreceptors, in concert with others, in suicidal behaviour. Two possible caveats should be borne in mind when interpreting the results presented here. The first is the choice of ligands. Under the conditions of the present experiment both a 2 receptors and imidazoline binding sites were probably labeled. The second is the probable diagnostic non-uniformity of the brains obtained from suicide victims. Suicide victims suffer from a variety of axis I diagnoses such as: major depression, psychoactive substance use disorders, schizophrenia, bipolar disorder and other disorders or a combination of disorders. Some of the control group brains may have gone undiagnosed, suffering from axis I disorders which overlap the ones characterizing the suicide group. This may have masked any differences in a 2 binding between the groups. It would be of interest in the future to study brains of suicide victims with a known psychiatric history. An ideal study would include groups of suicide plus major depression, suicide plus schizophrenia, suicide without suicide and healthy controls. Such a design, where feasible, would facilitate the extraction of the ‘suicide effect’.
Acknowledgements This study was supported by a grant from the US–Israel Binational Science Foundation to Anat Biegon.
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References Arango, V., Ernsberger, P., Sved, A.F., Mann, J.J., 1993. Quantitative autoradiography of a 1 - and a 2 -adrenergic receptors in the cerebral cortex of controls and suicide victims. Brain Res. 630, 271–282. Barr, M.L., 1974. In: The Human Nervous System. An Anatomical Viewpoint, Harper and Row, New York. Berlan, M., Montastruc, J.L., Lafontan, M., 1992. Pharmacological prospects for alpha 2 -adrenoceptor antagonist therapy. Trends Pharmacol. Sci. 13, 277–282. Bylund, D.B., 1992. Subtypes of a 1 - and a 2 -adrenergic receptors. FASEB J. 6, 832–839. Callado, L.F., Meana, J.J., Grijalba, B., Pazos, A., Sastre, M., GarciaSevilla, J.A., 1998. Selective increase of alpha2A-adrenoceptor agonist binding sites in brains of depressed suicide victims. J. Neurochem. 70, 1114–1123. Carpenter, M.B., 1976. In: Human Neuroanatomy, 7th Edition, Williams and Wilkins, Baltimore. Crow, T.J., Cross, A.J., Cooper, S.J., Deakin, J.F.W., Ferrier, I.N., Johnson, J.A., Joseph, M.H., Owen, F., Poulter, M., Lofthouse, R., Corsellis, J.A.N., Chambers, D.R., Blessed, G., Perry, E.K., Perry, R.H., Tomlinson, B.E., 1984. Neurotransmitter receptors and monoamine metabolites in the brains of patients with Alzheimer-type dementia and depression and suicides. Neuropharmacology 23, 1561– 1569. DeArmond, S.J., Fusco, M.M., Dewey, M.M., 1974. In: Structure of the Human Brain. A Photographic Atlas, Oxford University Press, New York. De Paermentier, F., Crompton, M.R., Katona, C.L.E., Horton, R.W., 1991a. a 2 -Adrenoceptor binding sites in cortical samples from depressed suicide victims. Br. J. Pharmacol. 102, 2. De Paermentier, F., Crompton, M.R., Katona, C.L.E., Horton, R.W., 1991b. Brain a 2 -adrenoceptor binding sites in antidepressant-treated suicides and controls. Br. J. Pharmacol. 104, 275. Dillon, K.A., Gross-Isseroff, R., Israeli, M., Biegon, A., 1991. Autoradiographic analysis of serotonin 5-HT 1A receptor binding in the human brain postmortem: effects of age and alcohol. Brain Res. 554, 56–64. Ferrier, I.N., McKeith, I.G., Cross, A.J., Perry, E.K., Candy, J.M., Perry, R.H., 1986. Postmortem neurochemical studies in depression. Ann. NY Acad. Sci. 487, 128–142. Fuchs, E., Kramer, M., Hermes, B., Netter, P., Hiemke, C., 1996. Psychosocial stress in the tree shrews: clomipramine counteracts behavioural and endocrine changes. Pharmacol. Biochem. Behav. 54, 219–228. Gobert, A., Rivet, J.M., Audinot, V., Newman-Tancredi, A., Cistarelli, L., Millan, M.J., 1998. Simultaneous quantification of serotonin, dopamine and noradrenaline levels in single frontal cortex dialysates of freely-moving rats reveals a complex pattern of reciprocal auto- and heteroreceptor-mediated control of release. Neuroscience 84, 413–429. Gonzales, A.M., Pasqual, J., Meana, J.J., Barturen, F., del Arco, C., Pazos, A., Garcia-Sevilla, J.A., 1994. Autoradiographic demonstration of increased a 2 -adrenoceptor agonist binding sites in the hippocampus and frontal cortex of depressed suicide victims. J. Neurochem. 63, 256–265. Gross-Isseroff, R., Biegon, A., 1988. Autoradiographic analysis of [ 3 H]imipramine binding in the human brain postmortem: Effects of age and alcohol. J. Neurochem. 51, 528–534. Gross-Isseroff, R., Israeli, M., Biegon, A., 1988. Autoradiographic analysis of [ 3 H]desmethylimipramine binding in the human brain postmortem. Brain Res. 456, 120–126. Gross-Isseroff, R., Dillon, K.A., Fieldust, S.J., Biegon, A., 1990a. Autoradiographic analysis of a 1 -noradrenergic receptors in the human brain postmortem: effect of suicide. Arch. Gen. Psychiatry 47, 1049– 1053.
271
Gross-Isseroff, R., Salama, D., Israeli, M., Biegon, A., 1990b. Autoradiographic analysis of age dependent changes in serotonin 5-HT 2 receptors of the human brain postmortem. Brain Res. 519, 223–227. Gross-Isseroff, R., Dillon, K.A., Israeli, M., Biegon, A., 1990c. Regionally selective increases in m opiate receptor density in brains of suicide victims. Brain Res. 530, 312–316. Gross-Isseroff, R., Biegon, A., Voet, H., Weizman, A., 1998. The suicide brain — a review of postmortem receptor / transporter binding studies. Neurosci. Biobehav. Rev. 22, 653–662. Jones, C.R., Palacios, J.M., 1991. Autoradiography of adrenoceptors in rat and human brain: alpha-adrenoceptor and idazoxan binding sites. Prog. Brain Res. 88, 271–291. Jones, S.B., Bylund, D.B., Rieser, C.A., Shekim, W.O., Nyer, J.A., Carr, G.W., 1983. a 2 -Adrenergic receptor binding in human platelets, alterations during the menstrual cycle. Clin. Pharmacol. Ther. 43, 90–96. Jhnwar-Uniyal, M., Leibowitz, S.F., 1986. Impact of circulating corticosterone on alpha 1 - and alpha 2 -noradrenergic receptors in discrete brain areas. Brain Res. 368, 404–408. Karkanias, G.B., Etgen, A.M., 1995. A thermodynamic analysis of estrogen regulation of alpha 2 -adrenoceptor binding. Brain Res. 593, 7–13. Mahoney, A., Seeley, H., 1990. Clonidine: old friend — new guises. Br. J. Hosp. Med. 44, 358–361. Meana, J.J., Garcia-Sevilla, J.A., 1987. Increased a 2 -adrenoceptor density in the frontal cortex of depressed suicide victims. J. Neural Transm. 70, 377–381. Meana, J.J., Barturen, F., Garcia-Sevilla, J.A., 1989. Characterization and regional distribution of a 2 -adrenoceptors in postmortem human brain using the full agonist [ 3 H]UK 14304. J. Neurochem. 52, 1210–1217. Meana, J.J., Barturen, F., Garcia-Sevilla, J.A., 1992. a 2 -Adrenoceptors in the brain of suicide victims: increased receptor density associated with major depression. Biol. Psychiatry 31, 471–490. Morrison, J.H., Foote, S.L., O’Connor, D., Bloom, F.E., 1982. Laminar, tangential and regional organization of the noradrenergic innervation of monkey cortex: dopamine-beta-hydroxylase immunohistochemistry. Brain Res. Bull. 9, 309–319. Nieuwenhuys, R., Voogd, J., van Huijzen, C., 1981. In: The Human Central Nervous System, 2nd Edition, Springer-Verlag, New York. Ordway, G.A., Widdowson, P.S., Smith, K.S., Halaris, A., 1994. Agonist binding to a 2 -adrenoceptors is elevated in the locus coeruleus from victims of suicide. J. Neurochem. 63, 617–624. Sastre, M., Garcia-Sevilla, J.A., 1993. Opposite age-dependent changes of alpha 2A -adrenoceptors and nonadrenoceptor [ 3 H]idazoxan binding sites (I2-imidazoline sites) in the human brain: strong correlation of I2 with monoamine oxidase-B sites. J. Neurochem. 61, 881–889. Sastre, M., Garcia-Sevilla, J.A., 1994. Density of alpha-2A adrenoceptors and Gi proteins in the human brain: ratio of high-affinity sites to antagonist sites and effect of age. J. Pharmacol. Exp. Ther. 269, 1062–1072. Sastre, M., Garcia-Sevilla, J.A., 1997. Densities of I2-imidazoline receptors, alpha2-adrenoceptors and monoamine oxidase B in brains of suicide victims. Neurochem. Int. 30, 63–72. Schildkraut, J.J., 1965. The catecholamine hypothesis of affective disorders: a review of supporting evidence. Am. J. Psychiatry 122, 509–522. Shimohama, S., Taniguchi, T., Fujiwara, M., Kameyama, M., 1986. Biochemical characterization of alpha-adrenergic receptors in human brain and changes in Alzheimer-type dementia. J. Neurochem. 47, 1294–1301. Zecevic, N., Verney, C., 1995. Development of the catecholamine neurons in human embryos and fetuses, with special emphasis on the innervation of the cerebral cortex. J. Comp. Neurol. 351, 509–535.