19
Psychiatry Research, 32: 19-28 Elsevier
Altered Pineal Serotonin
Binding in Some Suicides
D. Larry Sparks and Karley Y. Little Received February 7, 1989; revised version received December 4. 1989; accepted December 26. 1989. Abstract. Serotonergic abnormalities have been regularly of suicide victims. Because of evidence that pineal function subjects and that serotonin-releasing axons may innervate tritiated serotonin binding in a series of control and suicide
reported in the brains is altered in depressed the pineal, we assayed pineals. High affinity
binding
between
was present,
and there
were
apparent
differences
some suicides
and controls.
Key Words. Suicide,
melatonin,
serotonin,
receptors.
Previous post-mortem studies in suicide victims compared to controls have revealed considerable evidence that the concentrations of serotonin (5hydroxytryptamine, 5HT) and 5hydroxyindolacetic acid (SHIAA, main serotonin metabolite) are decreased in the brainstem (see Mann et al., 1986a, for a review). Frontal cortex serotonin type 2 binding (5HT,) has been found elevated in suicide victims by some, but not all, investigators (Stanley et al., 1986b; Cheatham et al., 1988; Arora and Meltzer, 1989). lmipramine binding in frontal cortex, thought to be a marker of presynaptic serotonin activity, has been found to be both decreased (Stanley et al., 1982) and normal (Arora and Meltzer, 1987). Serotonin type I binding (SHT,), measured by tritiated serotonin, has not been found abnormal (Crow et al., 1984; Mann et al., 19866), while 5HTIA binding, a subtype measured by tritiated 8hydroxy-para-amino tetralin (8-OH-DPAT), was found increased by Arora and Meltzer (1989). The level of P-noradrenergic binding in frontal cortex has been found increased in homogenate membrane (Mann et al., 19866) and autoradiographic studies (Arango et al., 1988; Biegon and Israeli, 1988). These studies were of subjects uncategorized by probable psychiatric diagnosis. To what extent these findings represent changes attributable to mood disorders is unclear since only about 50% of suicides are so diagnosable. Because melatonin levels may be altered in suicidal and depressed patients and are derived from serotonin, some attention has also been focused on the pineal gland, the site of most melatonin synthesis. Stanley and Brown (1988) recently reported a decreased melatonin concentration in the pineal of suicides vs. controls dying during
D. Larry Sparks, Ph.D., is Research Assistant Professor, Sanders-Brown Research Center on Agin - - and Departments of Pathology and Neurology, University of Kentucky Medical Center; and Medical Examiner’s Program, Kentucky Justice Cabinet, Lexington, KY 40536. Karlev Y. Little. M.D.. is Assistant Professor, Department of Psychiatry, Universityof North Carolina School of Medicine, Chapel Hill, NC. (Reprint requests to Dr. K.Y. Little, University of North Carolina at Chapel Hill, Department of Psychiatry, CB # 7160, Medical School Wing B, Chapel Hill, NC 27599-7160, USA,) 01651781/90/$03.50
@ 1990 Elsevier Scientific
Publishers Ireland
Ltd.
20 the hours 2200-0600h, the peak melatonin-production period. Clinically, melatonin levels in suicidal inpatients have been reported to be higher than those in nonsuicidal depressed inpatients (Beck-Friis, 1985). However, other studies have found that depressed patients may have low melatonin levels (Wetterberg, 1978) a decreased nocturnal melatonin rise (Brown et al., 1985), and an increased sensitivity to the inhibiting effects of light during the night (Lewy et al., 1985). Possible explanations include diminished precursor availability, decreased pinealocyte capacity, or diminished noradrenergic and other signal input. The third explanation is most consistent with the empirical evidence and prevailing conceptual models (although recent work suggests that 5-hydroxytryptophan availability may be an important variable in explaining serotonergic function; Delgado et al. 1989). Much evidence suggests that diminished and erratic noradrenergic function occurs in serious depressive illnesses. Noradrenergic innervation by the nervi conarii (sympathetic fibers that arise in the superior cervical ganglion) markedly influences the activity of pineal serotonin-N-acetyl transferase (SNAT), the major enzyme involved in the formation of melatonin (Axelrod, 1974). However, preliminary work in our laboratory has suggested serotonin receptor binding in the human pineal. Animal work has shown low affinity ( Kd 200-I ,000 nM) Ketanserin binding (5HT,) in bovine pineal (Govitrapong et al., 1984), and blockage of tryptophan effect on rat SNAT by cyproheptadine, a serotonin-receptor antagonist (King et al., 1982), but no effect of serotonin on isoproterenol-stimulated SNAT activity in rat pineal culture (Chan and Ebadi, 1981). Because of the substantial evidence of serotonin abnormalities in suicides, we characterized pineal 5HT, binding in subjects dying suddenly and compared this binding in a series of suicides and controls. Methods Portions of human pineal were saved as part of the normal coroner-authorized autopsy protocol. Acquisition of tissue was within 24 hours of death (Perry and Perry, 1983; Whitehouse et al., 1984). The tissue was frozen at -70 ‘C until use and coded so that the technician was blind to all details about the subject. Blood-alcohol levels and a toxicological screen were performed by the state toxicological laboratory in Frankfort, KY. Identification of cases as suicides was decided by the medical examiner. Control subjects were matched for age, sex, time of death, and, to the extent possible, alcohol use. The causes of death in I I suicides included gunshot wounds (n = X), carbon monoxide (n = 2), and hanging (n = I). Twenty-three controls died from either gunshot wounds (n = 5), automotive trauma (n = 7) sudden cardiovascular causes (n = 8) asphyxia (n = 2) or perforated ulcer (n = I). For binding studies, tissue was homogenized in 5 vol (W:V) of 0.05 M Tris, pH 7.7, at 37 “C, and centrifuged at Sl,OOOg for IO min. Supernatant was discarded and the process repeated twice to ensure that the membranes were cleansed of any drugs present at the time of death (Bennett et al., 1985). Serotonin binding (5HT,) was done by the method of Nelson et al. (1978) using tritiated serotonin. Ligand and crude tissue homogenate were incubated to equilibrium at 37 “C for IO min in glass tubes. Nonspecific binding was defined as the activity remaining in the presence of IO PM unlabeled 5HT. 5HT, binding was subtyped into 5HT,, by the addition of 100 nM and 1,000 nM 8-hydroxy-N, N-dipropyl-2and non-5HT,, aminotetralin (OH-DPAT, final concentrations) to some tubes before incubation (Peroutka. 1986). Final values were based on wet weight for the initial study and protein content for the suicide study, as quantified by the methods of Lowry et al. (195 I).
21 Binding from five young and five old control subjects was measured at six ligand concentrations in a range previously found significant in other brain regions (0.5 to 8 nM). These data were analyzed using a nonlinear, least-squares iterative technique from the computer programs EBDA and LIGAND (Munson and Rodbard, 1980). In the second series of controls and suicides, binding was measured at three ligand concentrations (I, 2 and 4 nM). Differences between old and young controls, and controls vs. suicides, were tested for significance with Student’s t test. Correlations between binding values and age, time of death, month of death, post-mortem interval, and blood-alcohol level were searched for using linear regression. Mean differences in these variables were also compared for significant differences using Student’s t test. Analysis of variance (ANOVA) was performed on mean data for three daily time periods and causes of death.
Results Binding in the human pineal was detected using tritiated
gland, with apparent serotonin. Saturation
high and low affinity components, occurred at the 4 nM and 6 nM
concentrations, but binding increased at 8 nM. An initial ligand analysis using a one- or two-site model did not converge on satisfactory binding parameters, undoubtedly due to a deficiency of points representing lower affinity binding. Since saturation appeared to occur between 4 nM and 6 nM, we dropped the 8 nM point for a second analysis. When this point was dropped, evidence emerged for a high affinity site with a K,, of 6.05 + 0.84 nM and B,,, of 366.6 + 50.9 fmol/mg wet weight. Correlation coefficients for each individual Scatchard plot varied from 0.80 to 0.99. There were no differences between five young controls (average age 33 + 6 years) vs. five old controls (average age 73 + 8 years). Competitive binding analysis with OH-DPAT (5HTIA ligand) at 100 nM and 1,000 nM concentrations added to 3H-5HT in samples from seven controls showed no inhibition, confirming these sites as 5HTta, 5HTIc, or 5HT,n subtypes. Among the suicides, several subjects had consistently low binding at the three ligand concentrations (Fig. I). At 1 nM, mean (zk SD) binding in suicides averaged 1.55 + 1.52 fmol/ mg protein vs. 2.11 f 1.35 fmol/ mg protein in controls (one-tailed
Fig. 1. Serotonin binding for each suicide 16.5 10
0
1nM
2nM
SEROTONINBINDINGFOR EACH SUICIDE
4nM
22 Wilcoxon rank sum, p < 0.06). Five of 11 suicides demonstrated values below the lowest of 23 controls (0.05, 0.08, 0.08, 0.20, and 0.28 vs. 0.60), as seen in Fig. 2. Similarly at 2 nM (Fig. 3), 4 of 11 suicides were lower than the lowest control (one-tailed Wilcoxon rank sum, p < 0.05),although the means (+ SD) were not significantly different (2.89 f 2.71 vs. 4.21 f 2.60). At 4 nA4 (Fig. 4), mean (I!I SD) values were similar (suicides 6.73 + 4.38 vs. 6.16 III 3.17 for controls), and there were no rank differences. Six of 11 suicides and 8 of 23 controls had measurable blood-alcohol levels. The mean (+ SD) level in the suicides was 65 + 80 mg/dl vs. 79 I!I 127 mg/dl in the controls. There were no correlations between alcohol level and 5HT binding in the Fig. 2. 3H-serotonin binding in
suicides and controls (1 nM concentration)
Fig. 3. 3H-serotonin binding in suicides and controls (2 nM concentration) 10.0 gi
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SUICIDES N-l 1
CONTROLS N=20
3-H SEROTONIN BINDING 1nM Cont.
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3-H SEROTONIN BINDINQ 2nM Cont.
23 Fig. 4. 3H-serotonin binding in suicides and controls (4 nM concentration) 16.0
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3-H SEROTONM BINDING 4nM Cont. combined sample of suicides and controls. However, among the five suicides with the lowest levels of 5HT binding, only one had a measurable ethanol level, as compared to five of the other six suicides. Mean (+ SD) ethanol level was 22.6 + 50.5 mg/dl (one at I 13 mg/dl) in the low subgroup, as compared to 101.7 + 88.8 mg/dl in the high subgroup (one-tailed t test, p < 0.06). Among suicides at the 2 nM 5HT concentration, there was a positive correlation between ETOH level and binding (r = 0.68, p = 0.02). These results suggest that those suicides with higher 5HT binding, equivalent to controls, might have shown abnormally lower binding if they had been free of ethanol. Mean time of death was 1llOh in controls and did not correlate with 5HT binding. In the five suicides with lower binding, times of death were OlOOh, 0400h, 0800h, 1600h, and 1800h. Mean 5HT binding did not differ among the three time periods, 0600-1400h, 1400-22OOh, and 2200-0600h. The mean (91 SD) post-mortem interval was similar between suicides and controls (13.8 + 5.3 hours vs. 14.2 + 7.6 hours) and was not correlated with 5HT binding. Among gunshot victims (suicides = 8, controls = 5) there were no differences in
24 5HT binding. Combined values for gunshot victims vs. subjects dying by other means showed no significant difference. Age was similar in suicides (36 + 15 years) vs. controls (40 + 17 years) and was not correlated with 5HT binding. Two suicides and two controls were female. By inspection, their binding values were unexceptional. Discussion To our knowledge, this is the first report of serotonin binding in the human pineal gland. The data presented strongly support the existence of a saturable high affinity site. We further believe our results suggest that two components of serotonin binding occur. If so, lower affinity 5HT binding would have contributed to an overestimate of & (6.05 nM) and B,,, (367 fmol/mg wet weight) for the high affinity site. Our point-to-point comparisons between suicides and controls were done at concentrations that we believe to be relevant to the high affinity site. If the high affinity Kd actually occurs in the 4- to 6-nM range as our computer-generated one-site model suggests, our finding at 4 nM showing suicides equivalent to controls is strong evidence that important differences do not exist. The diminished binding levels at I- and 2-nM concentrations would then be attributable to changes in affinity, most likely an artifact. If, however, the actual Kd is in the l-to 3-nM range, as we suspect, our group differences may indicate significant alterations in receptor function in the suicides. Unfortunately, the current studies did not anticipate overlapping binding at two sites and were not performed in a manner that would allow resolution of this question. Studies need to be done at more and higher 3H-5HT concentrations so that lower affinity binding can be characterized and an accurate two-site analysis can be performed. Clearer results should also emerge if the subtypes of serotonin binding involved are isolated and analyzed separately. Our experiment found that the high affinity component is not 5HTiA. Even if the grouped suicides are not distinct from the controls, there may be suicide subtypes with altered binding. Our data and clinical experience both support this conclusion. Analysis of the ethanol levels suggests that subjects alcohol free at the time of death (and perhaps not alcoholics) were more likely to have abnormal serotonin binding. These data might also be interpreted as suggesting that the suicide group with higher 5HT binding experienced ethanol-induced increases that obscured greater suicide-control differences. Against this interpretation, however, is the fact that among all subjects ethanol levels did not correlate with binding. Frequently, suicides and controls who die violently have been acutely ingesting ethanol. Other studies have not found an effect of acute ethanol on serotonin binding (Hunt and Dalton, 1982). However, the relationship between chronic alcoholism and serotonin receptor status, either as an etiological or secondary effect, remains unclear. In animal studies, chronic ethanol increased striatal 3H-5HT binding, but decreased hippocampal binding (Muller et al., 1980). Wong et al. ( 1988) found lower 5HT levels and increased 3H-5HT binding in alcohol-preferring rats. In humans, Banki (1981) has reported decreased cerebrospinal fluid SHIAA in alcoholic women. The regulation of serotonin receptor numbers and the characteristics of
25 autoreceptors are not so well understood and differ somewhat from those of norepinephrine (Peroutka, 1988). The 5HT, receptors appear to be down-regulated after presynaptic cell lesions or antagonist treatment (Conn and Sanders-Bush, 1987). In early studies, 5HT, receptors appeared to be up-regulated after lesions, but the regulation of specific subtypes and regions has not been sorted out yet (Nelson et al., 1978). If our findings reflect a decreased serotonin availability previously associated with suicidality, the apparent down-regulation suggests the 5HT, pattern of regulation. There is a close relationship between 5HT, receptors and 5HTtc receptors, which are heavily concentrated in the choroid plexus, as well as diffusely at lower concentrations in other brain regions (Pazos et al., 1987a, 1987b). The response of 5HTtc receptors to agonists or antagonists has not been clarified at this point. If the pineal 5HT receptors are of the 5HTlc type, it would be consistent with the notion of the pineal as a “periventricular” organ. The functional relevance of this binding is uncertain. Serotonin receptors in the pineal might be located on pinealocytes (possibly not associated with a synapse), presynaptically on serotonergic-releasing axons, or presynaptically on noradrenergic fibers. If 5HT receptors are located on the pinealocytes, they might regulate melatonin synthesis or release, or even the excretion of serotonin. (Evidence suggests the pineal releases serotonin into the cerebrospinal fluid diurnally, which may have a physiological role; Garrick et al., 1983). If synaptic, the origin and function of any serotonin-releasing axons are uncertain. Using both formaldehyde-induced fluorescence and peroxidase-antiperoxidase immunohistochemistry preparations, Matsurra and Sano (1983) documented in dogs the presence of serotonin-containing neurons entering the pineal anteriorly that were not affected by sympathectomy. In many species, including man, fiber tracks enter the pineal anteriorly through the habenular commissure and posterior commissure which originate in the habenula, stria medullaris, hypothalamus, and lateral geniculate body (Ronnekliev and Moller, 1979; Ronnekliev et al., 1980; Moller and Korf, 1983a, 19836). Along these lines, an interesting development is the recent finding of Demisch et al. (1987) that the specific serotonin uptake blocker fluvoxamine increased the level of serum melatonin, suggesting that serotonergic neurotransmission may contribute to the release of melatonin. In addition, serotonin is stored in the sympathetic axon terminals (Zweig and Axelrod, 1969), especially in the apical (or posterior) part of the gland (Sparks and Hunsaker, 1986). This neuronal pool of serotonin is probably the result of nonspecific uptake of serotonin “leaked” from the pinealocytes. The function of this stored serotonin is unknown, but serotonin-specific heteroreceptors on sympathetic neurons might be the substrate for the binding we have found (Raiteri et al., 1986). Our failure to find time-related changes is subject to Type II error. No effort was made to assure an even distribution of times of death in controls. Other evidence clearly shows a diurnal variation in /3-adrenergic receptors (Gonzalez-Brito et al., 1988). It may be, however, that pineal 5HT receptors do not vary daily. Longestablished data have indicated increases in suicide in spring (Durkheim, 1951; Pokorny, 1968). This suggests that local environmental perturbations might intensify circadian rhythm dysfunctions, including those of mood regulation. No effort was made to control for month or season of death in our study, and no relationships were found to 5HT binding.
26
The interpretation of our suicide data should be carefully formulated. Clearly, there is substantial overlap between binding in suicides and controls. As noted before, however, suicides are a heterogeneous group. There appeared to be a subset of suicides with reduced affinity or total numbers of 5HT receptors. Dependable and correlative clinical histories might have shed light on the significance of decreased binding in these individuals. Nevertheless, in contrast to our results, other postmortem brain studies have been able to demonstrate group differences comparing uncategorized suicides to controls. The “permissive” theory of mood disorders postulates that stress-induced noradrenergic changes cause depression only when modulatory serotonergic influences are lost (Prange et al., 1974). Supportive recent work has suggested that compensatory changes in both serotonergic and noradrenergic systems are necessary for full clinical response to antidepressants (Hsiao et al., 1987). On the basis of our findings and others’ animal work, it is possible the pineal gland may model coinnervation by serotonergic and noradrenergic axons. Work is underway to replicate and further characterize our findings and to explore the relationship between serotonin and norepinephrine receptor states in the pineal. Acknowledgments. The authors thank Dr. John Hunsaker, Associate Chief Medical Examiner, for his assistance; Dr. Norman Pedigo for his help with data analysis; Virginia Lynn Gift and Debbie Howard for typographical assistance; Kimberly Hall for administrative assistance; and the anonymous reviewers from this journal for their advice.
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27 Cheatham, S.C.; Cromptom, M.R.; Katona, C.L.E.; and Horton, R.W. Brain 5-HT, receptor binding sites in depressed suicide victims. Brain Research. 443:272-280, 1988. Conn, P.J., and Sanders-Bush, E. Central serotonin receptors: Effector systems, physiological roles and regulation. Psychopharmac,ol~~~y, 92:267-277, 1987. 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.; and Tomlinson, B.E. Neurotransmitter receptors and monoamine metabolites in the brains of patients with Alzheimer-type dementia and depression, and suicides. Neuropharmacology, 23( I2B): I56 I -I 569, 1984. Delgado, P.L.; Charney, D.S.; Price, L.H.; Goodman, W.K.; Aghajanian, G.K.; and Heninger, G.R. Behavioral effects of acute tryptophan depletion in psychiatric patients and healthy subjects. Abstract, Society for Neuroscience Annual Meeting, Phoenix, AZ, October, 1989. Demisch, K.; Demisch, L.; Nickelson, T.; and Ruth, R. The influence of acute and subchronic administration of various antidepressants on early morning melatonin plasma levels in healthy subjects: Increases following fluvoxamine. Journal of Neural Transmission, 68:257270, 1987. Durkheim, E. Suicide. Glencoe, IL: Free Press, 1951. Garrick, N.A.; Tamarkin, L.; Taylor, P.L.; Markey, S.P.; and Murphy, D.L. Light and propranolol suppress the nocturnal elevation of serotonin in the cerebrospinal fluid of rhesus monkeys. Science, 22 I ~474-476, 1983. Gonzalez-Brito, A.; Jones, D.J.; Ademe, R.M.; and Reiter, R.J. Characterization and measurement of I-l25-iodopindolol binding in individual rat pineal glands. Existence of a 24-hour rhythm in P-adrenergic receptor density. Brain Research. 938: 108-I 14, 1988. Govitrapong, P.; Murrin, L.C.; and Ebadi, M. Characterizations of dopaminergic receptor sites in bovine pineal gland. Journal qf Pineal Research, I :2 15-226, 1984. Hsiao, J.; Agren, H.; Bartko, J.J.; Rudorfer, M.V.; Linnoila, M.; and Potter, W.Z. Monoamine neurotransmitter interactions and the prediction of antidepressant response. Archives of General Psychiatry, 44: IO78- 1083, 1987. Hunt, W.A., and Dalton, T.K. Neurotransmitter-receptor binding in various brain regions in ethanol-dependent rats. Pharmacology. Biochemistry, and Behavior, 141733-739, 1982. King, T.S.; Steinlechner, S.; Steger, R.W.; Richardson, R.A.; and Reiter, R.J. Tryptophan loading inhibits serotonin N-acetyltransferase activity in the rat pineal gland. Transactions Q/ the Society/or Neuroscience. 10:763, 1982. Lewy, A.J.; Nurnberger, J.1.; Wehr, T.A.; Pack, D.; Becker, L.E.; Powell, R.L.; and Newsome, D.A. Supersensitivity to light. Possible trait marker for manic-depressive illness. American Journal of’ Psychiatry, 1421725-727, 1985. Lowry, 0.; Rosebrough, N.J.; Farr, A.L.; and Randall, R.J. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry, 193:263-274, 1951. Mann, J.J.; McBride, P.A.; and Stanley, M. Post-mortem monoamine receptor and enzyme studies in suicide. Annals qf’the New York Academy of’sciences, 487:1 14-121, 19864. Mann, J.J.; Stanley, M.; McBride, P.A.; and McEwen, B.S. Increased serotonin-2 and fl-adrenergic receptor binding in the frontal cortices of suicide victims. Archives of’ General Psychiatry, 431954-959, 1986h. Matsurra, T., and Sano, Y. Distribution of monoamine-containing nerve fibers in the pineal organ of untreated and sympathectomized dogs. Cell & Tissue Research, 234:5 19-53 I, 1983. Moller, M., and Korf, H.W. Central innervation of the pineal organ of the Mongolian gerbil: A histochemical and lesion study. Ceil & Tissue Research, 230:259-272. 19834. Moller, M., and Korf, H.W. The origin of central pinealopetal nerve fibers in the Mongolian gerbil as demonstrated by the retrograde transport of horseradish peroxidase. CeN & Tissue Research. 230:273-287, 19836. Muller, P.; Britton, R.S.; and Seeman, P. The effects of long-term ethanol on brain receptors for dopamine, acetylcholine, serotonin and noradrenaline. European Journal (?/ Pharmacology, 65:3 I-37, 1980.
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Psychiatry Research, 32: 19-28 Elsevier
Altered Pineal Serotonin
Binding in Some Suicides
D. Larry Sparks and Karley Y. Little Received February 7, 1989; revised version received December 4. 1989; accepted December 26. 1989. Abstract. Serotonergic abnormalities have been regularly of suicide victims. Because of evidence that pineal function subjects and that serotonin-releasing axons may innervate tritiated serotonin binding in a series of control and suicide
reported in the brains is altered in depressed the pineal, we assayed pineals. High affinity
binding
between
was present,
and there
were
apparent
differences
some suicides
and controls.
Key Words. Suicide,
melatonin,
serotonin,
receptors.
Previous post-mortem studies in suicide victims compared to controls have revealed considerable evidence that the concentrations of serotonin (5hydroxytryptamine, 5HT) and 5hydroxyindolacetic acid (SHIAA, main serotonin metabolite) are decreased in the brainstem (see Mann et al., 1986a, for a review). Frontal cortex serotonin type 2 binding (5HT,) has been found elevated in suicide victims by some, but not all, investigators (Stanley et al., 1986b; Cheatham et al., 1988; Arora and Meltzer, 1989). lmipramine binding in frontal cortex, thought to be a marker of presynaptic serotonin activity, has been found to be both decreased (Stanley et al., 1982) and normal (Arora and Meltzer, 1987). Serotonin type I binding (SHT,), measured by tritiated serotonin, has not been found abnormal (Crow et al., 1984; Mann et al., 19866), while 5HTIA binding, a subtype measured by tritiated 8hydroxy-para-amino tetralin (8-OH-DPAT), was found increased by Arora and Meltzer (1989). The level of P-noradrenergic binding in frontal cortex has been found increased in homogenate membrane (Mann et al., 19866) and autoradiographic studies (Arango et al., 1988; Biegon and Israeli, 1988). These studies were of subjects uncategorized by probable psychiatric diagnosis. To what extent these findings represent changes attributable to mood disorders is unclear since only about 50% of suicides are so diagnosable. Because melatonin levels may be altered in suicidal and depressed patients and are derived from serotonin, some attention has also been focused on the pineal gland, the site of most melatonin synthesis. Stanley and Brown (1988) recently reported a decreased melatonin concentration in the pineal of suicides vs. controls dying during
D. Larry Sparks, Ph.D., is Research Assistant Professor, Sanders-Brown Research Center on Agin - - and Departments of Pathology and Neurology, University of Kentucky Medical Center; and Medical Examiner’s Program, Kentucky Justice Cabinet, Lexington, KY 40536. Karlev Y. Little. M.D.. is Assistant Professor, Department of Psychiatry, Universityof North Carolina School of Medicine, Chapel Hill, NC. (Reprint requests to Dr. K.Y. Little, University of North Carolina at Chapel Hill, Department of Psychiatry, CB # 7160, Medical School Wing B, Chapel Hill, NC 27599-7160, USA,) 01651781/90/$03.50
@ 1990 Elsevier Scientific
Publishers Ireland
Ltd.
20 the hours 2200-0600h, the peak melatonin-production period. Clinically, melatonin levels in suicidal inpatients have been reported to be higher than those in nonsuicidal depressed inpatients (Beck-Friis, 1985). However, other studies have found that depressed patients may have low melatonin levels (Wetterberg, 1978) a decreased nocturnal melatonin rise (Brown et al., 1985), and an increased sensitivity to the inhibiting effects of light during the night (Lewy et al., 1985). Possible explanations include diminished precursor availability, decreased pinealocyte capacity, or diminished noradrenergic and other signal input. The third explanation is most consistent with the empirical evidence and prevailing conceptual models (although recent work suggests that 5-hydroxytryptophan availability may be an important variable in explaining serotonergic function; Delgado et al. 1989). Much evidence suggests that diminished and erratic noradrenergic function occurs in serious depressive illnesses. Noradrenergic innervation by the nervi conarii (sympathetic fibers that arise in the superior cervical ganglion) markedly influences the activity of pineal serotonin-N-acetyl transferase (SNAT), the major enzyme involved in the formation of melatonin (Axelrod, 1974). However, preliminary work in our laboratory has suggested serotonin receptor binding in the human pineal. Animal work has shown low affinity ( Kd 200-I ,000 nM) Ketanserin binding (5HT,) in bovine pineal (Govitrapong et al., 1984), and blockage of tryptophan effect on rat SNAT by cyproheptadine, a serotonin-receptor antagonist (King et al., 1982), but no effect of serotonin on isoproterenol-stimulated SNAT activity in rat pineal culture (Chan and Ebadi, 1981). Because of the substantial evidence of serotonin abnormalities in suicides, we characterized pineal 5HT, binding in subjects dying suddenly and compared this binding in a series of suicides and controls. Methods Portions of human pineal were saved as part of the normal coroner-authorized autopsy protocol. Acquisition of tissue was within 24 hours of death (Perry and Perry, 1983; Whitehouse et al., 1984). The tissue was frozen at -70 ‘C until use and coded so that the technician was blind to all details about the subject. Blood-alcohol levels and a toxicological screen were performed by the state toxicological laboratory in Frankfort, KY. Identification of cases as suicides was decided by the medical examiner. Control subjects were matched for age, sex, time of death, and, to the extent possible, alcohol use. The causes of death in I I suicides included gunshot wounds (n = X), carbon monoxide (n = 2), and hanging (n = I). Twenty-three controls died from either gunshot wounds (n = 5), automotive trauma (n = 7) sudden cardiovascular causes (n = 8) asphyxia (n = 2) or perforated ulcer (n = I). For binding studies, tissue was homogenized in 5 vol (W:V) of 0.05 M Tris, pH 7.7, at 37 “C, and centrifuged at Sl,OOOg for IO min. Supernatant was discarded and the process repeated twice to ensure that the membranes were cleansed of any drugs present at the time of death (Bennett et al., 1985). Serotonin binding (5HT,) was done by the method of Nelson et al. (1978) using tritiated serotonin. Ligand and crude tissue homogenate were incubated to equilibrium at 37 “C for IO min in glass tubes. Nonspecific binding was defined as the activity remaining in the presence of IO PM unlabeled 5HT. 5HT, binding was subtyped into 5HT,, by the addition of 100 nM and 1,000 nM 8-hydroxy-N, N-dipropyl-2and non-5HT,, aminotetralin (OH-DPAT, final concentrations) to some tubes before incubation (Peroutka. 1986). Final values were based on wet weight for the initial study and protein content for the suicide study, as quantified by the methods of Lowry et al. (195 I).
21 Binding from five young and five old control subjects was measured at six ligand concentrations in a range previously found significant in other brain regions (0.5 to 8 nM). These data were analyzed using a nonlinear, least-squares iterative technique from the computer programs EBDA and LIGAND (Munson and Rodbard, 1980). In the second series of controls and suicides, binding was measured at three ligand concentrations (I, 2 and 4 nM). Differences between old and young controls, and controls vs. suicides, were tested for significance with Student’s t test. Correlations between binding values and age, time of death, month of death, post-mortem interval, and blood-alcohol level were searched for using linear regression. Mean differences in these variables were also compared for significant differences using Student’s t test. Analysis of variance (ANOVA) was performed on mean data for three daily time periods and causes of death.
Results Binding in the human pineal was detected using tritiated
gland, with apparent serotonin. Saturation
high and low affinity components, occurred at the 4 nM and 6 nM
concentrations, but binding increased at 8 nM. An initial ligand analysis using a one- or two-site model did not converge on satisfactory binding parameters, undoubtedly due to a deficiency of points representing lower affinity binding. Since saturation appeared to occur between 4 nM and 6 nM, we dropped the 8 nM point for a second analysis. When this point was dropped, evidence emerged for a high affinity site with a K,, of 6.05 + 0.84 nM and B,,, of 366.6 + 50.9 fmol/mg wet weight. Correlation coefficients for each individual Scatchard plot varied from 0.80 to 0.99. There were no differences between five young controls (average age 33 + 6 years) vs. five old controls (average age 73 + 8 years). Competitive binding analysis with OH-DPAT (5HTIA ligand) at 100 nM and 1,000 nM concentrations added to 3H-5HT in samples from seven controls showed no inhibition, confirming these sites as 5HTta, 5HTIc, or 5HT,n subtypes. Among the suicides, several subjects had consistently low binding at the three ligand concentrations (Fig. I). At 1 nM, mean (zk SD) binding in suicides averaged 1.55 + 1.52 fmol/ mg protein vs. 2.11 f 1.35 fmol/ mg protein in controls (one-tailed
Fig. 1. Serotonin binding for each suicide 16.5 10
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2nM
SEROTONINBINDINGFOR EACH SUICIDE
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22 Wilcoxon rank sum, p < 0.06). Five of 11 suicides demonstrated values below the lowest of 23 controls (0.05, 0.08, 0.08, 0.20, and 0.28 vs. 0.60), as seen in Fig. 2. Similarly at 2 nM (Fig. 3), 4 of 11 suicides were lower than the lowest control (one-tailed Wilcoxon rank sum, p < 0.05),although the means (+ SD) were not significantly different (2.89 f 2.71 vs. 4.21 f 2.60). At 4 nA4 (Fig. 4), mean (I!I SD) values were similar (suicides 6.73 + 4.38 vs. 6.16 III 3.17 for controls), and there were no rank differences. Six of 11 suicides and 8 of 23 controls had measurable blood-alcohol levels. The mean (+ SD) level in the suicides was 65 + 80 mg/dl vs. 79 I!I 127 mg/dl in the controls. There were no correlations between alcohol level and 5HT binding in the Fig. 2. 3H-serotonin binding in
suicides and controls (1 nM concentration)
Fig. 3. 3H-serotonin binding in suicides and controls (2 nM concentration) 10.0 gi
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3-H SEROTONM BINDING 4nM Cont. combined sample of suicides and controls. However, among the five suicides with the lowest levels of 5HT binding, only one had a measurable ethanol level, as compared to five of the other six suicides. Mean (+ SD) ethanol level was 22.6 + 50.5 mg/dl (one at I 13 mg/dl) in the low subgroup, as compared to 101.7 + 88.8 mg/dl in the high subgroup (one-tailed t test, p < 0.06). Among suicides at the 2 nM 5HT concentration, there was a positive correlation between ETOH level and binding (r = 0.68, p = 0.02). These results suggest that those suicides with higher 5HT binding, equivalent to controls, might have shown abnormally lower binding if they had been free of ethanol. Mean time of death was 1llOh in controls and did not correlate with 5HT binding. In the five suicides with lower binding, times of death were OlOOh, 0400h, 0800h, 1600h, and 1800h. Mean 5HT binding did not differ among the three time periods, 0600-1400h, 1400-22OOh, and 2200-0600h. The mean (91 SD) post-mortem interval was similar between suicides and controls (13.8 + 5.3 hours vs. 14.2 + 7.6 hours) and was not correlated with 5HT binding. Among gunshot victims (suicides = 8, controls = 5) there were no differences in
24 5HT binding. Combined values for gunshot victims vs. subjects dying by other means showed no significant difference. Age was similar in suicides (36 + 15 years) vs. controls (40 + 17 years) and was not correlated with 5HT binding. Two suicides and two controls were female. By inspection, their binding values were unexceptional. Discussion To our knowledge, this is the first report of serotonin binding in the human pineal gland. The data presented strongly support the existence of a saturable high affinity site. We further believe our results suggest that two components of serotonin binding occur. If so, lower affinity 5HT binding would have contributed to an overestimate of & (6.05 nM) and B,,, (367 fmol/mg wet weight) for the high affinity site. Our point-to-point comparisons between suicides and controls were done at concentrations that we believe to be relevant to the high affinity site. If the high affinity Kd actually occurs in the 4- to 6-nM range as our computer-generated one-site model suggests, our finding at 4 nM showing suicides equivalent to controls is strong evidence that important differences do not exist. The diminished binding levels at I- and 2-nM concentrations would then be attributable to changes in affinity, most likely an artifact. If, however, the actual Kd is in the l-to 3-nM range, as we suspect, our group differences may indicate significant alterations in receptor function in the suicides. Unfortunately, the current studies did not anticipate overlapping binding at two sites and were not performed in a manner that would allow resolution of this question. Studies need to be done at more and higher 3H-5HT concentrations so that lower affinity binding can be characterized and an accurate two-site analysis can be performed. Clearer results should also emerge if the subtypes of serotonin binding involved are isolated and analyzed separately. Our experiment found that the high affinity component is not 5HTiA. Even if the grouped suicides are not distinct from the controls, there may be suicide subtypes with altered binding. Our data and clinical experience both support this conclusion. Analysis of the ethanol levels suggests that subjects alcohol free at the time of death (and perhaps not alcoholics) were more likely to have abnormal serotonin binding. These data might also be interpreted as suggesting that the suicide group with higher 5HT binding experienced ethanol-induced increases that obscured greater suicide-control differences. Against this interpretation, however, is the fact that among all subjects ethanol levels did not correlate with binding. Frequently, suicides and controls who die violently have been acutely ingesting ethanol. Other studies have not found an effect of acute ethanol on serotonin binding (Hunt and Dalton, 1982). However, the relationship between chronic alcoholism and serotonin receptor status, either as an etiological or secondary effect, remains unclear. In animal studies, chronic ethanol increased striatal 3H-5HT binding, but decreased hippocampal binding (Muller et al., 1980). Wong et al. ( 1988) found lower 5HT levels and increased 3H-5HT binding in alcohol-preferring rats. In humans, Banki (1981) has reported decreased cerebrospinal fluid SHIAA in alcoholic women. The regulation of serotonin receptor numbers and the characteristics of
25 autoreceptors are not so well understood and differ somewhat from those of norepinephrine (Peroutka, 1988). The 5HT, receptors appear to be down-regulated after presynaptic cell lesions or antagonist treatment (Conn and Sanders-Bush, 1987). In early studies, 5HT, receptors appeared to be up-regulated after lesions, but the regulation of specific subtypes and regions has not been sorted out yet (Nelson et al., 1978). If our findings reflect a decreased serotonin availability previously associated with suicidality, the apparent down-regulation suggests the 5HT, pattern of regulation. There is a close relationship between 5HT, receptors and 5HTtc receptors, which are heavily concentrated in the choroid plexus, as well as diffusely at lower concentrations in other brain regions (Pazos et al., 1987a, 1987b). The response of 5HTtc receptors to agonists or antagonists has not been clarified at this point. If the pineal 5HT receptors are of the 5HTlc type, it would be consistent with the notion of the pineal as a “periventricular” organ. The functional relevance of this binding is uncertain. Serotonin receptors in the pineal might be located on pinealocytes (possibly not associated with a synapse), presynaptically on serotonergic-releasing axons, or presynaptically on noradrenergic fibers. If 5HT receptors are located on the pinealocytes, they might regulate melatonin synthesis or release, or even the excretion of serotonin. (Evidence suggests the pineal releases serotonin into the cerebrospinal fluid diurnally, which may have a physiological role; Garrick et al., 1983). If synaptic, the origin and function of any serotonin-releasing axons are uncertain. Using both formaldehyde-induced fluorescence and peroxidase-antiperoxidase immunohistochemistry preparations, Matsurra and Sano (1983) documented in dogs the presence of serotonin-containing neurons entering the pineal anteriorly that were not affected by sympathectomy. In many species, including man, fiber tracks enter the pineal anteriorly through the habenular commissure and posterior commissure which originate in the habenula, stria medullaris, hypothalamus, and lateral geniculate body (Ronnekliev and Moller, 1979; Ronnekliev et al., 1980; Moller and Korf, 1983a, 19836). Along these lines, an interesting development is the recent finding of Demisch et al. (1987) that the specific serotonin uptake blocker fluvoxamine increased the level of serum melatonin, suggesting that serotonergic neurotransmission may contribute to the release of melatonin. In addition, serotonin is stored in the sympathetic axon terminals (Zweig and Axelrod, 1969), especially in the apical (or posterior) part of the gland (Sparks and Hunsaker, 1986). This neuronal pool of serotonin is probably the result of nonspecific uptake of serotonin “leaked” from the pinealocytes. The function of this stored serotonin is unknown, but serotonin-specific heteroreceptors on sympathetic neurons might be the substrate for the binding we have found (Raiteri et al., 1986). Our failure to find time-related changes is subject to Type II error. No effort was made to assure an even distribution of times of death in controls. Other evidence clearly shows a diurnal variation in /3-adrenergic receptors (Gonzalez-Brito et al., 1988). It may be, however, that pineal 5HT receptors do not vary daily. Longestablished data have indicated increases in suicide in spring (Durkheim, 1951; Pokorny, 1968). This suggests that local environmental perturbations might intensify circadian rhythm dysfunctions, including those of mood regulation. No effort was made to control for month or season of death in our study, and no relationships were found to 5HT binding.
26
The interpretation of our suicide data should be carefully formulated. Clearly, there is substantial overlap between binding in suicides and controls. As noted before, however, suicides are a heterogeneous group. There appeared to be a subset of suicides with reduced affinity or total numbers of 5HT receptors. Dependable and correlative clinical histories might have shed light on the significance of decreased binding in these individuals. Nevertheless, in contrast to our results, other postmortem brain studies have been able to demonstrate group differences comparing uncategorized suicides to controls. The “permissive” theory of mood disorders postulates that stress-induced noradrenergic changes cause depression only when modulatory serotonergic influences are lost (Prange et al., 1974). Supportive recent work has suggested that compensatory changes in both serotonergic and noradrenergic systems are necessary for full clinical response to antidepressants (Hsiao et al., 1987). On the basis of our findings and others’ animal work, it is possible the pineal gland may model coinnervation by serotonergic and noradrenergic axons. Work is underway to replicate and further characterize our findings and to explore the relationship between serotonin and norepinephrine receptor states in the pineal. Acknowledgments. The authors thank Dr. John Hunsaker, Associate Chief Medical Examiner, for his assistance; Dr. Norman Pedigo for his help with data analysis; Virginia Lynn Gift and Debbie Howard for typographical assistance; Kimberly Hall for administrative assistance; and the anonymous reviewers from this journal for their advice.
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