Psychological Medicine, 1977, 7, 599-605 Printed in Great Britain
Cyclic AMP in cerebrospinal fluid of manic and depressive patients ROBERT M. POST,1 HINRICH CRAMER AND FREDERICK K. GOODWIN From the Sections on Psychiatry and Psychobiology, National Institute of Mental Health, Bethesda, Maryland USA, and the Department of Neurology and Neurophysiology, University of Freiburg, Germany
Cyclic 3',5'-adenosine monophosphate (c-AMP) was measured in cerebrospinal fluid (CSF) of manic and depressive patients with and without probenecid administration both before and during treatment with various psychotropic drugs. Oral probenecid (100 mg/kg) produced substantial c-AMP accumulations in CSF suggesting a probenecid-sensitive transport mechanism for c-AMP. Baseline and probenecid-induced accumulations of c-AMP were not significantly different in manic and depressed patients, while baseline levels in depressed patients were higher than those in neurological controls. Imipramine, amitriptyline, lithium, tryptophan, and electroconvulsant therapies did not significantly alter levels or accumulations of c-AMP in CSF of depressed patients.
SYNOPSIS
INTRODUCTION Measurement of cyclic 3',5'-adenosine monophosphate (c-AMP) in psychiatric populations has been stimulated by a large body of data suggesting a key role for c-AMP in neuronal transmission (McAfee et al. 1971), especially receptormediated processes for the biogenic amines (Daly, 1976; Greengard, 1976). Shimiza etal. (1971) and Carenzi et al. (1975) reported a dopamine and norepinephrine-sensitive adenylate cyclase system in the human brain; this system was not sensitive to serotonin. Recently, Kebabian et al. (1975) suggested that cyclic AMP may be increased by dopaminergic transmission in bovine superior cervical ganglia. Stone et al. (1975) reported that in pyramidal cells in the cerebral cortex norepinephrine and cyclic AMP both produced inhibitory effects on cell firing. However, it appears that correlations between cyclic AMP metabolism and behaviour may vary widely in different areas of brain. For example, Skolnick & Daly (1974) reported positive correlations with motor activity and cyclic AMP in midbrainstriatal areas, but negative correlations in the cerebral cortex. Dopamine-induced increases in adenylate cyclase activity in animal systems appear to be 1 Address for correspondence: Dr Robert M. Post, Section on Psychobiology, Adult Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20014, USA.
inhibited by neuroleptic drugs in a close relationship to their clinical antipsychotic potency (Clement-Cormier et al. 1974; Iversen, 1975). Moreover, alterations in cyclic AMP activity have been related to the processes of sub- and supersensitivity of catecholamine receptors (Iversen, 1975; Palmer & Scott, 1974; Axelrod, 1974). Recently, alterations in cyclic nucleotides have been postulated to play a critical role in the development of tolerance to narcotic drugs (Collier et al. 1975; Sharma et al. 1975; Merali et al. 1975). Most current strategies for assessment of central nervous system neurotransmitter function in man, such as measurement of CSF amine metabolites, have reflected presynaptic amine mechanisms; to the extent that measurement of cyclic nucleotides in human CSF holds the potential for reflecting postsynaptic receptor function, study of cyclic AMP was initiated. Although several studies of urinary cyclic AMP have been reported, interpretation of the results is complicated by the fact that the nucleotide in that body fluid is largely of peripheral origin (Sutherland, 1970). Moreover, urinary cyclic AMP is altered by exercise (Eccleston et al. 1970), which in part may explain why the urinary data from patients with affective illness are inconsistent and controversial (Table 1). Abdulla & Hamadah (1970) and Paul et al. (1970) reported urinary cyclic AMP as low in depression, while Brown et al.
599
600
Robert M. Post Hinrich Cramer and Frederick K. Goodwin Table 1. Inconsistencies in studies of c-AMP in urine of psychiatric patients Investigators
State studied
Abdulla & Hamadah (1970) Paul etal. (1970,19716) Brown « a/. (1972) Jenner etal. (1972) Hamadah etal. (1972) Naylor era/. (1974) Paul et al. (1971a) Huliin etal. (1974) Perry etal. (1973) Eccleston etal. (1970)
Depression Depression Depression and mania Depression and mania Depression Depression Switch into mania Switch into mania Periodic catatonia Exercise
(1972) and Jenner et al. (1972) were unable to replicate this finding. Naylor et al. (1974) reported cyclic AMP levels in depressed patients increased with recovery and Hamadah et al. (1972) also found an increase following electroconvulsive therapy. While Paul et al. (1971a) reported increases in urinary cyclic AMP associated with the switch from depression into mania, Huliin et al. (1974) were unable to replicate this finding and Perry et al. (1973) reported no significant alterations in urinary cyclic AMP during episodes or the rapid switch in periodic catatonia. In an attempt to measure cyclic AMP of central nervous system origin more directly, Robinson et al. (1970) studied pooled samples of cerebrospinal fluid (CSF) and reported no marked differences in levels in the manic or depressive pool compared with a neurologic control group. In a preliminary communication (Cramer et al. 1972a), our group reported that cyclic AMP in CSF tended to be higher in depressed patients compared with manic patients or neurological controls, contrary to the earlier findings of lowered urinary cyclic AMP in some depressed patients. We now report data from a larger series of patients studied both prior to and during treatment with various psychotropic medications. In addition, accumulations of cyclic AMP in CSF following probenecid administration are reported; probenecid blocks the transport of various organic acids out of CSF and also produces large increases in cyclic AMP levels in CSF. METHOD Patients with primary affective illness diagnosed according to the criteria of Feighner et al. (1972) as revised by Spitzer et al. (1975) were studied on two in-patient metabolic research units at the
Finding Low Low Not significant Not significant Increase with recovery Increase with recovery Increase Not significant Not significant Increase
National Institute of Mental Health; informed consent was obtained prior to all procedures. Depressed patients were moderately to severely ill and were subclassified as unipolar or bipolar on the basis of the absence or presence of a prior history of mania. Most manic patients displayed symptomatology typical of hypomania or Stages I and II (Carlsson & Goodwin, 1973). Patients were rated twice daily on global items of depression, mania, psychosis, anxiety and anger on a 15-point scale derived from that of Bunney & Hamburg (1963). They were maintained on a low catecholamine diet and intake of non-prescribed medication, including caffeine, was restricted. Baseline lumbar punctures were performed at 9.00 a.m. after a night's bedrest, and again at 3 p.m. following 18 hours of probenecid administration (100 mg/kg) in 4 divided doses as previously described (Goodwin et al. 1973). Twelve to 15 cm 3 of CSF were removed for other clinical and biological studies, including measurement of amine metabolites, following which an aliquot of 1 cm3 was removed for analysis of cyclic AMP and frozen at - 70 °C until analysed. The patients were studied initially after a drug-free period of at least 2 weeks and again during the course of treatment with various drugs. Six patients received L-tryptophan (8-9 g daily); 10 either imipramine or amitriptyline at doses of 150-250 mg a day for 10-29 days; 9 patients were studied on lithium carbonate for 13-54 days maintaining a serum level of 0-7 to 1-15 mEq/1; 1 patient following 24 days treatment with alpha methyl-/>-tyrosine (AMPT) at peak doses of 4 g; and 6 heroin addict patients were studied while receiving methadone (80-90 mg/ day) as maintenance therapy. Six patients were studied before and after a short series of electroconvulsive treatment (mean = 8) for severe
601
Cyclic AMP in cerebrospinal fluid 20 r
15
10
\MP(t
Neurological controls
*
Depression
Mania
Depression
Mania
70 60 50 40 30
Probenecidinduced accumulations
20 10 0
Fio. 1. Cyclic AMP in CSF of manic and depressed patients.
depression unresponsive to prior pharmacological therapy. The procedures were also repeated in 5 depressed patients who were asked to participate in moderate to intense physical activity 4 hours prior to lumbar puncture study in order to assess the effects of psychomotor activity, roughly equivalent to that typically observed in mania (Post et al. 1973). In collaboration with T. N. Chase, drug-free, baseline (but not probenecid) LPs were obtained from neurological controls (Cramer ef al. 19726). Using the method of Gilman (1970), cyclic AMP was analysed directly in CSF, giving results identical to those obtained with prior chromatographic isolation of the nucleotide on anion exchange columns. Concentrations of cyclic AMP are expressed as picomoles per ml, each value representing the mean of at least 3 determinations. 39
Table 2. Lack of effect of A hours of motor activity on c-AMP in CSF Condition Bed rest, baseline (N = 5) Activity, baseline (N = 5)
c-AMP (p-mole/ml±s.E.M.) 13-4±2-3
11-6± 1-8
RESULTS Cyclic AMP levels in the CSF of patients with affective illness ranged from 3 to 26 picomoles/ml and increased approximately 300 % following 18 hours of probenecid administration. Fig. 1 illustrates values in patients with depression and mania compared with neurological controls. Depressed patients did not differ from manics in either baseline levels of cyclic AMP or probenecid-induced accumulations. Baseline levels of cyclic AMP were slightly, but significantly, higher in depressed patients than neurological controls PSM 7
Robert M. Post, Hinrich Cramer and Frederick K. Goodwin
602
(P < 005). Levels of c-AMP were not correlated with severity of rated depression (r = 004, NS) and did not differ according to diagnostic subgroups of unipolar or bipolar depression, or between agitated or retarded depression. Consistent with this was the finding that 4 hours of hyperactivity prior to lumbar puncture did not produce significant alterations in cyclic AMP levels (Table 2). Table 3. Lack of correlation of c-AMP in CSF of depressed patients with other variables Baseline r Age Severity of depression Severity of anxiety Severity of anger HVA 5HIAA MHPG
80
000 -004 002
003 -009 -010 010
Probenecid r -011 -0-28 0 02 -014 0-25 -005 0-33
Baseline or probenecid-induced accumulations of cyclic AMP did not correlate with severity of other symptoms, age, sex, or with the simultaneously obtained levels of the metabolites of serotonin (5HIAA), dopamine (HVA), or norepinephrine (MHPG) (see Table 3). A significant correlation was found between the probenecidinduced accumulation of cyclic AMP and the absolute dose of probenecid administered (r = 0-53, P < 001). Fig. 2 summarizes the findings in patients studied during treatment with tricyclic antidepressants (imipramine or amitriptyline), lithium, tryptophan, methadone, as well as immediately following electroconvulsive therapy. In both paired comparisons, or in relation to medication-free depressed patients (Student's t test for independent samples), these treatment strategies did not produce significant alterations in either baseline or probenecid-induced accumulations of cyclic AMP. The one exception was in
r
| ^^M
70
| Probenecid Baseline
60
50 o o. S
40
30 JV=23
N=1
Drugfree
Tricyclics
JV=4
N=3
N=S
20
10
Lithium
Tryptophan Methadone
ECT
FIG. 2. Lack of effect of psychotropic treatments on cyclic AMP in CSF.
603
Cyclic AMP in cerebrospinal fluid
the small number of patients studied on Ltryptophan who showed a lower level of probenecid-induced accumulation of cyclic AMP compared with the entire group of depressed patients. DISCUSSION Although little is known about the precise origin of cyclic nucleotides in cerebrospinal fluid and their transport characteristics, these findings and those of Cramer et al. (1972a, b) in CSF and Davoren & Sutherland (1963) in isolated erythrocytes suggest that c-AMP is eliminated by a probenecid-sensitive transport mechanism. Moreover, Sebens & Korf (1975) demonstrated that cyclic AMP administered intravenously did not penetrate into the cerebrospinal fluid. In their study of cyclic AMP in cisternal cerebrospinal fluid in the rabbit, they concluded that the nucleotide was of central origin, was eliminated by a probenecid-sensitive transport mechanism, and was increased by intracisternally-administered noradrenaline, isoprenaline, dopamine and histamine. Although Sebens & Korf (1975) suggest that cyclic AMP in CSF is released directly from central nervous system into CSF, clearly further work is needed to elucidate the anatomical and neurotransmitter specificity of cyclic AMP in CSF, including the possible contribution from lumbar spinal cord (Brooks et al. 1976). While the technique appears to hold some promise, it appears premature to consider that cyclic AMP in human lumbar cerebrospinal fluid is directly related to a specific central nervous system neurotransmitter or receptor function. The high correlation of probenecid-induced accumulations with the absolute dose of probenecid (administered on 100 mg/kg basis) suggests the possibility that transport characteristics may play a critical role in the concentration of c-AMP found in CSF. The results of the present study highlight that the findings of low urinary cyclic AMP in some studies of depressed patients (Table 1) are not paralleled by similar changes in cerebrospinal fluid. Baseline levels and probenecid-induced accumulations of cyclic AMP were not significantly different in depressed patients compared with manics, while depressed patients tended to have higher baseline levels compared with neurological controls. While this small increase in
mean c-AMP level is unlikely to be of physiological importance, it is of interest that, in one patient subsequently treated with AMPT for 24 days, high baseline (17-5) and probenecid-related accumulations (880) of c-AMP were noted. This could be consistent with the idea that catecholamine depletion and associated increased receptor sensitivity might be related to c-AMP levels in CSF. While these speculations may be of theoretical interest, the bulk of the findings in this study, including the correlational and probenecid data, do not support the idea of increased c-AMP in depression. This conclusion is consistent with the work of Robinson et al. (1970) and Geisler et al. (1977). Moreover, in further NIMH studies of cyclic nucleotides in CSF, there were not significant differences in either c-AMP or c-GMP among depressed, manic and schizophrenic patients and controls (Smith et al. 1976). A number of pharmacological and somatic treatments thought to affect brain cyclic AMP did not produce significant alterations in either baseline or probenecid-induced accumulations of cyclic AMP. These findings are consistent with those of Cramer et al. (1972Z>) and Heikkinen et al. (1974) in man, and Sebens & Korf (1975) in rabbits, showing that reserpine, haloperidol, L-dopa and tricyclic antidepressants (with the exception of maprotiline) did not affect probenecid-induced accumulations of cyclic AMP in cisternal CSF. Although systemic administration of these drugs did not produce significant alterations, intracisternally injected noradrenaline, isoprenaline, dopamine and histamine all produced increases in cyclic AMP in the rabbit. Intravenous administration of isoprenaline did increase CSF c-AMP (Sebens & Korf, 1975) and propanalol blocked L-dopa-induced increases in cisternal CSF c-AMP in the rat (Kiessling et al. 1975), suggesting a relationship between c-AMP in CSF to a beta adrenergic receptor mechanism, at least in these species. The lack of effect of lithium on baseline levels or probenecid-induced accumulations of c-AMP is of particular interest since lithium has been shown to inhibit transmitter- or hormonestimulated adenylase cyclase activity (Forn & Valdecasas, 1971; Forn, 1975; Ebstein et al. 1976). In these strategies lithium did not affect baseline (non-stimulated) levels of c-AMP and it may require a provocative agent to uncover 39-2
604
Robert M. Post, Hinrich Cramer and Frederick K. Goodwin
any potential effect of lithium on c-AMP concentrations in CSF. As discussed in the introduction, there is a growing body of data indicating that cyclic nucleotides are intimately related to basic processes of neural transmission and receptor alterations and may be associated with the phenomena of tolerance and supersensitivity. However, if further knowledge of these functions in man is to be gained from indirect measures of cyclic nucleotide levels in CSF, additional basic work on the relationship of CSF to central nervous system c-AMP will be necessary.
REFERENCES AbduUa, Y. H. & Hamadah, K. (1970). 3',5'-Cydic adenosine monophosphate in depression and mania. Lancet i, 378381. Axelrod, J. (1974). The pineal gland: a neurochemical transducer. Science 184, 1341-1348. Brooks, B. R., Sode, J. & Engel, W. K. (1976). Cyclic nucleotide metabolism in motor neuron disease. In Research Trends in Amyotrophic Lateral Sclerosis. UCLA Forum in Medical Sciences, vol. 19 (ed. J. M.Andrews and R. T. Johnson), pp. 101-118. Academic Press: New York. Brown, B. L., Salway, J. G., Albano, J. D. M., Hullin, R. P. & Ekins, R. P. (1972). Urinary excretion of cyclic AMP and manic-depressive psychosis. British Journal of Psychiatry 120,405^108. Bunney, W. E., Jr & Hamburg, D. A. (1963). Methods for reliable longitudinal observation of behaviour. Archives of General Psychiatry 9, 280-294. Carenzi, A., Gillin, J. C , Guidotti, A., Schwartz, M. A., Trabucchi, M. & Wyatt, R. J. (1975). Dopamine-sensitive adenyl cyclase in human caudate nucleus. Archives of General Psychiatry 32, 1056-1059. Carlsson, G. A. & Goodwin, F. K. (1973). The stages of mania: a longitudinal analysis of the manic episode. Archives of General Psychiatry 28, 221-228. Clement-Cormier, Y. C , Kebabian, J. W., Petzold, G. L. & Greengard, P. (1974). Dopamine-sensitive adenylate cyclase in mammalian brain: a possible site of action of antipsychotic drugs. Proceedings of the National Academy of Sciences of the USA 71, 1113-1117. Collier, H. O. J., Francis, D. L., McDonald-Gibson, W. J., Roy, A. C. & Saeed, S. A. (1975). Prostaglandins, cyclic AMP and the mechanism of opiate dependence. Life Sciences 17, 85-90. Cramer, H., Goodwin, F. K., Post, R. M. & Bunney, W. E., Jr (1972a). Effects of probenecid and exercise on cerebrospinal fluid cyclic AMP in affective illness. Lancet i, 1346— 1347. Cramer, H., Ng, L. K. Y. & Chase, T. N. (19726). Effect of probenecid on levels of cyclic AMP in human cerebrospinal fluid. Journal of Neurochemistry 19, 1601-1602. Daly, J. W. (1976). The nature of receptors regulating the formation of c-AMP in brain tissue. Life Sciences 18, 1349-1358. Davoren, P. R. & Sutherland, E. W. (1963). The effect of L-epinephrine and other agents on the synthesis and release of adenosine 3',5'-phosphate by whole pigeon erythrocytes. Journal of Biological Chemistry 238, 3009-3015. Ebstein, R., Belmaker, R., Grunhaus, L. & Rimon, R. (1976).
Lithium inhibition of adrenaline-stimulated adenylate cyclase in humans. Nature 259, 411-413. Eccleston, D., Loose, R., Pullar, I. A. & Sugden, R. F. (1970). Exercise and urinary excretion of cyclic AMP. Lancet ii, 612-613. Feighner, J. P., Robins, E., Guze, S. B., Woodruff, R. A., Jr, Winokur, G. & Munoz, R. (1972). Diagnostic criteria for use in psychiatric research. Archives of General Psychiatry 26, 57-63. Forn, J. (1975). Lithium and cyclic AMP. In Lithium Research and Therapy (ed. F. N. Johnson), pp. 485-497. Academic Press: New York. Forn, J. & Valdecasas, F. G. (1971). Effect of lithium on brain adenyl cyclase activity. Biochemical Pharmacology 20, 2773-2779. Geisler, A., Bech, P., Johannesen, M. & Rafaelson, O. J. (1977). Cyclic AMP levels in cerebrospinal fluid of manicmelancholic patients. Neuropsychobiology 2, 211-220. Gilman, A. G. (1970). A protein binding assay for adenosine 3',5'-cyclic monophosphate. Proceedings of the National Academy of Sciences of the USA 67, 305-312. Goodwin, F. K., Post, R. M., Dunner, D. L. & Gordon, E. K. (1973). Cerebrospinal fluid amine metabolites in affective illness: the probenecid technique. American Journal of Psychiatry 130, 73-79. Greengard, P. (1976). Possible role for cyclic nucleotides and phosphorylated membrane proteins in postsynaptic actions of neurotransmitters. Nature 260, 101-108. Hamadah, K., Holmes, H., Barker, G. B., Hartmen, G. C. & Parke, D. V. W. (1972). Effect of electric convulsion therapy on urinary excretion of 3',5'-cyclic adenosine monophosphate. British Medical Journal iii, 439-441. Heikkinen, E. R., Myllyla, V. V., Vapaatalo, H. & Hokkanen, E. (1974). Urinary excretion and cerebrospinal fluid concentration of cyclic adenosine-3',5'-monophosphate in various neurological diseases. European Neurology 11, 270-280. Hullin, R. P., Salway, J. G., Allsopp, M. N. E., Barnes, G. D., Albano, J. D. M. & Brown, B. L. (1974). Urinary cyclic AMP in the switch process from depression to mania. British Journal of Psychiatry 125, 457-458. Iversen, L. L. (1975). Dopamine receptors in the brain. A dopamine-sensitive adenylate cyclase models synaptic receptors, illuminating antipsychotic drug action. Science 188, 1084-1089. Jenner, F. A., Sampson, G. A., Thompson, E. A., Somerville, A. R., Beard, N. A. & Smith, A. A. (1972). Manicdepressive psychosis and urinary excretion of cyclic AMP. British Journal of Psychiatry 121, 236-237. Kebabian, J. W., Steiner, A. L. & Greengard, P. (1975). Muscarinic cholinergic regulation of cyclic guanosine 3\5'-monophosphate in autonomic ganglia: possible role in synaptic transmission. Journal of Pharmacology and Experimental Therapeutics 193, 474-488. Kiessling, M., Lindl, T. & Cramer, H. (1975). Cyclic adenosine monophosphate in cerebrospinal fluid effects of theophylline, L-dopa and a dopamine receptor stimulant in rats. Archiv fur Psychiatrie und Nervenkrankheiten 220, 325-333. McAfee, D. A., Schorderet, M. & Greengard, P. (1971). Adenosine 3\5'-monophosphate in nervous tissue: increase associated with synaptic transmission. Science 171, 11561158. Merali, Z. R., Singhal, L., Hrdina, P. D. & Ling, G. M. (1975). Changes in brain cyclic AMP metabolism and acetylcholine and dopamine during narcotic dependence and withdrawal. Life Sciences 16, 1889-1894. Naylor, G. J., Stansfield, D. A., Whyte, S. F. & Hutchinson, F. (1974). Urinary excretion of adenosine 3',5'-cyclic monophosphate in depressive illness. British Journal of Psychiatry 125,275-279.
Cyclic AMP in cerebrospinal fluid Palmer, G. C. & Scott, H. R. (1974). The cyclic AMP response to noradrenalin in young adult rat brain following postnatal injections of 6-hydroxydopamine. Experientia 30, 520-521. Paul, M. I., Ditzion, B. R., Pauk, G. L. & Janowsky, D. S. (1970). Urinary adenosine 3',5'-monophosphate excretion in affective disorders. American Journal of Psychiatry 126, 1493-1497. Paul, M. I., Cramer, H. & Bunney, W. E., Jr (1971 a). Urinary adenosine 3',5'-monophosphate in the switch process from depression to mania. Science 171, 298-303. Paul, M. I., Cramer, H. & Goodwin, F. K. (19716). Urinary cyclic AMP excretion in depression and mania. Archives of General Psychiatry 24, 327-333. Perry, T. L., Hemmings, S., Drummond, G. I., Hansen, S. & Gjessing, L. R. (1973). Urinary cyclic AMP in periodic catatonia. American Journal of Psychiatry 130, 927-929. Post, R. M., Kotin, J., Goodwin, F. K. & Gordon, E. K. (1973). Psychomotor activity and cerebrospinal fluid amine metabolites in affective illness. American Journal of Psychiatry 130, 67-72. Robinson, G. A., Coppen, A. J., Whybrow, P. C. & Prange, A. J. (1970). Cyclic AMP in affective disorders. Lancet ii, 1028-1029. Sebens, J. B. & Korf, J. (1975). Cyclic AMP in cerebrospinal fluid: accumulation following probenecid and biogenic amines. Experimental Neurology 46, 333-344.
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Sharma, S. K., Klee, W. A. & Nirenberg, M. (1975). Dual regulation of adenylate cyclase accounts for narcotic dependence and tolerance. Proceedings of the National Academy of Sciences of the USA 72, 3092-3096. Shimizu, H., Tanaka, S., Suzuki, T. & Matsukado, Y. (1971). The response of human cerebrum adenyl cyclase to biogenic amines. Journal of Neurochemistry 18, 1157-1161. Skolnick, P. & Daly, J. W. (1974). Norepinephrine-sensitive adenylate cyclases in rat brain: relation to behaviour and tyrosine hydroxylase. Science 184, 175-177. Smith, C. S., Tallman, J. F., Post, R. M., van Kammen, D. P., Jimerson, D. C , Brown, G. L., Brooks, B. R. & Bunney, W. E., Jr (1976). An examination of baseline and drug-induced levels of cyclic nucleotides in the cerebrospinal fluid of control and psychiatric patients. Life Sciences 19, 131-136. Spitzer, R. L., Endicott, J. & Robins, E. (1975). Clinical criteria for psychiatric diagnosis and DSM III. American Journal of Psychiatry 32, 1187-1192. Stone, T. W., Taylor, D. A. & Bloom, F. E. (1975). Cyclic AMP and cyclic GMP may mediate opposite neuronal responses in the rat cerebral cortex. Science 187, 845-847. Sutherland, E. W. (1970). On the biological role of cyclic AMP. Journal of the American Medical Association 214, 1281-1288.
Cyclic AMP in cerebrospinal fluid of manic and depressive patients ROBERT M. POST,1 HINRICH CRAMER AND FREDERICK K. GOODWIN From the Sections on Psychiatry and Psychobiology, National Institute of Mental Health, Bethesda, Maryland USA, and the Department of Neurology and Neurophysiology, University of Freiburg, Germany
Cyclic 3',5'-adenosine monophosphate (c-AMP) was measured in cerebrospinal fluid (CSF) of manic and depressive patients with and without probenecid administration both before and during treatment with various psychotropic drugs. Oral probenecid (100 mg/kg) produced substantial c-AMP accumulations in CSF suggesting a probenecid-sensitive transport mechanism for c-AMP. Baseline and probenecid-induced accumulations of c-AMP were not significantly different in manic and depressed patients, while baseline levels in depressed patients were higher than those in neurological controls. Imipramine, amitriptyline, lithium, tryptophan, and electroconvulsant therapies did not significantly alter levels or accumulations of c-AMP in CSF of depressed patients.
SYNOPSIS
INTRODUCTION Measurement of cyclic 3',5'-adenosine monophosphate (c-AMP) in psychiatric populations has been stimulated by a large body of data suggesting a key role for c-AMP in neuronal transmission (McAfee et al. 1971), especially receptormediated processes for the biogenic amines (Daly, 1976; Greengard, 1976). Shimiza etal. (1971) and Carenzi et al. (1975) reported a dopamine and norepinephrine-sensitive adenylate cyclase system in the human brain; this system was not sensitive to serotonin. Recently, Kebabian et al. (1975) suggested that cyclic AMP may be increased by dopaminergic transmission in bovine superior cervical ganglia. Stone et al. (1975) reported that in pyramidal cells in the cerebral cortex norepinephrine and cyclic AMP both produced inhibitory effects on cell firing. However, it appears that correlations between cyclic AMP metabolism and behaviour may vary widely in different areas of brain. For example, Skolnick & Daly (1974) reported positive correlations with motor activity and cyclic AMP in midbrainstriatal areas, but negative correlations in the cerebral cortex. Dopamine-induced increases in adenylate cyclase activity in animal systems appear to be 1 Address for correspondence: Dr Robert M. Post, Section on Psychobiology, Adult Psychiatry Branch, National Institute of Mental Health, Bethesda, Maryland 20014, USA.
inhibited by neuroleptic drugs in a close relationship to their clinical antipsychotic potency (Clement-Cormier et al. 1974; Iversen, 1975). Moreover, alterations in cyclic AMP activity have been related to the processes of sub- and supersensitivity of catecholamine receptors (Iversen, 1975; Palmer & Scott, 1974; Axelrod, 1974). Recently, alterations in cyclic nucleotides have been postulated to play a critical role in the development of tolerance to narcotic drugs (Collier et al. 1975; Sharma et al. 1975; Merali et al. 1975). Most current strategies for assessment of central nervous system neurotransmitter function in man, such as measurement of CSF amine metabolites, have reflected presynaptic amine mechanisms; to the extent that measurement of cyclic nucleotides in human CSF holds the potential for reflecting postsynaptic receptor function, study of cyclic AMP was initiated. Although several studies of urinary cyclic AMP have been reported, interpretation of the results is complicated by the fact that the nucleotide in that body fluid is largely of peripheral origin (Sutherland, 1970). Moreover, urinary cyclic AMP is altered by exercise (Eccleston et al. 1970), which in part may explain why the urinary data from patients with affective illness are inconsistent and controversial (Table 1). Abdulla & Hamadah (1970) and Paul et al. (1970) reported urinary cyclic AMP as low in depression, while Brown et al.
599
600
Robert M. Post Hinrich Cramer and Frederick K. Goodwin Table 1. Inconsistencies in studies of c-AMP in urine of psychiatric patients Investigators
State studied
Abdulla & Hamadah (1970) Paul etal. (1970,19716) Brown « a/. (1972) Jenner etal. (1972) Hamadah etal. (1972) Naylor era/. (1974) Paul et al. (1971a) Huliin etal. (1974) Perry etal. (1973) Eccleston etal. (1970)
Depression Depression Depression and mania Depression and mania Depression Depression Switch into mania Switch into mania Periodic catatonia Exercise
(1972) and Jenner et al. (1972) were unable to replicate this finding. Naylor et al. (1974) reported cyclic AMP levels in depressed patients increased with recovery and Hamadah et al. (1972) also found an increase following electroconvulsive therapy. While Paul et al. (1971a) reported increases in urinary cyclic AMP associated with the switch from depression into mania, Huliin et al. (1974) were unable to replicate this finding and Perry et al. (1973) reported no significant alterations in urinary cyclic AMP during episodes or the rapid switch in periodic catatonia. In an attempt to measure cyclic AMP of central nervous system origin more directly, Robinson et al. (1970) studied pooled samples of cerebrospinal fluid (CSF) and reported no marked differences in levels in the manic or depressive pool compared with a neurologic control group. In a preliminary communication (Cramer et al. 1972a), our group reported that cyclic AMP in CSF tended to be higher in depressed patients compared with manic patients or neurological controls, contrary to the earlier findings of lowered urinary cyclic AMP in some depressed patients. We now report data from a larger series of patients studied both prior to and during treatment with various psychotropic medications. In addition, accumulations of cyclic AMP in CSF following probenecid administration are reported; probenecid blocks the transport of various organic acids out of CSF and also produces large increases in cyclic AMP levels in CSF. METHOD Patients with primary affective illness diagnosed according to the criteria of Feighner et al. (1972) as revised by Spitzer et al. (1975) were studied on two in-patient metabolic research units at the
Finding Low Low Not significant Not significant Increase with recovery Increase with recovery Increase Not significant Not significant Increase
National Institute of Mental Health; informed consent was obtained prior to all procedures. Depressed patients were moderately to severely ill and were subclassified as unipolar or bipolar on the basis of the absence or presence of a prior history of mania. Most manic patients displayed symptomatology typical of hypomania or Stages I and II (Carlsson & Goodwin, 1973). Patients were rated twice daily on global items of depression, mania, psychosis, anxiety and anger on a 15-point scale derived from that of Bunney & Hamburg (1963). They were maintained on a low catecholamine diet and intake of non-prescribed medication, including caffeine, was restricted. Baseline lumbar punctures were performed at 9.00 a.m. after a night's bedrest, and again at 3 p.m. following 18 hours of probenecid administration (100 mg/kg) in 4 divided doses as previously described (Goodwin et al. 1973). Twelve to 15 cm 3 of CSF were removed for other clinical and biological studies, including measurement of amine metabolites, following which an aliquot of 1 cm3 was removed for analysis of cyclic AMP and frozen at - 70 °C until analysed. The patients were studied initially after a drug-free period of at least 2 weeks and again during the course of treatment with various drugs. Six patients received L-tryptophan (8-9 g daily); 10 either imipramine or amitriptyline at doses of 150-250 mg a day for 10-29 days; 9 patients were studied on lithium carbonate for 13-54 days maintaining a serum level of 0-7 to 1-15 mEq/1; 1 patient following 24 days treatment with alpha methyl-/>-tyrosine (AMPT) at peak doses of 4 g; and 6 heroin addict patients were studied while receiving methadone (80-90 mg/ day) as maintenance therapy. Six patients were studied before and after a short series of electroconvulsive treatment (mean = 8) for severe
601
Cyclic AMP in cerebrospinal fluid 20 r
15
10
\MP(t
Neurological controls
*
Depression
Mania
Depression
Mania
70 60 50 40 30
Probenecidinduced accumulations
20 10 0
Fio. 1. Cyclic AMP in CSF of manic and depressed patients.
depression unresponsive to prior pharmacological therapy. The procedures were also repeated in 5 depressed patients who were asked to participate in moderate to intense physical activity 4 hours prior to lumbar puncture study in order to assess the effects of psychomotor activity, roughly equivalent to that typically observed in mania (Post et al. 1973). In collaboration with T. N. Chase, drug-free, baseline (but not probenecid) LPs were obtained from neurological controls (Cramer ef al. 19726). Using the method of Gilman (1970), cyclic AMP was analysed directly in CSF, giving results identical to those obtained with prior chromatographic isolation of the nucleotide on anion exchange columns. Concentrations of cyclic AMP are expressed as picomoles per ml, each value representing the mean of at least 3 determinations. 39
Table 2. Lack of effect of A hours of motor activity on c-AMP in CSF Condition Bed rest, baseline (N = 5) Activity, baseline (N = 5)
c-AMP (p-mole/ml±s.E.M.) 13-4±2-3
11-6± 1-8
RESULTS Cyclic AMP levels in the CSF of patients with affective illness ranged from 3 to 26 picomoles/ml and increased approximately 300 % following 18 hours of probenecid administration. Fig. 1 illustrates values in patients with depression and mania compared with neurological controls. Depressed patients did not differ from manics in either baseline levels of cyclic AMP or probenecid-induced accumulations. Baseline levels of cyclic AMP were slightly, but significantly, higher in depressed patients than neurological controls PSM 7
Robert M. Post, Hinrich Cramer and Frederick K. Goodwin
602
(P < 005). Levels of c-AMP were not correlated with severity of rated depression (r = 004, NS) and did not differ according to diagnostic subgroups of unipolar or bipolar depression, or between agitated or retarded depression. Consistent with this was the finding that 4 hours of hyperactivity prior to lumbar puncture did not produce significant alterations in cyclic AMP levels (Table 2). Table 3. Lack of correlation of c-AMP in CSF of depressed patients with other variables Baseline r Age Severity of depression Severity of anxiety Severity of anger HVA 5HIAA MHPG
80
000 -004 002
003 -009 -010 010
Probenecid r -011 -0-28 0 02 -014 0-25 -005 0-33
Baseline or probenecid-induced accumulations of cyclic AMP did not correlate with severity of other symptoms, age, sex, or with the simultaneously obtained levels of the metabolites of serotonin (5HIAA), dopamine (HVA), or norepinephrine (MHPG) (see Table 3). A significant correlation was found between the probenecidinduced accumulation of cyclic AMP and the absolute dose of probenecid administered (r = 0-53, P < 001). Fig. 2 summarizes the findings in patients studied during treatment with tricyclic antidepressants (imipramine or amitriptyline), lithium, tryptophan, methadone, as well as immediately following electroconvulsive therapy. In both paired comparisons, or in relation to medication-free depressed patients (Student's t test for independent samples), these treatment strategies did not produce significant alterations in either baseline or probenecid-induced accumulations of cyclic AMP. The one exception was in
r
| ^^M
70
| Probenecid Baseline
60
50 o o. S
40
30 JV=23
N=1
Drugfree
Tricyclics
JV=4
N=3
N=S
20
10
Lithium
Tryptophan Methadone
ECT
FIG. 2. Lack of effect of psychotropic treatments on cyclic AMP in CSF.
603
Cyclic AMP in cerebrospinal fluid
the small number of patients studied on Ltryptophan who showed a lower level of probenecid-induced accumulation of cyclic AMP compared with the entire group of depressed patients. DISCUSSION Although little is known about the precise origin of cyclic nucleotides in cerebrospinal fluid and their transport characteristics, these findings and those of Cramer et al. (1972a, b) in CSF and Davoren & Sutherland (1963) in isolated erythrocytes suggest that c-AMP is eliminated by a probenecid-sensitive transport mechanism. Moreover, Sebens & Korf (1975) demonstrated that cyclic AMP administered intravenously did not penetrate into the cerebrospinal fluid. In their study of cyclic AMP in cisternal cerebrospinal fluid in the rabbit, they concluded that the nucleotide was of central origin, was eliminated by a probenecid-sensitive transport mechanism, and was increased by intracisternally-administered noradrenaline, isoprenaline, dopamine and histamine. Although Sebens & Korf (1975) suggest that cyclic AMP in CSF is released directly from central nervous system into CSF, clearly further work is needed to elucidate the anatomical and neurotransmitter specificity of cyclic AMP in CSF, including the possible contribution from lumbar spinal cord (Brooks et al. 1976). While the technique appears to hold some promise, it appears premature to consider that cyclic AMP in human lumbar cerebrospinal fluid is directly related to a specific central nervous system neurotransmitter or receptor function. The high correlation of probenecid-induced accumulations with the absolute dose of probenecid (administered on 100 mg/kg basis) suggests the possibility that transport characteristics may play a critical role in the concentration of c-AMP found in CSF. The results of the present study highlight that the findings of low urinary cyclic AMP in some studies of depressed patients (Table 1) are not paralleled by similar changes in cerebrospinal fluid. Baseline levels and probenecid-induced accumulations of cyclic AMP were not significantly different in depressed patients compared with manics, while depressed patients tended to have higher baseline levels compared with neurological controls. While this small increase in
mean c-AMP level is unlikely to be of physiological importance, it is of interest that, in one patient subsequently treated with AMPT for 24 days, high baseline (17-5) and probenecid-related accumulations (880) of c-AMP were noted. This could be consistent with the idea that catecholamine depletion and associated increased receptor sensitivity might be related to c-AMP levels in CSF. While these speculations may be of theoretical interest, the bulk of the findings in this study, including the correlational and probenecid data, do not support the idea of increased c-AMP in depression. This conclusion is consistent with the work of Robinson et al. (1970) and Geisler et al. (1977). Moreover, in further NIMH studies of cyclic nucleotides in CSF, there were not significant differences in either c-AMP or c-GMP among depressed, manic and schizophrenic patients and controls (Smith et al. 1976). A number of pharmacological and somatic treatments thought to affect brain cyclic AMP did not produce significant alterations in either baseline or probenecid-induced accumulations of cyclic AMP. These findings are consistent with those of Cramer et al. (1972Z>) and Heikkinen et al. (1974) in man, and Sebens & Korf (1975) in rabbits, showing that reserpine, haloperidol, L-dopa and tricyclic antidepressants (with the exception of maprotiline) did not affect probenecid-induced accumulations of cyclic AMP in cisternal CSF. Although systemic administration of these drugs did not produce significant alterations, intracisternally injected noradrenaline, isoprenaline, dopamine and histamine all produced increases in cyclic AMP in the rabbit. Intravenous administration of isoprenaline did increase CSF c-AMP (Sebens & Korf, 1975) and propanalol blocked L-dopa-induced increases in cisternal CSF c-AMP in the rat (Kiessling et al. 1975), suggesting a relationship between c-AMP in CSF to a beta adrenergic receptor mechanism, at least in these species. The lack of effect of lithium on baseline levels or probenecid-induced accumulations of c-AMP is of particular interest since lithium has been shown to inhibit transmitter- or hormonestimulated adenylase cyclase activity (Forn & Valdecasas, 1971; Forn, 1975; Ebstein et al. 1976). In these strategies lithium did not affect baseline (non-stimulated) levels of c-AMP and it may require a provocative agent to uncover 39-2
604
Robert M. Post, Hinrich Cramer and Frederick K. Goodwin
any potential effect of lithium on c-AMP concentrations in CSF. As discussed in the introduction, there is a growing body of data indicating that cyclic nucleotides are intimately related to basic processes of neural transmission and receptor alterations and may be associated with the phenomena of tolerance and supersensitivity. However, if further knowledge of these functions in man is to be gained from indirect measures of cyclic nucleotide levels in CSF, additional basic work on the relationship of CSF to central nervous system c-AMP will be necessary.
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