Life Sciences, Vol . 21, pp . 933-936 Printed in the II .S .A .
NEUROTRANSMITTER METABOLITES IN THS CEREBROSPINAL FLIIID OF MAN FOLLOWING PHYSOSTIGMINE Rennette L . Davis l , Leo $. Hollisterl Frederick K. Goodwin 2, Edna R. Cordon 2 1 Veterans Administration Hospital and Stanford University School of Medicine, Palo Alto, CA 2 National Institute of Mental Health, Hethesda, MD 20014
(Received in final form August 23, 1977 A 3 .0 mg dose of physostigmine or normal saline was given intravenously to 23 normal subjects who had been treated with probenecid. Homovanillic acid and 3-methoxy-4-hydroxyphenylglycol concentrations were significantly higher in the lumbar cerebrospinal fluid of subjects who received physoatigmine than subjects who received normal saline . This establishes biochemical evidence for a cholinergic link in the central nerwus system of man . Physostigmine, a short acting reversible acetylcholinesterase inhibitor, increases brain acetylcholine and cholinergic transmission . The drug has a brief beneficial action in manic depressive psychosis, Huntington's disease and tardive dyskineaia (1 ; 2, 3) . Hy altering cteolinergic activity, physostigmine might also affect the turnover and release of other neurotransmitters. If so, its mechanism of action in these neuropsychiatric conditions could be quite co~licated . in an attempt to determine the effect of physostigmine on brain dopamine, norepinephrine and aerotonin, the drug was administered to normal subjects and their lumbar cerebrospinal fluid assayed for homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and .'3-methoxy-4-hydroxyphenylglycol (MHPG) . METHODS Twenty-three normal wluateer subjects between the ages of 21 and 55 were recruitedt 17 mere men, six were women. None had any personal or family his tory of psychiatric illness . All were rigorously familiarized with the study design and gave their informed consent. Subjects did not know whether they would receive physoatigmine or placebo . Fourteen subjects received physostigmine and nine received a placebo infusion . Subjects were hospitalized at either the General Clinical Research Center at Stanford Medical Center or the Psychiatric Clinical Research Center at the Palo Alto Veterans Administration Hospital . All spent one afternoon and night in the hospital prior to the physoatigmine or placebo infusion that occurred the following morning. At 9 :30 AM on the infusion day all subjects received 1 .0 mg of methacopolamine bromide subcutaneously to block the peripheral cholinamiThis dose produced a tachycardia in all mstic properties of physoatigmine . Assignsubjects . At 10 AM either physostigmine or placebo infusion was begun . ment to treatments was ranäom, although more subjects were assigned to physostigmine . Physostigmine 0.5 mq in 0.5 ml of 0 .25 N saline, or only the saline 933
934 itself, After a ml were of 0 .25
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was administered intravenously every four minutes until 2 ml were given . ten minute pause for cognitive assessment, two additional doses of 0 .5 given five minutes apart . A total of 3 .0 mq of physostigmine or 3 .0 ml N saline was administered in approximately 30 minutes .
Subjects were pretrented with probenecid (100 mq/kq in four divided doses) . The first dose (30 mg/kg) was given at 10 PM on the day prior to the physostiqmine infusion . The following three doses of 23 mq/kg each were given at 3 AM, 8 AM and 1 PM on the day of the infusion . Probenecid, by inhibiting the active transport system required for the removal of the two acid metabolites (5-HIAA and HVA) from brain CSF to blood, results in their acoumulation in the CSF (4) . Animal (5) ae well as clinical (6) data suggest that this technique provides a measure which more closely reflects central nerwus system amine trunover than do baseline metabolite levels alone . Lumbar puncture was performed at 4 PM, with the removal of 25 ml of CSF . Subjects were at bed rest from 7 PM on day one until three hours after the lumbar puncture . The CSF was analyzed for homovanillic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and probenecid by gas chromatography-mass spectrometry methods (7) . Analysis of 5-hydroxyindoleacetic acid (5-HIAA) used spectroflurometric procedures (8) . RESULTS Values for HVA, MHPG, 5-HIAA and probenecid for the group of subjects who received physostigmine and for those data for the group who received saline are shown in Table 1 . The t-teat for unpaired data of unequal sample size was used to determine if the groups differed (Table 2) . Subjects who received physostigmine had significantly higher levels of HVA and MHPG in CSF than the group receiving saline . The two groups did not significantly differ in 5-HIAA concentrations or in probenecid levels, suggesting that the results with the catecholamine metabolites could not be attributed to non-specific transport differences or to differential effectiveness of probenecid blocade . HVA assays were done on every subject, and probenecid and HIAA assessments on all but one aubject~ the MHPG data were leas complete . TABLE 1 Concentrations of HVA, 5-HIAA, MHPG and Probenecid in Cerebrospinal Fluid of Subjects Treated with Physostigmine-Methscopolamine and Subjects Treated with Saline-Methscopolamine Ph soati N = z ~
= S .E . ~
e-Metha c~ lamine Group
14 195 .1 ng/ml 55 .2 14 .8
13 94 .3 ng/ml 32 .7 9 .1
11 11 .2 ng/ml 3 .1 1 .0
13 16 .0 uq/ml 4 .2 1 .2
Saline-Methacopolamine Group N = x~ d = S .E . a
9 148 .2 nq/ml 44 .3 14 .8
9 ~ 73 .1 nq/ml 41 .4 13 .8
5 5 .9 ng/ml 4 .9 2 .2
9 15 .0 ug/ml 2 .9 1 .0
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C3F Neurotraasaitter Metabolitae TABLE 2
Mean (Standard Deviation) Values for Concentrations of BVA, 5-SIAA, MBPG and Probenecid in Cerebrospinal Fluid
Phyaostigmine-Methscopolamine Saline-Methscopolamine p value
195 .1(55) 148 .2(44) .05
94 .3(26 .6) 73 .1(38 .5) n .e .
11 .2(7 .2) 5 .9(5 .7) < " 02
Probenecid 16 .0(0 .4) 15 .0(0 .3) n .s .
p values calculated by t-teat of unpaired data, taro tailed . SVA, 5-HIAA, MBPG values in aq/mlt Probenecid in uq/ml . DISCU38ION This is the first demonstration in humane of altered CSF HVA or MBPG folThese results are lowing systemic administration of a choh *+~++~i+~tic agent. consistent with studies in animals showing elevated SVA concentrations in brain following chol i~i~ tica (9, 10, 11) . Hoth physoatigmine and oxotremorine increa~e dopamine turnover in rat brain striatum and limbic system (9-11, 12-16) . Atropine has the opposite effect (17-20) . HVA in human lumbar CSF probably originates in ventricular pools of the metabolite, with little contribution from the cord (21) . Animal studies indicate that the majority of the HVA in the third sad fourth ventricles originates from dopamine metabolism in the striatum (22,23) . Thus elevated lumbar C3F SVA probably reflects elevated atriatal HVA. Anatomical, biochemical and pharmacologic data demonstrate an interaction between dopaminargic and cholinergic neurons in the neostriatum. However, the nature of this interaction is extremely complex. Cholinergic regulation of do pamine turnover at the nigral level is precisely the opposite of atriatal regulation (9-11, 12-16, 24, 25) . Furthermore, while stimulation of atriatal muacarinic cholinergic receptors may block dopamine release, stimulation of atriatal nicotinic receptors appears to augment dopamine release (26, 27) . Thus a non-specific cholinergic agent like phyeoetigmine could mediate both increased and decreased dopamine release by stimulating nicotinic and muscarinic receptors . The relationship between dopamine release, transmission and degradation, Either increased or deae reflected by HVA, is glso not straightforward . creased dopaminergic activity can cause increased production of SVA (28) . All of these factors complicate the interpretation of elevated HVA in lumbar CSF following systemic administration of phyeostigmine . Any interpretation should be consistent with the previously reported physostigmine-induced de crease in the frequency of abaormnl movements in Huntington's disease and tarIn general, both conditions are improved by drugs dive dyskinesia (29-31) . that decrease dopaminergic activity and worsened by agents that increase dopaminergic activity, possibly by blocking presynaptic release . Elevated HVA could reflect preeynaptic degradation . However, since there may be both excitatory and inhibitory dopamine receptors, increased dopaminergic activity at appropriate receptors could theoretically decrease the frequency of abnormal movements in Huntington's disease and tardive dyekineala (33) . The extent to which MSPG in lumbar C8F reflects brain aorepinephriae meSpinal cord perfusion studies demonstrate that part of tabolism ie unclear. the MHPG fro® lumbar CSF has its origins in the spinal cord (34), Thus, al though a cholinergic effect on norepinephrine release has been found in rat brain this study only yields indirect evidence for the existence of this inter-
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