Lumbar cerebrospinal fluid homovanillic acid concentrations are higher in female than male non-human primates

Brain Research, 334 (1985) 375-379

375

Elsevier BRE 20799

Lumbar cerebrospinal fluid homovanillic acid concentrations are higher in female than male non-human primates RICHARD F. SEEGAL Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, N Y 12201 (U.S.A.)

(Accepted December 18th, 1984) Key words: cerebrospinal fluid (CSF) - - homovanillic acid (HVA) - - non-human primates - - sexual dimorphism

Concentrations of homovanillic acid and 5-hydroxyindoleacetic acid were determined in lumbar cerebrospinal fluid from intact male and female Macaca nemestrina. Concentrations of homovanillic acid, but not 5-hydroxyindoleacetic acid were significantly higher in the females under baseline conditions. Following probenecid treatment, 5-hydroxyindoleacetic acid concentrations were significantly elevated over baseline conditions in both sexes although only homovanillic acid concentrations were significantly higher in the female monkeys. Recent evidence indicates that central neurotransmitters not only influence the release of hormones of pituitary originl0, but also respond to alterations in circulating concentrations of pituitary and gonadal hormones 1~.20.21,30. Evidence of this neuroendocrine interaction is most clear for dopamine ( D A ) which has been identified as the inhibitory factor controlling the release of prolactin and luteinizing h o r m o n e from the anterior pituitary 13. In turn, the activity of dopaminergic neurons in the tuberoinfundibular and arcuate regions of the hypothalamus have been shown to be influenced by circulating prolactin. The nigroneostriatal dopaminergic system may also be influenced by circulating hormones. Physiological modifications of neurotransmitter function induced by exposure to exogenously applied estradiol benzoate or valerate in ovariectomized female rats include: increased density of D A receptors6,7; altered apomorphine or amphetamine-induced rotation in unilaterally lesioned animals2,3,15; increased excitability of striatal neurons27; increased neuroleptic-induced catalepsy4; and changes in concentrations of dopamine metabolites and activity of dopaminergic neurons in rat striatum during the estrus cycle 5,1s,23. Because many of these changes are observed in hypophysectomized rats, estrogen probably directly af-

fects dopaminergic neurons rather than acting indirectly by increasing circulating levels of prolactin or other pituitary factors. One means of assessing central dopaminergic function, particularly in the human or non-human primate, is by measuring concentrations of metabolites of D A such as homovanillic acid ( H V A ) in cerebrospinal fluid (CSF). In addition to providing a means of repeatedly sampling neurochemical function, this technique is particularly well-suited to estimating dopaminergic activity in the striatum because CSFH V A originates primarily from that structure 25,31. This study was undertaken to determine whether dopaminergic activity, estimated by determination of CSF concentrations of H V A , differs between intact adult male and female non-human primates. In addition to baseline (no drug) determinations of H V A concentrations, probenecid was employed to inhibit a probenecid sensitive transport system which actively removes acid metabolites such as H V A and 5H I A A from the central nervous system. This test has been successfully applied to studies of central dopaminergic function in humans s. Subjects consisted of 5 male Macaca nemestrina (pig-tailed macaques) weighing between 7 and 9 kg each and 4 female M. nemestrina weighing between 4

Correspondence." R. F. Seegal, Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201, U.S.A.

0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

376 and 6 kg each. All animals were purchased from Charles River Primate Imports. Examination of dentition revealed that the animals were between 4 and 6 years of age at the beginning of the experiment. For baseline determination of metabolite concentrations, the animals were anesthetized with ketamine-HCl (Ketaset, 5 - 7 mg/kg) and two 1-ml aliquots of CSF were obtained by lumbar puncture within 15 rain of their receiving the Ketaset. It has recently been determined that ketamine-HCl induces no detectable change in central dopaminergic function if samples are collected within 15 min following admin-

istration of the anesthetic t. All taps wero made between 14.00 and 16.00 h in order to avoid any circadian variations in concentrations of biogenic amine metabolites. A minimum of two weeks was permitted between spinal taps. For determination of CSF-HVA concentrations under probenecid, the monkeys received an intravenous (i.v.) injection of 100 mg/kg probenecid (92.8 mg/ml) administered at the rate of 1 ml/min. Lumbar CSF-taps were carried out under Ketaset anesthesia 5 h after the animals received the probenecid. Concentrations of H V A and 5-HIAA in CSF were

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MINUTES Fig. 1. A: 50~1 of a 2:1 0.2 N perchloric acid diluted cerebrospinat fluid sample from a baseline (no drug) male Macaca nemestrina. B: 50#1 of a 2:1 0.2 N perchloric acid diluted cerebrospinal fluid sample from a baseline (no drug) female M. nernestrina. C: 50.ul of a 2:1 0.2 N perchloric acid diluted cerebrospinal fluid sample from the same male M. nemestrina as in A. Monkey treated with 100 mg/kg probenecid (i.v.). Cerebrospinat fluid sample collected 5 h after i.v. probenecid. D: 50kd of a 2:1 0.2 N perchloric acid diluted cerebrospinal fluid sample from the same female M. nernestrina as in B. Monkey treated with 100 mg/kg probenecid (i.v.). Cerebrospinal fluid sample collected 5 h after i.v. probenecid.

377 determined by high-performance liquid chromatography employing electrochemical detection (HPLC-ECD). The chromatographic system consisted of a Waters 6000A chromatography pump (Waters Assoc., Milford, MA), a Supelco 5/~ C-18 reverse phase column, a Waters 710 B WISP autosampler, a Bio-Analytical Systems LC-SA detection cell, a laboratory built amperometric detector and a Hewlett-Packard 3385A data automation system. The mobile phase consisted of 100 mM NaH2PO 4, 2.6 x 10 -3 M octyl sodium sulfate, 1 × 10-4 M (Na2)EDTA, 2.5 x 10 -4 M triethylamine and 16% M E O H at pH 4.15 and was pumped at a flow rate of 1 ml/min through the column which was heated to 35 °C. The thin-layer cell was maintained at 0.8 V with respect to a silver-silver chloride reference electrode. Examination of half-wave potentials of H V A and 5-HIAA in the standards and the putative peaks in CSF indicated that they were virtually identical. Thus, we are indeed measuring these compounds in CSF. A portion of the second I ml aliquot of CSF was diluted 2:1 with 0.2 N perchloric acid and centrifuged at 13,000 rpm to precipitate protein in the CSF. A 50 /~l aliquot was injected onto the column. The above described chromatographic system allowed baseline resolution of acid metabolites of biogenic amines at levels as low as 50 pg/ml. Chromatograms of 50/~l of 2:1 perchloric acid diluted CSF samples from a male and female monkey under both baseline and probenecid conditions are presented in Fig. 1 A - D . Employing the M a n n Whitney U-test, a powerful non-parametric equivalent of the Student's t-test, it was shown that baseline H V A concentrations were significantly higher in the females than the males (Fig. 2A; P < 0.05) with no significant differences in 5 - H I A A concentrations noted between the male and female monkeys. Similar sex-related increases in CSF-HVA concentrations were observed 5 h after treatment with 100 mg/kg of probenecid (Fig. 2B); H V A concentrations were significantly higher in females than males (P < 0.01). Although 5 - H I A A concentrations were elevated following exposure to probenecid, no sex differences in CSF concentrations of 5 - H I A A were noted. The data indicate that CSF-HVA concentrations, under both baseline and probenecid conditions, are significantly higher in the intact adult female monkey

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than in male monkeys of comparable age and are in agreement with results obtained by Young and Ervin 28. Because of the sharp rostral-caudal gradient of H V A concentrations in CSF, the high concentrations of H V A in lateral ventricular fluid and the dramatic increase in H V A concentrations in CSF following electrical stimulation of the striatum 24, most of the H V A in CSF almost certainly originates in the striatum. Measurement of dopamine metabolite concentrations or neurotransmitter/metabolite ratios have been used to gain an estimate of neuronal activity or turnover, with elevated concentrations of metabolite indicative of greater neuronal activity22. 29. Thus, the higher concentrations of H V A in spinal fluid of female monkeys would imply a higher basal level of central dopaminergic activity, occurring primarily in the striatum. The present experiment is not capable of distinguishing the agent(s) responsible for the observed sexual dimorphism in CSF concentrations of HVA, although the obtained results are not due to male/female differences in CSF concentrations of tyrosine, a finding supported by results obtained by Young and Ervin 28. However, several lines of evidence derived from rodent studies suggest that ovarian hormones, and in particular estrogen, are capable of modifying striatal dopaminergic function2-4,6, 7A5. One explanation for the observed results is based on the assumption that these neuromodulator(s), at least in the stri-

378 atum, may act as weak dopamine receptor blocking

line conditions. Finally, Ebert et al~~ demonstrated

agents, activating a normally inhibitory feedback

that, following varying doses of probcnecid in the~ n o n - h u m a n primate, increases m H V A concentrations in lumbar CSF closely parallel increases in ven-

loop that, in central dopamine neurons, regulates their activity, resulting in increased D A turnover and higher levels of D A metabolites 26. These results have been discussed in light of the central actions of ovarian or pituitary hormones. However, because dopaminergic metabolism, presumably originating from the striatum, is estimated from a remote site, it is conceivable that these results could be due either to mate/female differences in the efficiency with which acid metabolites are removed from the CNS or simply reflect biogenic amine metabolism in spinal cord. However, two related observations suggest that the elevated H V A concentrations in lumbar cerebrospinal fluid of females are not due to the above explanations. If the higher H V A concentrations in females, observed under probenecid, were due to greater sensitivity to this drug, than it would be expected that other acid metabolites, sen-

tricular CSF (thought to exclusively reflect striatal dopamine metabolism) whereas 5 - H I A A concentrations are elevated only in lumbar CSF (and thereby presumably reflect cord metabolisnt). Indeed, further indirect evidence that 5-H1AA, measured in lumbar CSF, reflects non-sexually dimorphic cord metabolism of 5-HT is provided by Young and Ervin 2~ who noted higher concentrations of 5 - H I A A in cisternal CSF of female than male v e r v e t monkeys. This disparity in obtained results is most fikely due to the fact that cisternal CSF, because of its location, more accurately measures cortical 5-HT metabolism s than does m e a s u r e m e n t of 5 - H I A A in lumbar CSE', These results are in the same direction as those re-

show a similar sex-linked increase. Although there was a significant increase in 5 - H I A A concentrations under probenecid, there were no significant differ-

ported in humans 19 and provide further supportive evidence for the role of endogenous ovarian or pituitary factors as modulators of central dopaminergic activity, It is hoped that estimation of central dopaminergic activity, by m e a s u r e m e n t of metabolite concentrations in CSF, will prove usefui in devel-

ences between male and female monkeys. Similarly, only H V A and not 5 - H I A A demonstrated significant differences between males and females u n d e r base-

oping a therapeutic regimen for reducing the frequency and severity of neuroleptic-induced tardive dyskinesias.

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