Effect of age on cisternal cerebrospinal fluid concentrations of monoamine metabolites in nonhuman primates

Neurochem. Int. Vol. 13, No. 3, pp. 353-357, 1988 Printed in Great Britain. All rights reserved

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EFFECT OF AGE ON CISTERNAL CEREBROSPINAL FLUID CONCENTRATIONS OF M O N O A M I N E METABOLITES IN N O N H U M A N PRIMATES STEVEN E. SHELTON) NED H. KALIN, 1 JOHN P. GLUCK,3 MICHAEL F. KERESZTURY,2 VICKI A. ~ E R ~ and MARK H. LEWIS2'* ~psychiatry Service, William S. Middleton Memorial Veterans Hospital, Madison, WI 53705 and Department of Psychiatry, University of Wisconsin Medical School, Madison, WI 53792, 2Biological Sciences Research Center and Department of Psychiatry, University of North Carolina, Chapel Hill, NC 27514 and 3Department of Psychology, University of New Mexico, Albuquerque, NM 8713I, U.S.A. (Received 25 January 1988; accepted 30 March 1988)

Almemct--There are conflicting reports of the effects of aging onhuman neurotrausmitter systems as estimated by monoamine metabolite concentrations in cerebrospinal fluid (CSF). These discrepancies may be due to sampling site, age or sex of the subjects or other variables that affect CSF mctabolite determinations. Cisternal CSF concentrations of homovanillic acid (HVA), 3-raethoxy-4-hydroxypbenylethylene glycol (MHPG) and 5-hydroxyindoleacetic acid (5-HIAA), major mctabolites of dopamine, norepinephrine and serotonin, respectively, were measured in rhesus monkeys (Macaca mulatta) of two age groups. Concentrations of HVA and MHPG were significantly lower in the older group of monkeys, whereas no changes in 5-HIAA were found. This supports the hypothesis that brain catecholamine concentrations decline with age.

Cerebrospinal fluid (CSF) concentrations of homovanillic acid (HVA), 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylethylene glycol (MHPG), major metabolites of dopamine (DA), serotonin (5-HT) and norepinephrine (NE), respectively, have been used to estimate central nervous system metabolism of these neurotransmitters (Moir et al., 1970). Alterations of these neurotransmitter systems have been reported to be associated with aging, and are implicated in numerous behavioral changes and disease states, including Parkinsonism (Chase et al., 1976), psychotic depression (Asberg and Ikrtilsson, 1978; Post and Goodwin, 1974; van Praag et al., 1973), Alzheimer's disease (Adolfsson eta/., 1979a; Bareggi et al., 1982) and schizophrenia (Lake et al., 1980; Sedvall and Wode-Helgodt, 1980). Knowledge of age-related changes in these neurotransmitter systems will contribute to an understanding of the consequences of aging and their

*Address all correspondence to: Mark H. Lewis, Biological Sciences Research Center, CB No. 7250, BSRC, University of North Carolina, Chapel Hill, NC 27599-7250, U.S.A.

relationship to age-related central nervous system disorders. However, these systems are difficult to study in normal humans because CSF samples are generally taken from the lumbar region, a site less reflective of brain activity than ventricular or cisternal sites. For example, rostral-caudal gradients have been demonstrated for HVA and 5-HIAA (Gordon et al., 1975; Guidberg eta/., 1966) and there is the likelihood of increased contribution of some acid metabolites to CSF from the spinal cord (Wood, 1980). In many studies, CSF is sampled as part of other diagnostic procedures in subjects who suffer from neurological or organic disorders. Such samples may not be representative of a normal population. Other factors that may affect CSF metabolite levels are difficult to control in human studies. These include the time of day when the sample is collected (perlow et al., 1977), diet (Eccleston et al., 1970) and activity (Post et al., 1973). The importance of these variables has been recently reviewed by Kraemer (1985). The purpose of the present study was to determine if there are age-related changes in cisternal CSF concentrations of HVA, 5-HIAA and M H P G in nonhuman primates. Rhesus monkeys were used because they are biologically similar to humans and

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because it is possible to control i m p o r t a n t sources of variability. EXPERIMENTAL PROCEDURE

Experimental animals Subjects were 69 single-cage housed rhesus monkeys (Macaca mulatta), all of which were in good health. They were normally fed at 06:00 h, received water ad libitum, and were maintained on a 24-h light/dark cycle with lights on from 06:00 to 18:00h. Subjects were divided into two groups based on age. The younger group included 34 subjects (13 female and 21 male), with a mean age (_+SEM) of 5.4 __+0.3 years (range 2.8-10.3). Older monkeys included 35 subjects (28 female and 7 mate), with a mean age of 18.5 + 0.3 years (range 15.5-23.1). According to Cutler (1978), rhesus monkeys reach sexual maturity at 4 years of age, and decline in health and vigor beginning at 15 years. In our experience, some features of aging are generally seen after 20 years, with perimenopausal changes appearing at 2(~25 years and ovulatory function ceasing by about 30 years. The maximum age reported for a rhesus monkey is 37 years (Bowden and Jones, 1979). Fifty-six of the experimental animals had previously been used in other studies. In one study (n = 21), 11 monkeys were exposed to 5ppt of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD) transplacentally throughout gestation and transmammary for the first 4 months of life from mothers exposed for the previous 3.5 years. TCDD exposure was discontinued 2.2 years prior to CSF sampling. In a second study (n = 17), 9 monkeys were exposed daily to lead acetate in their drinking water at a dose that ranged from 2 to 10 mg/kg/day. This was done to control blood lead levels. The subjects were exposed to lead for 6-7 years over a 9 year period. Lead exposure was discontinued 2-2.7 years prior to CSF sampling. A final study (n = 18) involved socially isolating 11 monkeys during the first year of life. All animals were reared at the Harlow Primate Laboratory at the University of Wisconsin, although 18 old monkeys (8 male and 10 female) have been housed at the University of New Mexico for approximately the last 15 years. Statistical analysis indicted that neither TCDD, lead nor social isolation had any significant effect on CSF monoamine metabelite concentrations. CSF determinations from animals housed at the University of New Mexico were included in the analysis to increase the number of old animals and to allow for an analysis of age by sex interactions. CSF sampling On the day CSF was obtained, the 06:00 h feeding was withheld. Between 09:30 and 10:30h the subjects were anesthetized with ketamine HC1 (10.0 mg/kg, i.m,) and held in the lateral decubitus position, and a 2 ml CSF sample was obtained by percutaneous puncture to the cisterua rnagna. Bacopoulos et al. (1979) have demonstrated that 15 min after administration, this dose of ketamine does not significantly alter the concentration of the acid metabolities in monkey CSF, but it does increase the concentrations after 4 h. CSF samples used in this study were taken from 5 to 28 min after ketamine administration and the time did not differ significantly between the groups (13.2 + 1.0min for the younger group and 13.3-t-0.9rain for the older monkeys). Samples were centrifuged, placed on dry ice and then stored at - 7 0 ° C until assayed. All cisternal sampling was conducted by the same investigator (SES).

CSF monoamine metabolite determinations Concentrations of HVA, 5-HIAA and MHPG were determined in each sample by a modification of the method of Van Bockstaele et al. (1983), using high performance liquid chromatography with electrochemical detection. The eluent was modified by addition of 0.05M NaH2PO4, 0.1 mM EDTA and 7.0% methanol, with pH adjusted to 4.2 with glacial acetic acid. Samples were deproteinized by adding 100/~1 of 2.4 M HC104 to 250/~ 1 of CSF. CSF samples were cooled for 30min and centrifuged for 10min at 10,000g, a 250-/~1 aliquot was mixed with 50~1 of 5.0M sodium acetate and 250/il of this was injected into the liquid chromatograph. Blank-corrected standard curves for the quantification of all compounds were prepared by analyzing a series of standard solutions containing a fixed amount of the internal standard (5-hydroxyindolecarboxylic acid) and varying amounts of each compound. The standard solutions, in appropriate volumes of mobile phase, were subjected to the identical preparative steps used to assay unknown CSF samples. Sample concentrations were determined mathematically from the area-under-the-curve relative to the appropriate internal standards, using the slope and intercept of the standard curve obtained by regression analyses (routinely, r/>0.99). Statistical analysis A two-factor (age and sex) between-groups analysis of variance was conducted for each of the three compounds under study. Because it was clear from our analyses that none of the treatments (TCDD, lead or social isolation) had any effect on CSF monoamine metabolite concentrations, these determinations were pooled within age groups: Indeed, with one exception (t = 1.6 for 5-HIAA concentrations in lead treated and control animals), t values for these treatments were substantially less than 1.0. However, in addition to analyzing these pooled data, separate analyses were conducted using CSF determinations from monkeys serving only as control subjects, or from monkeys housed only at the University of Wisconsin. RESULTS Old m o n k e y s were f o u n d to have significantly lower cisternal H V A c o n c e n t r a t i o n s t h a n y o u n g m o n k e y s (see Fig. 1). This was true w h e t h e r all animals were included in the analysis ( F = 16.3, d.f. = 1,65, P < 0 . 0 0 1 ) , or w h e t h e r d e t e r m i n a t i o n s from control subjects only were used ( F = 12.3, d.f. = 1,33, P < 0.01). In b o t h cases, no significant m a i n effect o f sex was observed, n o r was there any evidence o f a n age by sex interaction. Additionally, n o significant difference was observed in H V A conc e n t r a t i o n s between m o n k e y s housed in N e w Mexico a n d those housed in Wisconsin ( F = 1.31, d.f. = 1,67). A significant inverse c o r r e l a t i o n was f o u n d for H V A a n d age (r = 0.49, d.f. = 67, P < 0.01), In c o n t r a s t to H V A , n o significant differences were f o u n d between age groups for the m a j o r metabolite o f serotonin, 5 - H I A A (see Fig. 2). This was true

CSF neurotransmitter metabolites and age in monkeys 35

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Fig. 1. Cbternal CSF HVA concentrations (ng/ml) in young and old rhesus monkeys of each sex.

Fig. 3. Cisternal CSF MHPG concentrations (ng/ml) in young and old rhesus monkeys of each sex.

whether subjects from all the treatment groups ( F = 0 . 9 3 , d.f. = 1,62) or only control subjects (F = 1.07, d.f. = 1,32) were used. As was the case for HVA, there was neither a si~itlc, ant main effect for sex nor a significant age-by-sex interaction. Comparable 5-HIAA values were observed in animals housed in both locations (F -- 2.24, d.f. = 1,67), although a subset of monkeys housed in New Mexico (3 of 18) exhibited low 5-HIAA concentrations. These three animals were not included in the analyses of 5-HIAA as their values ranged from 7.8 to 10.6 ng/ml compared to a mean of 36.3 (SD = 16.7) for the larger group of 18 animals. Significantly lower cisternal M H P G concentrations were found in old vs young monkeys (see Fig. 3), regardless of whether all animals were included in the analysis (F =44.7, d.f. = 1,65, P < 0.001), or only

control monkeys were included (F = 9.47, d.f. = 1,32, P < 0.01). As with HVA and 5-HIAA, no main effect was present for sex. A significant age-by-sex interaction was found, however (F =4.21, d.f. = 1,65, P < 0.05), with old males showing the lowest M H P G concentrations (see Fig. 3). The magnitude of the mean age difference was influenced considerably by location effects. M H I ~ values associated with monkeys housed at the University of New Mexico were substantially lower than those obtained from monkeys housed at the University of Wisconsin (F = 71.1, d.f. = 1,67, p < 0.001). Therefore, an additional analysis was conducted using only monkeys housed in Wisconsin. This analysis also suggested that M H P G values were lower in old monkeys ( F = 5 . 3 4 , d.f.=l,49, P < 0.05). Finally, a significant inverse correlation was also found for M H P G and age (r = 0.46, d.f. = 67, P < 0.01).

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Fig. 2. Cistemal CSF 5-HIAA concentrations (ng/ml) in youn8 and old rhesus monkeys of each sex. NC.I. 13/3~F

DISCUSSION

This is the first report of the effects of aging on CSF monoamine metabolites in a nonhuman primate species. Our finding that the catecholamine metabolltes HVA and M H P G decrease with age in rhesus monkeys, whereas the indoleamine metabofite 5-HIAA remains unchanged, is supported by some human data. However, interpretation of the conflicting results from human studies is complicated by the difficulty in controlling potentially confounding variables, such as site and time of sampling and presence of neurologic or organic disorders. In addition, it is ditficult to compare these studies because

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they involve different assay methods, and different age ranges and sex distributions in the study subjects. The few studies including control subjects without neurologic or organic disorders report mixed results. Gottfries et al. (1971) and Bareggi et al. (1982) found higher levels of CSF HVA in older subjects, whereas the work of Gerner et al. (1984) supports our finding that CSF HVA decreases with age. There are also conflicting findings for age-related changes in CSF 5-HIAA levels in subjects without neurological disorders. Gottfries et al. (1971) found a significant increase in CSF 5-HIAA levels in older subjects, but Bareggi et al. (1982) found no correlation between 5-HIAA and age. None of these studies reported levels of MHPG. There have been many similar studies in subjects with neurologic and organic disorders, primarily sampling lumbar CSF. Some investigators have found age-dependent increases in CSF HVA (Bowers and Gerbode, 1968; Gottfries et al., 1971; Bareggi et al., 1982). Conversely, others have reported agedependent decreases (Seifert et al., 1980), and even a U-shaped relationship (Bowers and Gerbode, 1968) has been reported. Young et al. (1980) reported no relationship between 5-HIAA levels and age in lumbar samples, but after repeated sampling (which was thought to result in inclusion of some CSF of recent origin in the lateral ventricles), a quadratic relationship with very small changes was found. Such conflicting results also have been reported for MHPG. A positive correlation for CSF M H P G with age was reported by Gerner et al. (1984), whereas Seifert et al. (1980) found no correlation. A problem consistently encountered when studying human subjects is the difficulty in obtaining samples reflective of brain activity. Leckman et al. (1980) attempted to overcome this problem by using the probenecid technique. Probenecid blocks active transport of metabolites out of the CSF and is believed to increase the concentration of metabolites in CSF proportionely to the turnover rate of the parent amine (Neff et al., 1967; Post and Goodwin, 1974). After oral probenecid loading in 154 neuropsychiatric patients, Leckman et al. (1980) found that age was negatively correlated with CSF HVA levels but was not correlated with CSF 5-HIAA. It is of interest that most CSF HVA originates from sources above the spinal cord (Curzon, 1975), and a steep gradient for HVA has been reported in rhesus monkeys from lateral ventricular CSF to lumbar CSF (Gordon et al., 1975). As a result, lumbar samples taken by Leckman et aL (1980) may be more reflective of central DA metabolism, as measured by

HVA, than samples obtained in other human studies done without the benefit of probenecid blockade. This could explain the similarity of findings between the Leckman et al. (1980) study and ours There is general agreement that central DA content is decreased in brains from both old primates and rodents (Adolfsson et al., 1979b). Studying rhesus monkeys, 2-18 years old, Goldman-Rakic and Brown (1981) reported a decrease of more than 50% in DA content in the prefrontal cortex associated with advancing age. Less dramatic decreases were found in other cortical regions and only minor changes in NE and 5-HT were found. Wenk et al. (1987) have also reported decreases in cortical concentrations of DA and its acidic metabolites, HVA and DOPAC. A recent study (Elsworth et al., 1987) supports the conclusion that changes in cortical DA systems could be reflected in cisternal CSF. These investigators reported that, in vervet monkeys, CSF HVA concentrations were found to correlate with HVA concentrations in the dorsal frontal cortex, but not with HVA concentrations in the basal ganglia. We observed no significant differences in monoamine metabolite concentrations between male and female monkeys. This is not in agreement with an earlier finding by Seegal (1985), who reported significantly higher HVA concentrations in lumbar CSF samples from female pig-tail macaques. He reported that no such sex difference was observed for 5-HIAA. Finally, the marked difference in M H P G values observed between monkeys housed in New Mexico versus Wisconsin is not readily interpretable. Explanations such as diet and/or activity are not easily employed as no location differences in HVA or 5-HIAA were observed. Acknowledgements--This research was supported by Veter-

ans Administration Medical Research funds, by grant MH42938 from the National Institute of Mental Health, and by Center grants HD03110 and MH33127. The authors gratefully acknowledge the editorial assistance of Carol Steinhart. REFERENCES

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