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Locomotor activity induced by d-amphetamine was found to be potentiated by food deprivation, a tryptophan-free diet, p-chlorophenylalanine and drugs proposed to antagonize serotonin receptors in brain. Administration of l-tryptophan 1 hour prior to d-amphetamine injection was found to antagonize the enhanced response to d-amphetamine in starved rats and in rats which had tryptophan removed from their diet. However, tryptophan did not block the potentiated response to d-amphetamine in animals pretreated with p-chlorophenylalanine. These findings suggested that the antagonism of d-amphetamine-induced activity by tryptophan in starved rats and rats fed a tryptophan-free diet was not due to a nonspecific depressant effect of the amino acid. Since accumulation of d-amphetamine and its metabolites was not affected by any of the treatments which enhanced its activity, it seems unlikely that an alteration in the metabolism of d-amphetamine can explain these findings. The present work provides additional support for the view that serotonergic fibers play an important role in the actions of d-amphetamine.
Current evidence suggests that the effects of d-amphetamine on locomotor activity involve an interaction between monoamine neural systems in brain. The locomotor stimulant action of d-amphetamine has been associated with the indirect release of catecholamines in brain, and particularly with the release of dopamine (Carlsson, 1970; Costa et al., 1972; Thornburg and Moore, 1973; Fibiger et al., 1973; Hollister et al., 1974a). More recently studies have suggested that d-amphetamine-induced locomotor activity is antagonized by central serotonergic pathways. Evidence in support of a serotonergic inhibitory system includes the potentiation of amphetamine-induced activity after lesions of the raphe nuclei (Neill et al., 1972), treatments with p-chlorophenylalanine (Mabry and Campbell, 1973, 1974), 5,6- and 5,7-dihydroxytryptamine (Breese et al., 1974; Hollister et al., 1974b) and serotonin receptor blocking agents (Rothlin, 1957). Other studies have shown that elevation of brain serotonin with pargyline antagonizes d-amphetamine-induced activity, providing additional evidence for this view (Breese et al., 1974; Hollister et al., 1974b).
The purpose of the present study was to investigate further the relationship of serotonergic neural systems in brain to the locomotor stimulant effects of d-amphetamine. Since food deprivation has been shown to potentiate the stimulating effects of d-amphetamine (Campbell and Fibiger, 1971; Simpson, 1974) and dietary tryptophan has been shown to affect serotonin synthesis in brain (Fernstrom and Wurtman, 1971, 1972; Perez-Cruet et al., 1972, 1974; Biggio et al., 1974; Gessa et al., 1974), one goal of the present study was to determine if the potentiation of d-amphetamine produced by starvation could be related to a lack of dietary tryptophan. In addition, the effects of p-chlorophenylalanine and serotonin receptor blocking agents on the locomotor stimulant effects of d-amphetamine and on the metabolism of d-amphetamine were investigated. Evidence will be presented which provides further support for the view that central serotonergic systems play an inhibitory role in the locomotor stimulant action of d-amphetamine.
Male Sprague-Dawley rats (Zivic-Miller Laboratories, Pittsburgh, Pa.) weighing 250 to 450 g were used in these studies. Animals were housed in 12-hour light and 12-hour dark environment with free access to food and water except where otherwise noted. Animals were habituated for 1 hour to the apparatus before they received d-amphetamine sulfate intraperitoneally.
Locomotor activity was recorded in “doughnut”-shaped activity cages housed in a dark, soundproof room as previously described (Hollister et al., 1974a). Each cage contained six photocell sensors equally spaced in the outer wall of a 9-cm wide runway. Interruptions of light beams were automatically recorded at 15-minute intervals over a 4-hour period. Previous experience (Hollister et al., 1974a) has shown that this recording device measures linear locomotion in a dose-related fashion at the doses of d-amphetamine sulfate used in the present studies. Doses of d-amphetamine sulfate greater than 3 mg/kg, which produced stereotyped behaviors as described by Taylor and Snyder (1971), consistently resulted in fewer lightbeam interruptions. Animals in this study were monitored for stereotyped behaviors, and elevated activity levels were not accompanied by increased licking, gnawing or repetitive movements in place.
Animals receiving p-chlorophenylalanine (PCPA, Chas. Pfizer & Co., Inc., Groton, Conn.) were given 150 mg/kg by oral intubation 48 and 24 hours before activity testing with d-amphetamine sulfate (d-amphetamine; Sigma Chemical Co., St. Louis, Mo.). All doses of d-amphetamine are expressed as milligrams per kilogram of the sulfate salt. l-Tryptophan (Calbiochem, San Diego, Calif.) suspended in 5% carboxymethylcellulose and cyproheptadine HCl (Merck & Company, Inc., West Point, Pa.) or methysergide maleate (Sandoz Pharmaceuticals, Hanover, N.J.) dissolved in sterile water were given i.p. 1 hour before the administration of d-amphetamine sulfate. 3H-d-amphetamine sulfate (general label, New England Nuclear, Boston, Mass.) was diluted with cold d-amphetamine and 2 mg/kg were given i.p. 1 hour before sacrifice.
Some animals were food deprived for a 48-hour period before they received d-amphetamine. Other animals on a diet deficient in l-tryptophan (Teklad Mills, Chagrin Falls, Ohio) were given a normal diet of powdered food for 3 days prior to the initiation of the tryptophan-free diet which contained 276.3 g/kg of salt-free acid hydrolysate of casein with the amino acids (in percent) arginine 2.9, histidine 3.1, isoleucine 5.3, leucine 5.3, lysine 8.5, methionine 1.4, phenylalanine 2.2, threonine 2.9, tryptophan none and valine 4.5; 150 g/kg of cornstarch; 433.7 g/kg of sucrose; 80.0 g/kg of hydrogenated cottonseed oil; 50 g/kg of salt mix (Rogers and Harper, 1965); and 10 g/kg of vitamin mix containing (in milligrams per kilogram of diet) 110.13 p-aminobenzoic acid, 1016.6 ascorbic acid, 0.44 biotin, 66.08 calcium pantothenate, 3596.92 choline dihydrogen citrate, 1.98 folic acid, 110.13 inositol, 49.56 vitamin K, 99.12 nicotinic acid, 22.03 pyridoxine HCl, 22.03 riboflavin, 22.03 thiamine HCl, 29.74 vitamin B12, 4446.88 cornstarch, and, in international units per kilogram of diet, 19,824 vitamin A, 2,203 vitamin D2, and 121.15 vitamin E. The control diet of powdered food was the tryptophan-free diet to which 1.74 g/kg of l-tryptophan were added. All animals had free access to water.
For the measurement of brain concentrations of norepinephrine and dopamine, brains were homogenized in 10 ml of ice-cold 0.4 N perchloric acid and kept frozen at −20°C until analyzed within the next 24 to 48 hours. After the homogenate was thawed and centrifuged, an aliquot of the supernatant was stirred with alumina and transferred to a column as described previously (Breese and Traylor, 1970). Norepinephrine and dopamine were eluted from the alumina with 0.2 M acetic acid and analyzed spectrofluorometrically (Anton and Sayre, 1962, 1964; Häggendal, 1963). After treatment of some animals with 50 mg/kg of pargyline (Abbott Laboratories, North Chicago, Ill.) 1 hour prior to killing and homogenization of brains in 1 M HCl with 0.1% ascorbic acid, serotonin was isolated and measured via native fluorescence by the method of Bogdanski et al. (1956). Brain 5-hydroxy-indoleacetic acid was measured by the method of Curzon and Green (1970) 1 hour after treatment with 200 mg/kg of probenecid (Merck Sharp & Dohme, West Point, Pa.).
In order to determine whether various treatments altered the metabolism of d-amphetamine, animals were injected with 2 mg/kg of 3H-d-amphetamine sulfate (30 µCi/rat). One hour after treatment, rats were killed and brains and livers were removed for analysis of d-amphetamine, p-hydroxyamphetamine, p-hydroxynorephedrine and p-hydroxyamphetamine glucuronide (liver only). Tissues were homogenized in acetone–formic acid (85:15) (8 ml/g of brain, 6 ml/g of liver) and centrifuged and the supernatant was decanted. The pellet was rehomogenized in one-half the original volume and centrifuged and the supernatants were combined. A standard solution containing 30 nmol each of d-amphetamine, p-hydroxyamphetamine (Smith Kline and French Laboratories, Philadelphia, Pa.) and p-hydroxynorephedrine (Aldrich Chemical Co., Milwaukee, Wisc.) was added to the supernatants. After this addition, 0.1 ml of concentrated formic acid and 25 ml of heptane–chloroform (4:1) were added. The mixture was shaken and then centrifuged. The organic layer was discarded, and the aqueous layer was washed twice with 25 ml of heptane–choloroform and then three times with 10 ml of benzene. To the aqueous layer, 0.5 ml of 10 M NaOH, 8 ml of ethyl acetate and 1.5 g of NaCl were added. This mixture was shaken vigorously for 10 minutes. After centrifugation, the ethyl acetate fraction was transferred to a tube containing 1 ml of acidified methanol (9:1; methanol–1 M HCl) and dried under nitrogen. The ethyl acetate extraction of the aqueous layer was repeated three more times. The combined dry extract was dissolved in 1 ml of 0.4 M HClO4 saturated with sodium carbonate, and 2 ml of dansyl chloride in acetone (4 mg/ml) were added. In order to hydrolyze excess dansyl chloride, 0.1 ml of proline (100 mg/ml) was added and the samples were stored for at least 1 hour. Excess acetone was evaporated under nitrogen, the dansylated amines were extracted into 3 ml of benzene and the benzene was separated and dried under nitrogen. The dried samples were redissolved in 40 µl of benzene, applied to a Silica Gel G plate (Beckman Instruments, Inc., Palo Alto, Calif.) and developed in isopropyl ether-triethylamine (3:1) followed by cyclohexane-ethyl acetate (1:1) and chloroform–triethylamine (5:1). Authentic standards were run on each plate. The spots were localized under ultraviolet light and scraped into scintillation vials. The dansylated amines were eluted from the gel with 10 ml of benzene–acetone (9:1) and recovery was estimated by fluorescence (excitation 350–emission 490, uncorrected). Toluene scintillation fluid (10 ml) was then added and the endogenous amphetamine and metabolites were measured by liquid scintillation spectrometry. Recoveries averaged 85% for d-amphetamine, 75% for p-hydroxyamphetamine and 60 to 75% for p-hydroxynorephedrine. For estimation of liver p-hydroxyamphetamine glucuronide, a 20-µl aliquot of the original aqueous layer (before ethyl acetate extraction) was applied to a Silica Gel G plate, overspotted with d-amphetamine, p-hydroxyamphetamine and p-hydroxynorephedrine standards and was developed in isopropanol–ammonia (4:1). The dried plates were sprayed with diazotized p-nitroaniline, and the zone below the nonconjugated metabolites was scraped into a scintillation vial and counted by liquid scintillation.
Statistical comparisons between treatment conditions were made via Student’s t test with two-tailed probability values reported. Correlational analyses were performed by regression analyses.
Food deprivation has been shown previously to increase the locomotor response to d-amphetamine (Campbell and Fibiger, 1971; Simpson, 1974). Consistent with these reports, animals deprived of food for 48 hours displayed a locomotor response 50% greater than control animals when treated with 2.0 mg/kg of d-amphetamine sulfate (fig. 1). The administration of 25 and 100 mg/kg of l-tryptophan (122 and 490 mmol/kg) or of 25 and 75 mg/kg of 5-hydroxytryptophan (5-HTP) (114 and 341 mmol/kg) antagonized this enhanced response to d-amphetamine in food-deprived animals in a dose-related fashion. These data suggested that the potentiation of d-amphetamine-stimulated activity induced by food deprivation might be related to changes in brain serotonin metabolism.
Since the activity response to d-amphetamine in food-deprived animals was reduced by l-tryptophan and 5-HTP, it was predicted that removal of tryptophan from the diet would potentiate d-amphetamine-induced locomotor activity. In accord with this view, rats maintained on a tryptophan-free diet exhibited an enhanced locomotor response to d-amphetamine (fig. 2). A significant potentiation of locomotor activity occurred after 2, 4, 8 and 14 days of tryptophan-free diet during a 3-hour period after administration of 2 mg/kg of d-amphetamine sulfate. Because the responses to d-amphetamine in animals fed either a control diet (experimental diet + tryptophan) or the same weight of the control diet as that consumed by animals on a tryptophan-free diet over a period of 4 days (pair-fed) was not different, they were combined (pair-fed, 3972 ± 572 counts/180 min; control, 4341 ± 237 counts/180 min; see “0” day). The activity of all groups of animals after saline injection (solid bars) was not significantly different from control.
As had been observed in food-deprived rats, the administration of l-tryptophan (25 or 100 mg/kg) abolished the enhanced effect of d-amphetamine in animals fed the tryptophan-free diet for 1, 2 or 14 days and had no significant effect on the activity response to d-amphetamine in animals fed the control diet (fig. 3).
Table 1 shows the effect of 1, 2, 4, 8 and 14 days of tryptophan-free diet on brain monoamine levels. Animals pair fed for 4 days are also included. Neither whole brain norepinephrine nor dopamine levels were significantly altered by the tryptophan-free diet, but whole brain serotonin concentrations fell progressively. By correlation analysis, whole brain serotonin levels were closely associated in a negative fashion to the locomotor response elicited by d-amphetamine (r = −0.989, n = 7, P < .001). Neither brain norepinephrine nor dopamine was significantly correlated with the d-amphetamine-induced activity (r = −0.234 and 0.179, respectively; P > .1).
The effect of 2 days of tryptophan-free diet and 2 days of food deprivation on an estimate of the turnover of brain serotonin is shown in table 2. Both food deprivation and a tryptophan-free diet caused a reduction in the accumulation of 5-HIAA after probenecid. However, since probenecid administration elevates brain tryptophan levels and thereby may increase brain serotonin turnover (Tagliamonte et al., 1971), the accumulation of serotonin after pargyline was also used to assess serotonin turnover. Data from this procedure also indicated that brain serotonin turnover was reduced after 2 days of food deprivation or tryptophan-free diet.
In order to examine the possibility that l-tryptophan might directly or nonspecifically decrease the activity response to d-amphetamine, the locomotor response was examined in rats treated with PCPA in combination with l-tryptophan or 5-HTP. Figure 4 illustrates the effects of the blockade of sertonin synthesis and/or the administration of serotonin precursors on the locomotor activity stimulated by 3 mg/kg of d-amphetamine sulfate. Whereas 75 mg/kg of l-5-HTP (341 mmol/kg) and 100 mg/kg of l-tryptophan (490 mmol/kg) did not significantly alter the response to d-amphetamine, PCPA administration potentiated locomotor stimulation by d-amphetamine. In PCPA-treated animals, 5-HTP significantly reduced the response to d-amphetamine but tryptophan did not. Since PCPA presumably blocks the formation of serotonin from l-tryptophan but not from 5-HTP, this effect appears to be due to an hydroxylated metabolite of l-tryptophan rather than to a direct effect of the amino acid.
Table 3 gives the concentrations of brain monoamines in each of the above treatment paradigms. Whereas 5-HTP produced a significant elevation of brain serotonin content in control and PCPA-pretreated groups, l-tryptophan increased serotońin content only in control rats. Brain serotonin levels were significantly correlated with the locomotor response to d-amphetamine in a negative fashion (r = −0.837, n = 6, P < .05). Neither brain norepinephrine nor dopamine shows a significant association with locomotor activity (r = 0.472, n = 6, P > .1 and r = 0.663, n = 6, P > .1, respectively.) Therefore, on the basis of brain monoamine content, serotonin levels are closely associated with the antagonism of the locomotor activity stimulated by d-amphetamine.
Both methysergide and cyproheptadine were found to potentiate the locomotor response to d-amphetamine in a dose-related fashion (fig. 5) when given 1 hour before the injection of 2 mg/kg of d-amphetamine sulfate. Cyproheptadine appeared to be somewhat more potent than methysergide in this action, showing more potentiation of d-amphetamine at milligram equivalent doses. The highest dose of these serotonergic blocking agents did not significantly alter the activity of animals given an injection of saline rather than d-amphetamine (control, 752 ± 70 counts/180 min, n = 24; methysergide, 764 ± 87, n = 7; cyproheptadine, 573 ± 60, n = 6).
In order to examine the possibility that the enhanced response to d-amphetamine was due to an altered amphetamine metabolism, brain levels of d-amphetamine, p-hydroxyamphetamine and p-hydroxynorephedrine and liver concentrations of d-amphetamine, p-hydroxyamphetamine and p-hydroxyamphetamine glucuronide were measured after pretreatment with most of the agents or conditions in this study. The brain content of d-amphetamine and p-hydroxynorephedrine and liver concentrations of p-hydroxyamphetamine glucuronide are shown in table 4. Drug pretreatment and diet alteration did not produce a significant change in the brain levels of d-amphetamine. However, the liver concentration of p-hydroxyamphetamine glucuronide was increased by 2 days of food deprivation and significantly increased by 2 days of tryptophan-free diet. This result suggests an increased catabolism of d-amphetamine. Nevertheless, these changes were not accompanied by large decreases in the brain levels of d-amphetamine and would not appear to be sufficient to account for the large differences in behavioral response.
Recent work has demonstrated an increased locomotor response to d-amphetamine in animals deprived of food (Campbell and Fibiger, 1971; Simpson, 1974). In their work, Campbell and Fibiger (1971) suggested that these increases were due to either an impaired metabolism of d-amphetamine or to food deprivation acting as a stressor, thereby increasing brain catecholamine activity. However, the present study indicates no change in brain levels of d-amphetamine and no impaired production of its metabolites at a time when locomotor stimulation is at a maximum. Furthermore, the administration of 5-HTP or l-tryptophan produced a dose-related reduction in d-amphetamine-stimulated activity in food-deprived animals, thereby suggesting that dietary deficiencies in serotonin precursors may also be involved in the potentiation of d-amphetamine by food deprivation. When combined with the observed decrease in brain serotonin turnover, these findings suggest that changes in serotonin metabolism in food-deprived animals may account for the enhanced locomotor response to d-amphetamine.
Further evidence that a dietary deficiency can affect the locomotor response to d-amphetamine was provided by the finding that rats deprived of dietary tryptophan also show an enhanced response to d-amphetamine. The sequential decline in brain concentration of serotonin found after the tryptophan-free diet is similar to that reported by Culley et al. (1963). Since the synthesis rate of brain serotonin is proposed to depend on brain tryptophan concentration (Jequier et al., 1969; Tagliamonte et al., 1971) and brain tryptophan levels are determined by the plasma ratio of free tryptophan to plasma neutral amino acids (Fernstrom and Wurtman, 1972; Biggio et al., 1974; Perez-Cruet et al., 1974), the administration of a tryptophan-free diet provided a physiological method for manipulating brain serotonin content and turnover. Like the food-deprived animals, the animals on this tryptophan-free diet had nearly normal activity responses to d-amphetamine when pretreated with l-tryptophan. However, animals fed the tryptophan-free diet did appear to be more sensitive to the effects of l-tryptophan, perhaps indicating that other mechanisms, in addition to the serotonergic system, are involved in the potentiation of d-amphetamine-induced activity in food-deprived animals. Nonetheless, the alterations in serotonin turnover and the strong correlation between brain serotonin content and d-amphetamine-induced activity provide further support for the concept that brain serotonin has an inhibitory function in the locomotor effects of d-amphetamine.
Previous workers using PCPA, midbrain raphe lesions or the dihydroxytryptamines to eliminate serotonin-containing systems in brain have found an increased locomotor response to d-amphetamine (Mabry and Campbell, 1973; Neill et al., 1972; Breese et al., 1974; Hollister et al., 1974b). Since the increased response to d-amphetamine after treatment with PCPA is antagonized by the administration of 5-HTP (Mabry and Campbell, 1973; Breese et al., 1974; Hollister et al., 1974b; fig. 4) but not by l-tryptophan, serotonin may mediate this inhibitory effect. Furthermore, since l-tryptophan fails to decrease activity in PCPA-pre-treated animals, the efficacy of l-tryptophan in antagonizing the increased d-amphetamine response in food-deprived and tryptophan-deprived animals is probably not due to a nonspecific effect of l-tryptophan.
Previous studies suggested that elevated content of serotonin induced by administration of pargyline inhibited locomotor activity of d-amphetamine and methylphenidate (Breese et al., 1974, 1975). Therefore, elevation of serotonin by tryptophan might be expected to reduce the locomotor response to d-amphetamine in otherwise untreated animals. In contrast to this expectation, it was observed that l-tryptophan did not reduce the control response to d-amphetamine but did lower potentiated responses to normal. The reason for this paradox is unknown and requires further investigation.
Lysergic acid diethylamide has been shown to potentiate the locomotor activity stimulated by d-amphetamine (Rothlin, 1957). The present study extends these findings to serotonin blocking agents devoid of psychotomimetic and/or serotonin receptor stimulating effects (Stone et al., 1961). Moreover, the relative potency of cyproheptadine and methysergide in enhancing the locomotor response to d-amphetamine parallels that found in another test for central antiserotonergic potency (Clineschmidt and Lotti, 1974). This similarity in central antiserotonergic effect and in the potentiation of d-amphetamine-induced motor activity lends further support to the concept of an inhibitory role for brain serotonin in the actions of d-amphetamine.
The major mechanism for inactivation of d-amphetamine in the rat is hydroxylation followed by conjugation with glucuronic acid (Dring et al., 1970). In addition, a small percentage of the unconjugated p-hydroxyamphetamine is converted to p-hydroxynorephedrine (Lewander, 1971; Brodie et al., 1968). Since many psychoactive drugs inhibit the hepatic metabolism of d-amphetamine (Clay et al., 1971; Consolo et al., 1967; Creaven and Barbee, 1969; Freeman and Sulser, 1972; Jonsson and Lewander, 1973; Lal et al., 1974; Shoeman et al., 1974), potentiation of the behavioral effects of amphetamine by several of these agents has been attributed to elevated tissue amphetamine content (Rand and Trinker, 1968; Sulser et al., 1966). Food deprivation has also been shown to alter the activity of many drug metabolizing systems (Kato and (Gillette, 1965). Therefore, brain levels of amphetamine and its major metabolite in brain, as well as liver p-hydroxyamphetamine glucuronide, were measured in most of the experimental conditions in this study. The finding that no experimental condition employed in this study results in a significant change in brain amphetamine content 1 hour after administration suggests that altered metabolism does not explain the behavioral responses observed. Failure of these regimens to change tissue content of p-hydroxynorephedrine or p-hydroxyamphetamine glucuronide also supports this conclusion. The elevation of liver p-hydroxyamphetamine glucuronide in food-deprived and tryptophan-free diet animals suggests a small change in the rate of amphetamine metabolism similar to that found in the metabolism of other aromatic amines after starvation (Furner and Feller, 1971). However, brain levels of amphetamine and p-hydroxynorephedrine are not changed by these treatments. Thus, it is unlikely that these small changes account for the larger differences in behavioral response of these animals.
In summary, the results of this study indicate that antagonism of brain serotonergic function increases the locomotor response to d-amphetamine and support the importance of dietary tryptophan in the regulation of serotonergic functions. Since d-amphetamine-stimulated locomotor activity has been shown to be dependent upon dopaminergic systems, these results also suggest an interaction between dopaminer-gic-stimulatory and serotonergic-inhibitory systems in brain. Finally, this demonstration of an inhibitory role for brain serotonergic systems in the actions of d-amphetamine, and recently of methylphenidate (Breese et al., 1975), may have importance to the therapeutic effectiveness of these drugs in the treatment of hyperactive children (Brase and Loh, 1975).
We are grateful to Marcine Kinkead, Susan Hollister, Edna Edwards and Joseph Farmer for their excellent technical assistance.
1This project was supported by U.S. Public Health Service Grants MH-16522, HD-03110, MH-13688 and ES-01104.