The effect of para-chlorophenylalanine on spontaneous locomotor activity in the rat

Neuropharnracology,

1971.10,25-32

Pergamon Press.

Printed in Gt. Britain.

THE EFFECT OF PARA-CHLOROPHENYLALANINE ON SPONTANEOUS LOCOMOTOR ACTIVITY IN THE RAT* H. C. FIBIGER~and B. A. CAMPBELL Department

of Psychology, Princeton University, Princeton, New Jersey 08540 (Accepted 30 June 1970)

Summary-The effect of p-chlorophenylalanine (p-CPA), a depletor of serotonin, on spontaneous locomotor activity was investigated in rats. It was found that p-CPA induced large and reproducible increases in locomotor activity in a variety of situations. This hyperactivity could be reversed by 5_hydroxytryptophan, a precursor of serotonin. The heightened behavioral arousal was not simply a reflection of the insomnia known to be produced by p-CPA, since hyperactivity occurred during both phases of the normal diurnal cycle and because baseline activity returned to normal at different rates depending upon the type of behavioral measure used. It is suggested that one function of serotonin is to modulate arousal thresholds and that the hyperactivity followingp-CPA was due to a decrease in these thresholds.

P~TU-CHLOROPHENYLALAN~NE (p-CPA), a potent depletor of brain 5hydroxytryptamine (5-HT), has minimal effects on other biogenic amines at a time during which maximum serotonergic effects are observed (KOE and WEISSMAN,1966). This has permitted a more exact delineation of the role of serotonin in behavior. Several recent papers have reported that depletion of serotonin by p-CPA results in behavioral hypoactivity (TENEN, 1967; VOLICER,1969). Insofar as this treatment also produces insomnia (MOURETet al., 1968), this is a somewhat unexpected finding. That is, animals which do not sleep might be expected to display increased levels of behavioral arousal. A furthel complication arises from the fact that administration of 5hydroxytryptophan (5-HTP), a precursor of 5-HT which increases 5-HT levels in brain (UDENFRIENDet al., 1957), also elicits behavioral depression (APRISONand FERSTER,1961; JOYCEand MROSOVSKY,1964). In agreement with these latter findings is the observation that electrical stimulation of the raphC nuclei, whose activities are mediated at least in part by 5-HT (for review see JOUVET,1969) decreases locomotor activity in rats (KOSTOWSKIet al., 1969). It appears therefore that both increases and decreases in the activity of central serotonergic mechanisms induce behavioral inhibition (HINGTGENand APRISON,1963). The present experiments were designed to investigate this apparent inconsistency. In the studies examining the effects of serotonin depletion on locomotor activity, short term measures of behavioral arousal were used (TENEN, 1967; VOLICER,1969) while in those investigating the effects of 5-HTP, longer measures were employed (JOYCEand MROSOVSKY, 1964). It is possible, therefore, that serotonin may exert one effect on exploratory behavior while having different or even opposite effects on more chronic measures of behavioral arousal. *This research was supported in part by Grant MH-08501 from the National Institute of Mental Health. TPresent address: Department of Psychiatry, University of British Columbia, Vancouver 8, British Columbia, Canada. 25

H. C. FIBIGERand B. A. CAMPBELL

26

EXPERIMENT

I

The purpose of this experiment was to investigate the effects of p-CPA in rats on two forms of locomotor activity, namely stabilimeter cages and running wheels. Method Adult male Sprague-Dawley rats (Perfection Breeders, Douglassville, Pa.) weighing 250-275 g at the beginning of the experiments were used in these experiments. The apparatus consisted of either stabilimeter cages or activity wheels. The stabilimeter cages are fully described in a previous paper (CAMPBELL, 1964). Briefly, they consisted of a wire mesh cage (17.5 x 20 x 37.5 cm) mounted on a central axis. The number of crossings over the central axis was recorded on electromagnetic counters located in a separate room from the cages. The wheels were standard Wahmann activity cages equipped with micro-switches which activated electromagnetic counters in an adjacent room. Both activity measuring devices were located in a sound-shielded room with an 80 dB white noise background. The ambient temperature was 22-23°C. The light-dark cycle was 12 hr on, 12 hr off, with the lights coming on at 9 a.m. Rats were randomly assigned to stabilimeter cages and running wheels for habituation (5 days for stabilimeter and 10 days for wheels). Food and water were available throughout the experiment. Activity was recorded automatically at 3 hr intervals throughout the experiment. From 9 a.m. to 12 p.m. each day, maintenance procedures were carried out during which data were not recorded. Daily activity therefore represents data collected during a 21 hr period. At 11.30 a.m. on the sixth day for the stabilimeter groups, and at 11.30 a.m. on the eleventh day for the running wheel groups, animals were lightly anaesthetized with ether and stomach-loaded with p-CPA (150 mg/kg and 300 mg/kg) in a 30mg/cc suspension, or an equal volume of saline. Activity was then measured for a 1Cday period subsequent to intubation. In the stabilimeter experiment each group contained 15 rats, while in the running wheel experiments each group consisted of 10 rats. Results The main results of these experiments are seen in Figs. 1 and 2. It is readily apparent that o-oSALlNE **P-CPA A-AP-CPA 0-J g

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FIG. 1. Effect ofparu-chlorophenylalanine

on daily activity in stahilimeter cages.

p-chlorophenylalanine

and locomotor activity

27

p-CPA elicited large dose-related increases in daily activity in both stabilimeter cages and running wheels. In the stabilimeter cages, p-CPA at 300 mg/kg resulted in maximum activity increases from 48 to 72 hr after administration, while the maximum effect of 150 mg/kg was observed from 24 hr to 48 hr post-intubation. No effect on activity was observed during the 24 hr immediately after drug administration, suggesting that depletion of 5-HT rather than a direct action of the drug was responsible for the observed effects. Activity returned to predrug levels 5-6 days after drug administration, depending upon the dosage used.

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FIG.2. EA‘ect ofpara-chlorophenylalanine

on daily activity in running wheels.

In the wheels, p-CPA produced an initial depression in activity during the first 24 hr after drug administration, As adrenergic mechanisms are known to be involved in determining levels of behavioral arousal (for review, see CARLSSON,1965) and as p-CPA depletes norepinephrine during this period (KOE and WEISSMAN,1966), this depression may have been due to the effect of p-CPA on catecholaminergic neurons. Subsequent to the initial depression, however, a gradual increase in running wheel activity was observed. This hyperactivity returned to pre-drug levels after 6 days at 150 mg/kg, and after 10 days at 300 mg/kg. The pattern of daily activity in wheels and stabilimeter cages is presented in Fig. 3. Here the activity on the day before intubation is compared with the activity pattern obtained from 48 to 69 hr after-p-CPA (300 mg/kg), a period during whichp-CPA exerted its maximum depleting effects on 5-HT (KOE and WEISSMAN,1966). p-CPA elevated locomotor activity throughout the entire day-night cycle. Significant psychomotor stimulation was observed when the lights were on (9.00 a.m.-9.00 p.m.), a period during which little if any activity occurred in the untreated animal, and during darkness when the typical nocturnal activity was greatly augmented by p-CPA. p-CPA therefore does not obscure the diurnal cycle displayed by normal animals, but rather, it produces an overall elevation of this pattern.

H. C. FIBIGERand B. A. CAMPBELL

28

I

STABILIMETER dark

TIME

OF

DAY

FIG. 3. Effect of para-chlorophenylalanine (300 mg/kg) on stabilimeter and running wheel activity on the third day after drug administration. The control data show the activity of the same animals on the day before intubation of pnm-chlorophenylalanine.

EXPERIMENT

II

The purpose of this experiment was to determine whether the observed locomotor stimulation induced byp-CPA was due to its depleting action on serotonin. If this was the case, then repletion of serotonin by administration of 5-HTP should counteract the depletioninduced hyperactivity. A4ethod

The animals were male Sprague-Dawley rats weighing 250-275 g at the beginning of the experiment. The apparatus consisted of the stabilimeter cages described in Experiment 1. Thirty-six rats were adapted in the stabilimeter cages for 5 days. On the sixth day at noon, rats were lightly etherized and given either p-CPA (300 m&kg) (n= 18) or an equal volume of saline (n= 18) orally, after which they were returned to the stabilimeter cages. At 6 p.m. on the eighth day (54 hr after-p-CPA or saline) half of the saline group (n=9) and half of the p-CPA group (n=9) were given i.p. injection of racemic 5-HTP (75 mg/kg) while the other half of each group was injected with the same volume of 0.9% saline. The 5-HTP (Sigma Chemical Company) suspension consisted of 25 mg 5-HTP/ml distilled water. A small amount of Tween 80 was added to both the 5-HTP suspension and the 0*9’% saline. Immediately after injection, the rats were returned to the stabilimeter cages, and activity was recorded at 3 hr intervals. Results

The results of this experiment are presented in Fig. 4. The left panel indicates thatp-CPA greatly augmented daily locomotor activity, which is in accordance with the findings of Experiment I. It is also clear, however, that this effect could be reversed by administration of 5-HTP. As seen in the right hand panel of Fig. 4, the depressant action of 5-HTP was maximal during the 6 hr immediately after its administration,

p-chlorophenylalanine

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29

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(75 mg/kg) on normal activity and ongara-chlorophenylalanine (300 mg/kg) induced hyperactivity in stabilimeter cages.

which the activity of these animals slowly began to return to that of the p-CPA saline group. This reversal of p-CPA hyperactivity by 5-HTP was statistically significant during the first three, 3 hr periods after 5-HTP ( 6 p.m.-9 p.m.: t=2*85, df=16, P O-05). 5-HTP also caused a depression in the activity of the saline pretreated group, a finding which is consistent with previous reports (APRISON and FERSTER, 1961; JOYCE and MROSOVSKY, 1964). The left hand panel of Fig. 4 indicates that the 5-HTP groups had returned to-.their pre-5-HTP activity levels by day 10, the day after 5-HTP administration.

after

EXPERIMENT

III

Insofar as the results of Experiments I and II failed to confirm those reported by both (1967) and VOLICER (1969) it was of interest to analyze this discrepancy. One major difference is that the animals in the present experiments were habituated to the apparatus, while they were not in Tenen’s and Volicer’s experiments. The purpose of this experiment was to examine the effects ofp-CPA on habituation to a novel environment. TENEN

Method

Male Sprague-Dawley rats weighing 250-275 g at the beginning of the experiment were used. The apparatus consisted of circular wire mesh cages (35 cm diameter, and 45 cm high). Two photocells were mounted at 90” angles from each other, 2.5 cm off the floor. The apparatus was programmed such that each time a photobeam was interrupted, a count was registered on a print-out counter. These counters printed the number of photobeams interrupted at 6-min intervals. Each wire mesh cage was housed individually in a fan-ventilated wooden box. The boxes were dark except for the light given off by the photocells. Temperature ranged from 22 to 23°C.

30

H. C. FIBIGER and B. A. CAMPEELL

Rats were intubated with p-CPA (300 mg/kg) or an equivalent volume of saline under light ether anaesthesia. Twelve animals were used in each group. They were then individually housed with food and water ad libitum in the animal colony for 48 hr. At the end of this period, they were placed in the photocell apparatus for 2 hr (2 p.m.4 p.m.) during which activity was measured. Results

The results are shown in Fig. 5.

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PERIODS

on locomotor activity in a novel environment.

During the first half hour, the p-CPA treated animals displayed no hyperactivity compared to controls. In fact, during the first 6 min the saline group was slightly more active than the p-CPA group but this effect was not statistically significant (t=0*91, P >0*05). This result is inconsistent with VOLICER(1969) who reported a significant decrease during a 5 min measure. The results are consistent, however, with those reported by TENEN(1967) who found no difference in activity between p-CPA and saline treated rats over a 30-min period. The present results indicate however, that serotonin depletion increases behavioral arousal after 30 min of habituation to a novel environment. DISCUSSION The results of these experiments indicate that p-chlorophenylalanine produces large increases in behavioral arousal in several situations. The gradual increase in hyperactivity and return to normal activity corresponds strikingly with the depletion and subsequent repletion of whole brain serotonin as reported by KOE and WEISSMAN(1966) and MOURET et al. (1968) in the rat. The results are consistent with the theory that serotonin has a trophotropic or sedative action in the central nervous system (BRODIEet al., 1959; BRODIEand REID, 1968). They are also in agreement with the observation that lesions of the raphe in rats produce increases in locomotor activity (KOSTOWSKIet al., 1968). The present procedures suggest that the process of habituation to a novel environment is not influenced by serotonergic mechanisms. No differences in spontaneous activity as measured by photocell interruptions were found during the first half hour, but after that

p-chlorophenylalanine

and locomotor activity

31

time, when both groups appeared to be habituated, thep-CPA group displayed considerably more activity than the controls. The observation that the increased locomotor activity which is elicited by a novel environment is not potentiated by serotonin depletion is consistent with data which suggest that arousal produced by amphetamine is not potentiated by p-CPA (MARTENSand VANROSSUM,1969). It is noteworthy, however, that AGHAJANIAN and SHEARD(1968) have recently reported that habituation to a startle response may be mediated by a mid-brain serotonergic mechanism. Thus serotonin may influence some forms of habituation while not affecting others. The rats in stabilimeter cages resumed pre-drug activity levels several days before running wheel activity returned to baseline. This is of particular interest in view of several recent reports. First, LYNCH(1970) has demonstrated that the neural substrates underlying running wheel activity and stabilimeter cage activity are distinct. Secondly, FUXE(1965) and DAHLSTROMet al. (1965) have reported that rates of depletion and repletion of monoamine levels after reserpine treatment vary in the different monoamine systems of the brain. FAITH et al. (1968) have verified these results behaviorally by demonstrating that rate of recovery of function after reserpine varies significantly depending on the behavioral measure used. These results indicate both at the neurochemical level and at the behavioral level that different systems in the brain may recover at different rates after drug treatment. Within this context, it is possible that the neurochemical mechanisms which serve to modulate stabilimeter activity recover more quickly afterp-CPA than do the substrates of running wheel activity. A variety of experimental procedures suggest that sensory thresholds may be mediated by serotonergic mechanisms. TENEN(1967) and LINTS and HARVEY(1969), for example, have demonstrated lowered jump thresholds in response to shock after depletion of 5-HT by p-CPA or by medial forebrain bundle lesions. Furthermore, p-CPA facilitates avoidance conditioning at low but not high shock levels (TENEN, 1967) and facilitates brightness but not position discrimination (STEVENSet al., 1967). The present findings indicate that thresholds for behavioral arousal may also be decreased during serotonin depletion. Thus, environmental and internal stimuli which were below the level required to elicit arousal in the normal untreated animals may have been sufficient to maintain high levels of locomotor activity in p-CPA treated animals (CAMPBELLand SHEFFIELD,1953). Acknowledgement-We chlorophenylalanine.

would like to thank the Chas. Pfizer Company for their generous supply of pavu-

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