P-chlorophenylalanine attenuates tonic immobility duration in chickens

Physiology & Behavior, Vol. 36, pp. 427-430. Copyright~ Pergamon Press Ltd., 1986. Printed in the U.S.A.

0031-9384/86 $3.00 + .00

P-Chlorophenylalanine Attenuates Tonic Immobility Duration in Chickens RICHARD A. HUGHES

Iowa State University, D e p a r t m e n t o f Psychology, A m e s , IA 50010 R e c e i v e d 9 M a y 1985 HUGHES, R. A. P-ehlorophenylalanine attenuates tonic immobility duration in chickens. PHYSIOL BEHAV 36(3) 427-430, 1986.---The results of two experiments demonstrated a dose-dependent reduction in tonic immobility (T1) duration by p-chlorophenylalanine (PCPA) in White Leghorn cockerels. In Experiment 1, five-day old chicks were given five intraperitoneal injections of either saline or 75 or 150 mg/kg of PCPA at a rate of one injection a day for five days. The effects of these injections were evaluated one day after the last injection. A total of 750 mg/kg of PCPA significantly reduced TI duration but 375 mg/kg did not. In Experiment 2, chicks were given either two saline injections, two 375 mg/kg PCPA injections, or one 375 mg/kg PCPA injection and one saline injection. The effects of these injections were evaluated either one or three days after injections. A total of 750 mg/kg of PCPA significantly reduced TI both one and three days after injections but 375 mg/kg of PCPA did not. Previous failures to obtain significant PCPA effects on TI may reflect insufficient drug amounts. Tonic immobility

p-Chlorophenylalanine

PCPA Serotonin

MOST animals will struggle vigorously when they are restrained. With continued restraint an animal will often stop struggling and may remain in a state of tonic immobility (TI) for several seconds to several minutes or more after restraint is ended. The species generality of this transient immobile state is well-documented as is a direct correspondence between TI duration and operationally defined states of fear [6]. Evidence concerning the potential adaptive significance of TI suggests that it may reflect a terminal defense response to predation [20,22]. Moreover, the striking similarity of TI characteristics to those of catatonic schizophrenia prompted the suggestion that TI may provide an infrahuman analog of that abnormal human state [8]. Recent research has focused on the neuropharmacology of TI. Although evidence indicates that altered adrenergic [4, 13, 14, 23], cholinergic [16, 18, 24, 31], and dopaminergic [5, 27, 28] activity can affect TI, other evidence suggests that serotonergic (5HT) systems play a pivotal role in TI phenomena [2, 3, 7, 9, 10, 11, 12, 17, 19, 25, 26, 27, 29, 30, 31]. The apparent correspondence between 5HT activity and T1 duration is weakened considerably by the failure of p-chlorophenylalanine (PCPA), a relatively specific depletor of 5HT, to affect TI duration [1, 2, 3, 17, 19, 30, 31]. However, the failure of PCPA to significantly affect TI may be dose-dependent. Although approximately 300 mg/kg of PCPA can produce a substantial and long-lasting depletion of brain 5HT in the rat, about 750 mg/kg of PCPA is required to produce a similar depletion of brain 5HT in domestic fowl [21]. Domestic fowl were used in all of the research in which PCPA did not affect TI [1, 2, 3, 17, 19, 30, 31]. Moreover, the amounts of PCPA used in the majority of these TI studies was about 300 mg/kg and in no case equaled or exceeded the 750 mg/kg that may be required to substantially deplete 5HT in domestic fowl. This evidence supports the suggestion that

427

Chicken

Domestic fowl

Cockerels

the failure of PCPA to affect TI in domestic fowl may reflect an insufficient reduction of 5HT in this species as opposed to a qualitative failure of PCPA to affect TI [2, 7, 29].

EXPERIMENT I This experiment was designed to examine the effects of 375 and 750 mg/kg of PCPA on TI in chickens tested one day after a series of five injections. The 750 mg/kg PCPA dose, test time, and injection procedures were patterned after methods reported to produce a substantial depletion of 5HT in the avian brain [21]. The lower PCPA dose was selected because it approximates the amount used in previous TI research [1, 2, 3, 17, 19, 30, 31]. METHOD

Subjects Forty-five White Leghorn cockerels (Welp-Line No. 937-A) were obtained at one-day posthatch from Welp Inc., (Bancroft, IA). The birds were housed in a temperature controlled brooder (Brower Mfg. No. 1680-1) until five days posthatch when they were coded for individual identification and housed five animals per cage in stainless steel wire mesh cages (62x25x18 cm). Free access to water and food (Wayne pullet starter) was provided under both housing conditions. The colony room was illuminated by overhead fluorescent lights from 0700 to 1900 hr.

Apparatus Immobility was induced in sound attenuating plywood chambers. The details of these chambers have been described [18]. Placement of a photobeam directed at a photosensor across the short axis of a shallow oval depression in

42~

t{ U G H ES

FABLE I

TABILE 2

MEAN TI DURA'I'I()N (SECI AS A FUNCTION ()f: DRUG T R E A T M E N T CONDITION

MI-.AN "11 I)URATION (SEC) AS A F'UNC I'ION Ol~ DRUG IREA ['MIEN] CONDITION AND T E S F TIME

Mean S.E.M.

DIST-H20

PCPA-375

PCPA-750

308. I 59.8

385.6 102. I

115.3 14.9

Test Day I Test Day 3

1) 1ST- H 2 0

P(' PA-375

PC P A - 7 5 0

3 1 2 . 9 (69.71" 2 8 5 . 9 (59.01

321.2 ~75.3) 300.8 173.71

110.4 (15.21 !~5.8 t26.61

*Number in parentheses=standard error of the mean. the floor of each chamber and the use of solid state programming equipment permitted automated measurement of TI duration. Each chamber was placed in a separate darkened room. Pro(.(,dllF( ~

At five days posthatch each animal was randomly assigned to one of three treatment groups (n= 15 per group). Each animal received five 2 cc/kg intraperitoneal injections at the rate of one per day for five consecutive days. Animals in group DIST-H20 were given distilled water injections. those in group PCPA-375 were given injections containing 75 rag/2 cc of PCPA (Sigma; d-l-chlorophenylalanine methyl ester hydrochloride dissolved in distilled water), and those in group PCPA-750 were given injections containing 150 mg/2 cc of PCPA. On each of the five injection days an animal was removed from its cage and carried by hand to an adjacent room where the animal was weighed, injected, and immediately returned to its cage. All animals were tested in a randomly determined order one day after the last injection ti.e., at ten days posthatch). On the test day, a chick was removed from its cage, placed into a small lidded container, and taken to one of the five rooms where TI was induced. For T1 induction a chick was held upright over the oval depression in the chamber floor. Five sec later the animal was inverted and gently but firmly restrained on its back in the oval depression. After 15 sec the chick was slowly released, a silent timer was started, and a cloth flap was lowered over the access hole to the interior of the chamber. If the chick righted itself at release or within ten sec of release, the induction procedure was immediately repeated. After successful TI induction the experimenter left the room. The timer stopped timing when the chick moved its body enough to permit the photobeam to activate the photosensor or until a criterion of 900 sec occurred. After the test each chick was returned to its home cage. All tests were conducted by an experimenter who was unaware of the treatment conditions. RESULTS

One chicken in Group PCPA-375 died before the last two PCPA injections were administered. The groups were equated (n=14) by randomly discarding one animal from each of the remaining groups. Drug treatment did not systematically affect susceptibility to TI induction. Only five chickens required more than one induction attempt. The distribution of these animals across treatment groups was 2, 2, and 1, in group DIST-H20, PCPA-750, and PCPA-375 respectively. There was some evidence of a systematic drug treatment effect on body weights across the five treatment days. Mean body weights did not decline but the order of weight gain was PCPA-750
Mean TI durations as a function of drug treatment are presented in Table 1. Drug treatment significantly affected TI duration, F(2,39)=5.28, p<0.01. Moreover, the dosedependent reduction in TI duration that is suggested by these data was confirmed by Duncan's test. The mean TI duration of group PCPA-750 was significantly shorter than the TI duration of group PCPA-375 (p<0.01) and group DIST-H20 (o<0.05). The difference in mean TI durations of the latter two groups was not significant. EXPERIMENT 2 In the first experiment TI duration was reduced significantly by 750 but not by 375 mg/kg of PCPA. This result was obtained on tests given just one day after a series of injections that were administered over five successive days. In rats the effect of PCPA on 5HT increases from one to three days after injection. The purpose of this second experiment was to examine the effects of 375 and 750 mg/kg of PCPA administered within one day on TI duration one and three days after injections. METHOD

Subjects Apparatus and Procedure Ninety White Leghorn cockerels were obtained at one day posthatch. With the exception of some differences to be described below, the subject characteristics, housing, maintenance, apparatus, and general procedures were as described in the first experiment. The design of this experiment was a 2 × 3 factorial. The animals were randomly assigned to one of two injection to test-time groups (one or three days) and one of three injection groups (D1ST-H20, PCPA-375, or PCPA750). At nine days posthatch the animals were given two IP injections of 2 cc/kg each. The first injection was administered between 0900 and 1100 hr and the second between 1400 and 1600 hr. The animals in group DIST-H20 received two injections of distilled water, those in group PCPA-375 received one 375 mg/kg injection of PCPA followed by a distilled water injection, and those in group PCPA-750 received two 375 mg/kg injections of PCPA. Testing occurred either one or three days later. RESULTS

Two chickens died before the TI tests (one from group PCPA-750 and one from group D1ST-H20). The number of animals per group was reduced to 14 by random procedures. As in the first experiment, drug treatment did not systematically affect susceptibility to TI induction. Only four animals required two induction attempts (one each in groups PCPA750 and DIST-H20 on tests one day after injections and one each in groups PCPA-375 and PCPA-750 on tests three days after injections). The summary data for TI duration as a function of drug treatment and injection-to-test interval are

PCPA A T T E N U A T E S IMMOBILITY

429

presented in Table 2. Analysis of variance revealed a significant main effect of drug treatment, F(2,78)=6.22, p<0.01. Neither the main effect of test-time nor the interaction term was significant. Duncan's test demonstrated that mean TI duration for group PCPA-750 was significantly shorter than TI duration for group PCPA-375 and group D1ST-H20 at both test intervals (,0<0.05). No other comparison of mean differences was significant. DISCUSSION In the present research a total of 375 mg/kg of PCPA did not significantly affect TI duration either one day after a series of injections given over five days (Experiment 1), or one or three days after injections when the injections were given within one day (Experiment 2). This absence of significant PCPA effects on TI duration is consistent with previous research in which similar PCPA amounts were used [1, 2, 3, 17, 19, 30, 31]. Collectively, these data suggest that PCPA does not affect TI. The apparent failure of PCPA to significantly affect TI is not congruent with the extensive evidence, cited earlier, that serotonergic systems play a pivotal role in TI phenomena. This discrepency, however, is evidently dose-dependent. In the present research a total of 750 mg/kg significantly reduced TI duration both in Experiment 1 on tests one day after injections and in Experiment 2 on tests one and three days after injections. The magnitude of these PCPA effects was relatively unaffected by the differences in injection schedule between the two experiments and by the different injection-test intervals of the second experiment (cf. mean TI durations of the PCPA-750 groups in Tables 1 and 2). This evidence suggests that 750 mg/kg of PCPA may exceed the amount required to affect TI duration. Moreover, since there is evidence that as much as 600 mg/kg of PCPA does not significantly affect TI duration [3], this further suggests that PCPA amounts in excess of 600 mg/kg and approaching 750 mg/kg may be required to significantly affect TI duration. The finding that PCPA, a relatively specific depletor of 5HT, can affect TI duration is clearly consistent with the central role that serotonergic systems seem to play in TI phenomena. The results of the present research, however, are not consistent with conceptual models of the way in which TI duration and serotonergic systems are related. One early attempt to describe the relationship between 5HT activity and TI specified that TI duration was inversely related to the electrical activity of serotonergic midbrain raphe neurons [29]. According to this model, PCPA did not affect TI because this drug does not affect midbrain raphe electrical activity. The present finding of reduced TI duration by PCPA is clearly incompatible with this model. A revised version of the serotonergic midbrain raphe model, however, was prompted by an earlier contradictory finding that the combined administration of the 5HT precursor tryptophan and the monoamine oxidase inhibitor pargyline, two drugs that were expected to increase TI duration [29], decreased TI duration [2].

The revised midbrain raphe model emphasizes postsynaptic phenomena and specifies that TI duration is inversely related to serotonergic midbrain raphe postsynaptic inhibition. Thus, any manipulation that can increase raphe postsynaptic inhibition should decrease TI duration and any manipulation that can decrease raphe postsynaptic inhibition should increase TI duration. The descriptive and predictive potential of the revised model is impressive. It incorporates the evidence on which the original model was based [29] and provides an explanation for the opposite effects on TI of tryptophan and pargyline when these drugs are administered separately and when they are combined [2]. Moreover, the recent findings that the 5HT agonist quipazine and the 5HT releasing drugs p-chloroamphetamine and fenfluramine can decrease TI duration are in accord with the revised model because each of these drugs should increase serotonergic midbrain raphe postsynaptic activity and thereby decrease TI [3, 25, 26, 27]. Although the revised model correlates well with a substantial amount of evidence, it cannot accommodate the results of the present research. In this research, amounts of PCPA that can reduce 5HT in the avian brain by about 71Y~ [21] reduced TI duration. According to the revised model, if reduced brain 5HT by PCPA affects TI the result should be an increase, not decrease, in TI duration. Further, the finding that centrally administered 5HT increases TI duration [10] is also incompatible with the revised model since this manipulation should increase raphe postsynaptic inhibition and thereby decrease TI duration. The decrease in TI duration produced by PCPA (Experiments 1 and 2) and the increase in TI duration produced by 5HT I10] are directly opposite to effects predicted by the revised serotonergic midbrain raphe model [2]. The basis of these contradictory findings is not readily apparent and additional research is clearly needed before the revised raphe model is either revised again or abandoned. One direction that such research might take is to further evaluate the effects on TI of drugs that can reduce 5HT activity. Various drug treatments that can reduce 5HT activity have been reported to have no significant effect on TI. These drug treatments include the neurotoxin 5,6-dihydroxytryptamine [31], and the 5HT receptor antagonists cinanserin [27], methysergide [15], and cyproheptadine [15]. In each of these instances, however, the drug effects on TI were evaluated at only one dose level. Considering the substantial involvment of 5HT systems in TI phenomena, and in view of the present findings, it is possible that a more extensive dose response determination of the effects of 5HT antagonists might reveal significant effects of those drugs on TI. If reduced 5HT activity by 5HT receptor antagonists is found to decrease TI duration, then this finding would suggest that TI duration and 5 HT activity are directly related, as is suggested by the present findings of decreased TI by PCPA and by the finding of increased TI by central application of 5HT [10]. If, on the other hand, reduced 5HT activity by these drugs is found to increase TI duration, then this finding would lend additional support to the revised serotonergic midbrain raphe model.

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