Parachlorophenylalanine and habituation to repetitive auditory startle stimuli in rats

Physiology atut Behavior. Voi. 5, pp. 1215-1219. Pergamon Press, 1970. Printed in Great Britain

Parachlorophenylalanine and Habituation to Repetitive Auditory Startle Stimuli in Rats' R O B E R T L. C O N N E R , J O N M. S T O L K , J A C K D. B A R C H A S ~ A N D S E Y M O U R L E V I N E 3

Department o.f P,s:vchiatr)', Stanford University School of Medicine, Stanford, California 94305, U.S.A. (Received 29 April 1970)

R. L., J. M. STOLK, J. D. BARCHASAND S. LEVINE. Parachlorophenylalan&eand habituation to repetitive atMitory startle stimuli hz rats. PHYSIOL.BEnAV. 5 (11) 1215-1219, 1970.--The relationship between brain serotonin levels and CONNER,

habituation of a skeletal-motor startle response was studied using parachlorophenylalanine (PCPA), a drug which inhibits the formation of serotonin. Depletion of brain serotonin by PCPA slows down, but does not prevent, habituation. PCPA given to rats that were habituated before starting drug treatment causes a transitory increase in startle response magnitude. Whether PCPA is administered before or after habituation, the treated rats exhibit heightened reactivity to startle stimuli following exposure to novel stimuli. These results suggest that brain serotonin plays a role in inhibitory processes. Parachlorophenylalanine

Brain serotonin

Habituation to auditory stimuli

HISTOCHEMiCALand neuroanatomical studies have established the discrete localization of serotonin (5HT) containing nerve fiber tracts within the brain. The recent introduction of parachlorophenylalanine (PCPA) by Koe and Weissman [5] has provided a relatively specific pharmacological tool for studying the behavioral and neurophysiological importance of 5HT. The relationship between brain 5HT and behavior, however, remains elusive. A number of reports, commencing with Koe and Weissman, have described a gross hyperreactivity of rats to environmental stimuli following reduction of brain 5HT levels. More discrete measures of altered reactivity indicate that rats depleted of 5HT by PCPA treatment [13] or by brain lesions [6] exhibit lowered shock thresholds. Based on these observations, it might be expected that PCPA treated rats would exhibit elevated levels of shock-induced fighting behavior [14], in which shock intensity is a major variable controlling the behavior. However, results from several studies in this laboratory failed to provide any evidence supporting this expectation [2]. During the course of the latter studies, however, it was observed that the PCPA treated rats did show greater reactivity to the shock, even though no quantitative change in the frequency of fighting behavior after drug treatment was noted. This observation suggests that brain 5HT may play a role in modulating overall levels of respon-

Skeletal-motor startle response

sivity to dynamic changes in tile level of environmental stimulation. Some support for this hypothesis has been reported by Aghajanian and Sheard [1]. These investigators found that electrical stimulation of the midbrain raphe area, the region containing most of the 5HT nerve cell bodies in the brain, causes dishabituation of a habituated skeletal-motor startle response. Prior treatment of animals with PCPA abolished the effects of raphe stimulation on startle response rnagnitude, but normal responding was restored by administration of the 5HT precursor 5-hydroxytryptophan. These data suggest that brain 5HT is related to the dishabituating effects of raphe stimulation. In the experiments described below, the relationship between brain 5HT levels and habituation to a skeletalmotor startle response was investigated further using PCPA. METHODS

Apparatus A stabilimeter, consisting of a small animal chamber mounted in a manner such that small movements of the chamber are electronically detected, amplified, and recorded, was enclosed in a ventilated chamber (IAC, Model AC-1). The animal chamber (21.5 × 8 × 9 cm) was constructed from 3.2 mm clear Plexiglas perforated with 3.2 m m holes on

~This study was supported by Research Grant NICH & HD 02881 from the National Institutes of Health, ONR 102-715 and NASA 05-020-168. 2Supported by Career Development Award MH 24,161 from the National Institute of Mental Health. 3Supported by USPHS Research Scientist Award 1-K05-MH-19,936-01 from the National Institute of Mental Health. 1215

1216

CONNER, STOLK, BARCHAS AND LEVINE

approximately 6.4 mm centers. The floor of the chamber consisted of 3.2 mm brass rods on 9.5 mm centers. The chamber weighed approximately 500 g. The chamber was supported at each end by parallel 30.5 cm cantilever arms and this structure was held in the horizontal plane by a 1.9 kg capacity compression spring positioned under a cross member between the arms and 7.6 cm from the fulcrum. The tension on the crosspiece was manually adjustable to compensate for differences in animal weights. Vertical deflections of the cage were mechanically linked to a transducer (Harvard Apparatus, Model 352-2) by a 5.1 cm lever. The output of the transducer was amplified and written-out by an electronic recording module (Harvard Apparatus, Model 350). The stabilimeter was calibrated by loading the animal chamber with 300 g in order to simulate the average weight of the animals used in the present experiments. A 10-g weight was then dropped onto the top of the chamber from various heights and the relationship between distance dropped and pen deflection was plotted. Pen deflections were measured to the nearest whole mm with no attempt to correct for arc distortions of pen movements. This calibration procedure indicated that cage deflections were approximately linearly related to pen deflections. The startle stimulus was a 6 kHz square wave, produced by an audio generator (Eico, Model 37), amplified (Lafayette, Model LA-85T), and delivered through two diffraction horn speakers (University, Model 4401) positioned 13 cm from the animal chamber. Stimulus duration was 50 msec, timed by an electronic interval timer (Grason-Stadler, Model E5350A) and gated through the contacts of high speed mercury-wetted reed relays. Rise time of the stimulus was estimated by monitoring the output of a small microphone placed inside the animal chamber on an oscilloscope. By this procedure rise time was determined to be less than 3 msec. The amplitude of the stimulus was determined empirically as that level sufficient to reliably evoke a startle response from an experimentally naive rat. Once determined, the stimulus amplitude remained fixed throughout and was estimated to be approximately 112 dB. A 10.2 cm cone speaker, positioned approximately 15.2 cm from the animal chamber, was connected to the output of a white noise generator (Foringer, Model 1291) through a manually operated switch. EXPERIMENT 1

Animals The animals were 10 Long-Evans male rats obtained from Simonsen Laboratories (Gilroy, California) at 70 days of age. The rats were individually housed in a temperature-regulated room with free access to food and water. Lights were on daily from 0700 to 1900. Procedure The animals were initially allowed 10 days to adapt to the laboratory environment, During this period, the animals were handled and weighed daily. Each animal was then placed in the stabilimeter chamber and permitted 12 rain of free exploration. On the following day, daily experimental sessions were started. Each session consisted of a 5 min quiet period followed by 10 presentations of the 6 kHz startle stimulus on a variable interval schedule of 45 sec (range: 30-60 sec). Immediately following the completion of the third session, one-half of the animal population was selected

randomly for subcutaneous injections of 300 mg/kg PCPA. The remaining animals were injected with vehicle solution. Thereafter injections were administered at the end of each daily experimental session. PCPA solutions were prepared by suspending the free amino acid in 0 . 9 ~ saline containing Tween-80 (one drop per 10 ml saline). Vehicle solutions lacked only PCPA. During the seventh and the eighth experimental sessions, a white noise stimulus was introduced into the chamber through the cone speaker. The novel auditory, stimulus was delivered for approximately 3 sec between the fifth and sixth presentations of the startle stimulus; onset of the novel stimulus was gradual so as to avoid elicitation of a startle response. After the ninth experimental session, conducted as described above without the white noise stimulus, the animals were sacrificed and their brains were removed. Each brain was dissected into telencephalon and brain stem fractions using procedures described previously; sections were then frozen and stored, and subsequently assayed for 5HT and norepinephrine [12]. Results Brain 5HT and norepinephrine levels are shown in Table 1. In each brain part, 5HT levels in P C P A treated rats were depleted to less than 10 per cent of control values (p < 0.01), while norepinephrine concentrations remained at 75 per cent of normal (p < 0.01).

TABLE ! BRAIN SEROTONINAND NOREPINEPHRINECONCENTRATIONS FOLLOWING TREATMENT WITH PARACHLOROPHENYLALANINE, AS DESCRIBED IN THE PROCEDURE FOR EXPERIMENT 1. ALL VALUES FOR THE AMINES ARE THE MEAN LEVELS, IN [/,g PER g OF BRAIN, -k S.E.M. FOR THE 5 RATS IN EACH TREATMENT GROUP

Amine Concentration in: Amine Serotonin Norepinephrine

Treatment

Brain Stem

Telencephalon

Vehicle PCPA Vehicle PCPA

0.436 ~iz 0.034 0.037 ~ 0.009 0.515 :j__0.019 0.387 7~ 0.013

0.385 ~ 0.012 0.033~ 0.01! 0.350 3- 0.024 0.259 ~ 0.0i0

Despite the variability apparent in a trial-by-trial presentation of the data, a characteristic pattern of habituation of the startle response was observed during the three sessions preceding the initiation of PCPA injections (Fig. 1). Response magnitude, initially high, decreased both within and between sessions, and some degree of spontaneous recovery of response magnitude was observed during the initial trials on the second and third sessions. There was no effect of PCPA treatment on startle response magnitude 24 hr after the initial injection (Day 4). However, 24 hr after the second drug injection (Day 5) the drug treated rats exhibited significantly higher (p < 0.05) response magnitude during initial presentations of the startle stimulus. A response decrement was observed during subsequent stimulus presentations. Despite continued injections, the response magnitude of the PCPA treated rats was not significantly greater than the control animals on the subsequent test day (Day 6) or during the initial trials on Day 7.

PARACHLOROPHENYLALANINE

I !

A N D HABITUATION

1217

t,, ....

"\

VEH(!

E

\

I + [

\

'

I

•

,0

I

•

I0

/

i

I0

TRI;4 S

FIG• 1. Mean magnitude of startle response in each group. From the uppermost left and across, each panel sequentially depicts the response mean on each of the l0 trials on each of the 9 testing days. PCPA injections were begun after the 3rd session. The novel white noise stimulus was presentedduring Day 7 (bottom left panel) and Day 8 (bottom center panel) as indicated by the arrow between the 5th and 6th trials•

After the brief presentation of the novel white-noise stimulus during the seventh session, the PCPA treated rats again exhibited a significantly higher overall level of startle responses than did the control rats (p < 0.05)• This increment in response magnitude persisted 24 hr, carrying over into the eighth test day during the initial trials given before readministering the white-noise stimulus. The second presentation of the novel auditory stimulus (Day 8) did not affect this change. However, this difference in startle response magnitude between the PCPA and vehicle treated rats was transitory; there was no overall difference between the two groups on Day 9.

statistically reliable during the initial trials of the eighth experimental day. Following presentation of the white-noise stimulus during the eighth session, the PCPA injected animals again exhibited significantly greater startle responses than did vehicle treated rats (p < 0.05); this difference was not observed during the ninth session conducted 24 hr later. The combined results of Experiments 1 and 2 clearly demonstrate that PCPA causes a transitory increment in the

EXPERIMENT 2

Method The animals were 18 Long-Evans male rats 70 days of age. The stabilimeter and associated apparatus previously described were employed. The procedure was identical to that used in Experiment 1 except that the novel white-noise stimulus was presented only during the eighth session of the present experiment. This procedure is essentially a replication of the previous experiment, necessitated by the small sample of animals therein.

v

,~J

~ ' , L d

I ~ -....j.\

f ,

CLE

/

'1'J i I

i:

i

LU

,t

;

~,

\

Results i

After initiating PCPA injections, a significant increment in the magnitude of the habituated startle response was initially observed in the treated rats after the third injection day (Fig. 2; Day 6, p < 0.05). This effect also was present on the seventh session (p < 0.05). This response increment was no longer

FIG. 2. Mean magnitude of startle response for each group on each trial on Day 3 (upper left panel), Day 6 (upper right panel), Day 7 (lower left panel) and Day 8.

1218

CONNER, STOLK, BARCHAS AND LEVINE

magnitude of the habituated startle response, an effect which appears two or three days after starting the daily drug injections. It may be important to note that this is the same time required for the maximum reduction in brain 5HT levels by daily injections of the drug. In addition, PCPA causes an increment in responding to presentations of the startle stimulus on trials immediately following the delivery of a brief novel stimulus. These data suggest that brain 5HT levels may play a role in the habituation process. If this hypothesis is correct then it would be expected that depletion of brain 5HT levels before habituation would effect a change in the time course of habituation. This prediction is evaluated in the next experiment. EXPERIMENT

magnitude over the first 5 trials on Days 2 and 3 (t's :- 2.41 and 2.72, respectively, df's .... 34, p's <: 0.05), as well as over the last 5 trials on Day 3 (t : : 2.17, d f .... 34, p < 0.05). There were no significant differences between the groups on Day 4, nor over the first 10 trials on Day 5. However, when administered 5 additional trials on Day 5, the PCPA treated rats showed a significantly greater overall response magnitude than did the vehicle treated controls (t 3.46, d)':~ 26, p < 0.01). Further, the difference in overall response levels between trials 6-10 and trials 11-15 was highly significant for the PCPA treated rats (t = 4.0t, d/~ t3, p --- 0.0t), while this statistical comparison did not approach significance for the vehicle treated rats (t -:-_ 1.00).

3

DISCUSSION

Method

The results of the present experiments suggest that brain 5HT plays a role in modulating the responses of the organism to environmental stimulation. Specifically with regard to the skeletal-motor startle response, normal concentrations of brain 5HT are not essential for normal levels of responding. Thus, PCPA treated rats, the brains of which contain about 90 per cent less 5HT than do normal controls (Table 1), do exhibit habituation of the startle response to intense auditory stimuli--however, they require more trials than normal to suppress responses to the repetitive stimuli (Experiment 3: Fig. 3). Similarly, the drug treated rats are hypersensitive to the disrupting effects of altered environmental input, whether it be a novel auditory stimulus (Experiments t and 2) or an alteration in the test session length (Experiment 3). While the changes in response levels observed after these experimental procedures are reliable and reproducible, they are transient. and normal response patterns are reestablished during subsequent testing. The present data cannot be evaluated in terms of the report of Aghajanian and Sheard [1] because of the very different experimental procedures employed. At first glance the effects of PCPA on habituation processes seemingly are quite remote from the drug's effect on other types of behavior. However. a more detailed consideration

The animals were 36 Long-Evans male rats 70 days of age. The apparatus previously described was used. In the present experiment the procedure differed in that the daily drug or vehicle injections were started four days before the first experimental session. Presentation of the startle stimuli was initiated the first time the animals were placed in the chamber. On the last day of testing, 14 randomly selected rats from each group were administered 5 additional trials on the same schedule (VI-45 sec), making a total of 15 trials in that session. This procedure was employed to determine whether the PCPA animals were sensitive to a change in the usual testing routine.

Results A trial-by-trial presentation of the results is shown in Fig. 3. Prior to analysis of these data, the 10 trials on each day were collapsed into 2 blocks of 5 trials each (Trials 1-5 and 6-10, respectively). An overall analysis of variance yielded a significant interaction between groups and blocks of testing trials (F ----8.89, df = 1/26, p < 0.01). Comparisons between the groups on each testing day indicated that the P C P A treated animals exhibited a significantly greater response

\

DAY Z

OA.Y I

DAY

q,

E

Z t9

l

I0

q

p.. W

I0

DAY ]5

DAY 4 tU .J Ja,-

I 0---0

q,

\

PCPA - VEHICLE

A

4[ lU

I

IO

I

I0

TRIALS

FIG. 3. Mean magnitude of startle response on each trial for each group. Fourteen rats from each group were administered 5 additional trials on Day 5,

PARACHLOROPHENYLALANINE A N D HABITUATION of these effects reveals what may be a c o m m o n mechanism of action of P C P A in many behavioral test situations. Both Tenen [13] and Schlesinger, Schreiber and Pryor [8] report that P C P A improves acquisition of an active avoidance task in rats. Tenen also reports an increased sensitivity to footshock (cf. [6]) as well as a decrease in emotionality following the drug, changes which could account for the effects on avoidance acquisition. The possibility that sensory modalities other than shock threshold are affected by P C P A treatment was entertained by Stevens, Resnick and Krus [10], who described an enhanced rate of acquisition of a brightness discrimination task after PCPA. An alteration in sensory thresholds, however, cannot explain the impaired acquisition of a passive avoidance response [9], or the increased responding for reward during a CS-paired punishment period [7] alter P C P A treatment, since an increased sensitivity to footshock would be expected to facilitate such behaviors. In each of the above situations (with the exception of the Aghajanian and Sheard report [1]) the reduction of brain 5HT with P C P A appears to cause an impairment in the organism's capacity to suppress a response. Reduced emotional reactivity after PCPA, an effect inferred by a number of investigators [7, 9, 13], could facilitate active, or retard passive, avoidance learning by reducing the probability of a

1219 competing response emerging. Thus, lowered 5HT levels in brain may decrease the tendency of freezing behavior to occur, resulting in the observed effects on active and passive avoidance behaviors. Finally, with regard to the present experiments, there is neurophysiological evidence for neuronmediated feedback suppression of sensory input (e.g [3, 4]) which might account for the effects of P C P A on habituation. Although P C P A has other effects on brain amines [11], the postulate that 5HT-containing neurons in the brain stem are capable of suppressing incoming sensory input and that brain 5HT thereby modulates the responses of the organism to its environment is consistent with the above data. With respect to habituation to startle stimuli, brain serotonergic neurons may play an accessory role since the observed effects of 5HT depletion by P C P A are transitory, and other brain mechanisms eventually are able to compensate for the drug-induced changes in habituation. Whether this role is peculiar to response suppression to auditory stimuli, or can be generalized to many types of response situations as postulated above, awails further experimentation. The authors gratefully acknowledge the assistance of Mr. GEORGE MUNDY, Mr. HUMBERTOGARCIA,Mrs. PAMELAANGWIN and Mrs. ELIZABETHERDELYI.

REFERENCES

I. Aghajanian, G. K. and M. H. Sheard. Behavioral effects of midbrain raphe stimulation--dependence on serotonin. Communs. behac. Biol. 1: 37-41, 1968. 2. Conner, R. L., J. M. Stolk, J. D. Barchas, W. C. Demcnt and S. Levine. The effect of parachlorophenylalanine on shockinduced fighting behavior in rats. Physiol. Behaz~. 5 : 1221-1224 (1970). 3. Galambos, R. Supprcssion of auditory nerve aclivity by stimulation of efferent fibers to cochlea. J. Neurophysiol. 19: 424--437, 1956. 4. Hernandez-Peon, R., H. Scherrer and M. Jouvet. Modification of electric activity in cochlear nucleus during attention in unanesthetized cats. Science 123: 331-332, 1956. 5. Koe, B. K. and A. Weissman. p-Chlorophenylalanine: a specific depletor of brain serotonin. J. Pharmac. exp. Ther. 154: 499-516, 1966. 6. Lints, C. E. and J. A. Harvey. Altered sensitivity to footshock and decreased brain content of serotonin following brain lesions in the rat. J. comp. physiol. Psychol. 67: 23-31, 1969. 7. Robichaud, R. C. and K. L. Sledge. The effects of p-chlorophenylalanine on experimentally induced conflict in the rat. Life Sci. 8" 965-969, 1969.

8. Schlesinger, K., R. A. Schreiber and G. T. Pryor. 12ffects of p-chlorophenylalanine on conditioned avoidance learning. Psychonom. Sci. 11: 225-226, 1968. 9. Stevens, D. A., L. D. Fechter and O. Res~fick. The effects of p-chlorophenylalanine, a depletor of brain serotonin, on behavior: II. Retardation of passive avoidance learning. Li[e Sci. 8: 379-385, 1969. 10. Stevens, D. A., O. Resnick and D. M. Krus. The effects of p-chlorophenylalanine, a depletor of brain serotonin, on behavior: I. Facilitation of discrimination learning. Life Sci. 6: 2215-2220, 1967. 11. Stolk, J., J. Barchas, W. Dement and S. Schanberg. Brain catecholamine metabolism following para-chlorophenylalanine {pCP) treatment. Pharmacologist 11 : 258, 1969. 12. Stolk, J. M., W. J. Nowack, J. D. Barchas and S. R. Platman. Brain norepinephrine: enhanced turnover after rubidium treatment. Science 168: 501-503, 1970 13. Tenen, S. S. The effects of p-chlorophenylalanine, a serotonin depletor, on avoidance acquisition, pain sensitivity and related behavior in the rat. Psychopharmacologia 10: 204-219, 1967. 14. Ulrich, R. E. and N. Azrin. Reflexive fighting in response to aversive stimulation. J. exp. Analysis Behav. 5:511-520, 1962.