Habituation of the startle response in adrenalectomized rats

Physiology and Behavior, Vol. 8, pp. 579-584, Brain Research Publications Inc., 1972. Printed in Great Britain.

Habituation of the Startle Response in Adrenalectomized Rats' MICHAEL DAVIS AND ANDREW

J. Z O L O V I C K

Yale University School o f Medichze and The Connecticut Mental Health Center, New Haven, Connecticut 06519, U.S.A. (Received 6 O c t o b e r 1971) M. AND A. J. ZOLOVICK. Habituation of the startle response in adrenalectomized rats. PHYSIOL.BEHAV. 8 (4) 579-584, 1972.--In a series of three experiments adrenalectomized, sham-operated and non-operated rats (N = 15)were presented with 27 tones at each of three intensities on two successive days or 60 tones at a 60 sec interstimulus interval with each tone being followed by another tone either 2, 4, 8 or 16 sec later, or 50 tones at a 30 sec interstimulus interval on each of five successive days. All groups had equivalent initial startle amplitudes and showed equivalent rates of response decrement at each test intensity and each interstimulus interval as well as over successive days of exposure. The results indicate that the adrenal glands are not crucial for habituation of the acoustic startle response and suggest that those aspects of stress adaptation that require an intact adrenal-pituitary system are not important for habituation of the startle.

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Habituation

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THE ACOUSTICstartle response in rats is being increasingly used to study the process of habituation. Laws derived from the study of startle habituation have been shown to apply to other forms of habituation [6] and have provided a basis for important distinctions that have been made concerning the parametric characteristics of habituation [3, 4]. In order to elicit a measurable startle response, it is necessary to present an intense auditory stimulus that has an abrupt onset [5]. Tone intensities of 120 or 130 db are commonly used. Such intensities, when presented to a human observer, are highly noxious or even stressful. If repetitive presentation of high intensity stimuli is stressful to the rat, then habituation to these stimuli might be viewed as a form of adaptation to stress. Since the pituitary-adrenal system has been shown to be important for stress adaptation, interference with this system might be expected to affect startle habituation. Thus removal of the adrenal glands, which severely retards stress adaptation in other situations [16] should also retard startle habituation if stress is an important consequence of a startleeliciting stimulus. To date, there is little evidence to support the view that the pituitary-adrenal system is important for startle-response habituation. Neither injection of adrenalin [13], nor adrenal demedullation [14], nor bilateral adrenalectomy [12] had any measurable effect on startle amplitude. Bilateral adrenalectomy also failed to affect the extent to which the startle response could be enhanced by presenting the eliciting stimulus in the presence of a cue that had previously been paired with shock [1]. Since in all of these studies the index of startle amplitude was based on the average amplitude over stimulus exposures, which can be viewed as a short habitua-

tion session, and since differences in rate of response decrement over observations would influence the average amplitude across all observations, the lack of differences within these studies suggests that differences in rate of habituation probably did not occur. However, since these experiments were not explicitly designed to study the effects of adrenalectomy on habituation, and since this information would be important for future theoretical treatments of habituation, the present series of experiments sought to evaluate this question more fully. Accordingly, startle responsiveness and rate of habituation of adrenalectomized and non-adrenalectomized rats were compared by repetitively presenting tones at several different intensities (Experiment 1), or interstimulus intervals (Experiment 2), or over several days (Experiment 3). EXPERIMENT 1

The purpose of Experiment 1 was to evaluate whether adrenalectomized and non-adrenalectomized rats would differ in their initial startle sensitivity. Several different intensities were employed to allow comparison at different levels of startle amplitude and these intensities were presented several times on each of two days to evaluate also whether the groups might differ in their rate of response decrement both within a day and across a 24-hr retention interval.

Method Animals. Forty-five adult male albino rats of the SpragueDawley strain weighing between 350--400 g were used. Upon receipt from the supplier (Charles River Co.) the animals

aThis research was supported by USPHS Grants MH 17856 and MH 07114 and by NSF Grant GB-23685 and the State of Connecticut. Our thanks to Drs. Philip Applewhite and Frank Gardner for their suggestions, Harry Bear for assistance in operating, and especially to Lee Schulhof for running the animals and scoring the data. 579

580 were housed in a large colony room in group cages of 5 animals each and maintained on a 12-hr light cycle. Apparatus. Five separate stabilimeter devices were used to record the amplitude of the startle response. Each stabilimeter consisted of a 3.5 × 6 × 6 in. Plexiglas and wire mesh cage suspended within a 10 × 8 × 8 in. wooden frame. Within this frame, the cage was sandwiched between four compression springs, above, and a 2 × 2 in rubber cylinder, below, with an accelerometer (M.B. Electronics Type 302) located between the bottom of the cage and the top of the rubber cylinder. Cage movement resulted in displacement of the accelerometer and the resultant voltage was fed through a matched accelerometer amplifier (M.B. Electronics Model N504), the output of which was proportionate to the velocity of accelerometer displacement. The amplified signal then was fed to a specially designed sample and delay circuit. Basically this circuit consisted of five channels, one for each stabilimeter, and was used to sample the peak accelerometer voltage that occurred during a 25 msec time band, 15 msec after the onset of the startle-eliciting stimulus. Immediately prior to this sample period, each channel was discharged so that any spontaneous activity occurring between stimulus exposures was erased. In this way, the amplitude of the startle response of five animals was recorded simultaneously and stored in one of each of the five channels. Immediately after the 25 msec sample period, the five channels were discharged sequentially into one channel of a Honeywell Model 2106 Visicorder. The output appeared as a series of five rectilinear galvanometer spikes, one for each cage, separated in time to allow discrimination of each. This system thus obviated any possibility of overlap among the five channels and, since it permitted use of the full 100 mm width of the paper, allowed appreciable resolution for each amplitude measurement. The amplitude of the startle response was defined as the mm of galvanometer deflection and was measured by hand to the nearest mm. The five stabilimeters were housed in one corner of an 8 × 8 × 7 ft ventilated, sound-attenuated chamber (Industrial Acoustics, Co.). The cages were placed in a semicircle, 45 in from an Altec, high-frequency loud-speaker which was used to provide a 4000 Hz, 90 msec tone, which was shaped through an electronic switch (Grason-Statler #E829E) with a rise-decay time of 5 msec. The intensity of the tone, which was varied across experiments was measured with a General Radio Model 1551-C Sound Level Meter (20 Hz setting) byplacing the microphone in each cage and positioning the cages so as to have comparable db-readings in each. Throughout all phases of the experiments a background level of 60 db white noise was maintained. Background illumination consisted of the light provided by the two 150-W bulbs in the ceiling of the IAC room. Operations. Bilateral adrenalectomies were performed on 15 animals under ether anesthesia using the suprarenal approach. An additional 15 animals were sham-operated. F o r these animals all features of the above operation were performed except for removal of the adrenals. Another 15 animals served as non-operated controls. Following successful removal of the adrenals, the peritoneum and skin were sewn with discontinuous sutures. Following these procedures all animals were returned to the colony room and maintained on food and water, ad lib. The adrenalectomized animals were given a 1 Y/oosaline drinking solution to supplement the loss of sodium. Procedure. On the first experimental day (Day 1) 15 of the 45 animals, five from each of the three groups, were placed

DAVIS AND ZOLOVICK in the stabilimeters and presented with 27 tones at each of three different intensities (100, 110, and 120 db). The various intensities were presented in an irregular order with the restrictions that each intensity followed itself and every other intensity equally often and that the various intensities were distributed uniformly over the total series of 81 tones. On the second experimental day (Day 2), the procedure was identical and was included to evaluate the degree to which the various operations might affect retention of startle habituation over a 24 hr interval. The above procedures were carried out in a series of three replications with each replication requiring two days. The first experimental day occurred 9 days after the operations in Replication 1, 11 days after in Replication 2, and 13 days after in Replication 3. The replications were identical except that the order in which the three groups were run within a day was varied across replications so that the average time of day each group received tone exposure was the same for all groups across replications. Data analysis. The data were analyzed with multifactor analyses of variance with repeated measures using type of operation (groups) as a between animals factor and tone intensity (intensity), blocks of nine tones (blocks), and days (days) as within animals factors.

Results and Discussion Figure 1 presents the mean amplitude of the startle response over blocks of nine tones at test intensities of 120 db (solid circles), 110 db (open circles), and 100 db (open triangles) on Days 1 and 2 for the adrenalectomized (left panel), sham-operated (middle panel), and non-operated groups (right panel). The results have been combined over replications since the results within each replication were highly similar. Figure 1 indicates that the amplitude of the startle response was greater, the higher the intensity of the tone. This was true on both Days 1 and 2 and was highly reliable (F = 109.00, df= 2/84, p < 0.001; and F ---- 87.10. df= 2/84, p < 0.001; respectively). Most important, however, was that startle amplitude at each intensity was essentially identical for all three groups since neither the groups factor nor the groups by intensity interaction approached statistical significance. Thus adrenalectomy did not alter startle sensitivity. Figure 1 also illustrates that substantial response decrement across the three blocks of nine tones occurred on Day 1 (F = 9.15, d r : 2/84, p < 0.001) with a faster rate of decrement the higher the intensity of the tone as witnessed by a reliable blocks by intensity interaction (F : 9.35, df= 4/168, p < 0.001). Again, however, all three groups had similar rates of response decrement at each of the three test intensities since neither the groups by blocks interaction nor the groups by blocks by intensity interaction was statistically significant. Thus adrenalectomy did not alter response decrement to repetitive tone exposure. Finally, Fig. 1 illustrates that startle habituation was retained over the 24 hr interval, since the overall level of startle on Day 2 was lower than on Day 1. This retention effect, which was highly significant ( F : 24.98, d f : 1/42, p < 0.001) was, however, evident in each group and no reliable differences among the groups were found either with regard to their overall startle on Day 2 or on the degree of change from Day 1 to Day 2, since neither the groups factor on

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Day 2 nor the groups by days interaction approached statistical significance. Thus adrenalectomy did not alter retention of startle habituation over a 24 hr interval. EXPERIMENT 2

If startle decrement to repetitively presented tones is considered to be a result of a progressive increase in some inhibitory process, then the results of Experiment 1 suggest that the adrenals are not crucial for this kind of inhibition to develop. Another inhibitory-like phenomenon that is readily demonstrable within the startle system is one in which the likelihood of response to a tone can be depressed by preceding it at short intervals by another, single tone, where, in general, the degree of suppression is greater, the shorter the interval between the two tones. This relationship has been shown over a wide range of intervals, ranging from 40 msec [7] to 15 sec [2], and has been consid~a'ed as a measure of the decay of short-term habituation [3]. The purpose of Experiment 2, therefore, was to evaluate whether adrenalectomized and non-adrenalectomized animals might differ on the degree to which a previous single tone could depress responsivity to another tone presented shortly thereafter.

Method Animals. The animals were the same 45 animals used in Experiment 1. Apparatus. All features were identical to those in Experiment 1. Procedure. Twenty-four hr after Experiment 1, a group of 5 animals was placed in the stabilimeters and after 15 min presented with 60 tones. These tones, which will be called

the conditioning tones, were presented once every 60 see. Either 2, 4, 8 or 16 sec after each conditioning tone, another tone, which will be called the test tone, was presented. The four different conditioning-test intervals were presented in an irregular order with the restrictions that each interval followed itself and every other interval equally often and the various intervals were presented uniformly over the entire session. The intensity of both the conditioning and test tones was 120 db. Identical to Experiment 1, 5 animals from each of the three groups were run on a day over three replications with the order in which the particular groups were run within a replication varied so that the average time of day each group received tone exposure was the same for all groups across replications.. Data analysis. The data were analyzed with analyses of variance with repeated measures using type of operation (Groups) as a between animal factor and conditioning-test interval (intervals) or blocks of ten conditioning tones (blocks) as within animal factors.

Results and Discussion Figure 2 shows the mean amplitude of the startle response at each of the four conditioning-test intervals for each of the three groups combined over all replications. Figure 2 illustrates that the amplitude of the startle was highly dependent on the time since a previous stimulus, with a higher amplitude, the longer the conditioning-test interval. This relationship was seen in every subject and was highly reliable (F-----32.72, d r = 31126, p < 0.001). Most important, however, was that the slope of this function was highly similar for all groups and neither the groups factor nor the groups by intervals interaction was statistically significant.

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The purpose of Experiment 3 was to evaluate whether differences between adrenalectomized and non-adrenalectomized animals might emerge if several days of stimulus exposure were used. Also, in view of the lack of differences among the groups in Experiments 1 and 2, the success of the present operative technique was checked by measuring either stress-induced plasma levels of corticosterone between operated and non-operated groups following Experiment 3.

Method Animals. The animals were 30 male albino rats that weighed between 350-400 g. Apparatus. All features of the apparatus were identical to those in Experiment 1. Operations. The same operative technique was used as that in Experiment I, except that only two groups of 15 adrenalectomized and 15 non-operated animals were included in Experiment 3.

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Procedure. On the first experimental day, which occurred nine days after the operations, each of 5 animals was placed in a stabilimeter and after 15 rain presented with 50, 120 db tones at 30 sec ISI. This procedure was then repeated on the remaining 25 animals so that all 30 animals were run on the same day. Animals from the adrenalectomized and nonoperated groups were distributed uniformly across the six squads of 5 animals each, so that the average time of day both groups were run was the same. These procedures were then repeated for the next four days, creating a total of five days of exposure. One week following the last exposure day, the animals were returned to the stabilimeters and given one additional series of 50 tones to evaluate the degree to which the adrenalectomy might affect long-term retention of startle habituation. Immediately following the final experimental session the animals were individually placed in an ether-saturated chamber for 30 see. Fifteen see after removal from the chamber the animals were decapitated and blood collected in heparinized tubes. Plasma was obtained by centrifugation and then frozen for later analyses. Plasma levels of corticosterone (supplied by Up John Co.) were determined fluorometrically according to the methods of Peron and Dorfman [15] and Moncloa, Peron and Dorfman [11]. Data analysis. The startle data were analyzed with analyses of variance with repeated measures using type of operation (groups) as the between animal factor and blocks of five tones (blocks) and days (days) as the within animals factors.

HABITUATION IN ADRENALECTOMIZED RATS

583

Results and Discussion

their rate of response decrement over repetitive presentation of each of these intensities, their rate of decrement over a 24 hr interval at each of these three intensities, their level of startle over different intervals following a previous tone exposure, their rate of response decrement over tones presented at a 60 see ISI, their rate of decrement over tones presented at a 30 sec ISI, their rate of decrement over five days of periodic tone exposure, or their degree of retention one week following five days of tone exposure. Recently [10] it has been suggested that the adrenal glands may be important for habituation of the startle response. This suggestion was based, in part, on the results of a study by Johnson [9] who reported that the combined degree of response decrement of chronic and acutely adrenalectomized rats was less than that of non-operated rats when 10, 130 db tones were presented on each of six days. This conclusion was based on the finding that the overall level of startle on Day 4 was higher for the adrenalectomized animals, as compared to the controls. While the difference on Day 4 was significant at the 5 % level of confidence when startle amplitudes were transformed to log scores, a number of factors raise some question as to the generality of the finding and of the conclusion that the adrenals are necessary for startle response habituation. First, of the 15 original chronic adrenalectomized animals, only the 8 animals that showed the lowest corticosteroid response to ether stress were included in the study, so that difficulties inherent in animal selection might have been operating. Secondly, data were not presented for Days 5 and 6 since more than 10 % of the responses on these days were not measurable because the animal was walking around the chamber at the time the startle stimulus was presented, or because the startle was c~mposed of an initial downward movement of the chamber. Presumably, the startle response on the other days was therefore recorded as an initial upward movement of the cage. Since the acoustic startle response in rats is typically recorded as an initial downward movement of the cage [8] it is unclear what response Johnson was measuring. In the present experiment, the recorded startle was seen as an initial downward movement

Figure 4 shows the mean amplitude of the startle response over blocks of five tones on each of the five days for the adrenalectomized and non-operated groups. Figure 4 indicates that substantial habituation occurred across days ( F = 14.60, d f : 4/112, p < 0.001) and across blocks of tones (F = 6.22, df= 9/252, p < 0.001). More importantly, the rate of decrement both across days and across blocks of tones was similar for both groups and neither the overall difference (groups) nor the groups by blocks, nor the groups by days, nor the groups by blocks by days interaction was statistically significant. Finally, both groups showed equivalent amounts of retention one week following their last day of exposure. The mean startle amplitude across all 50 tones on this retention day (not shown in Fig. 4) was 21.4 for the non-operated group and 19.8 for the adrenalectomized group. Since these values were almost identical to those of Day 5, both groups showed essentially perfect retention over the 7-day interval and the lack of difference between the adrenalectomized and nonoperated groups indicates that the adrenal glands were not necessary for this retention to occur. The lack of differences in habituation between the adrenalectomized and non-operated animals was not due to an unsuccessful removal of the adrenal glands. Thus ether stress evoked a mean plasma corticosterone concentration of 36.6 :k 5.1 ~tg/100 ml in the non-operated animals but only 2.7-4-2.5 ~g/100 ml in the adrenalectomized animals and this difference was highly reliable ( t = 23.04, d f : 28, p < 0.001).

GENERAL DISCUSSION

The present results indicate that adrenalectomized rats were indistinguishable from sham-operated and non-operated rats over a wide variety of forms of startle habituation. Adrenalectomized animals did not differ from controls on their initial levels of startle at each of three test intensities,

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DAVIS AND ZOLOVICK

of the cage, followed by an upward deflection at a longer latency. Most compelling, however, was the fact that within her own study, analyses of variance failed to find either a significant overall difference between the adrenalectomized and control groups, or a significant operation by trials interaction, or a significant operation by days interaction. The lack of significance of both of these interactions, which should have been sensitive to different rates of habituation

between the various groups, makes it difficult to conclude that adrenalectomized rats showed slower rates of habituation. The present series of studies, as well as the available literature, indicate therefore that an intact adrenal-pituitary system is not crucial for habituation of the acoustic startle response and suggest that those aspects of stress adaptation that are mediated by the adrenal-pituitary system are not critical for habituation of the startle.

REFERENCES 1. Applezweig, M. H. and F. D. Baudry. The pituitary-adrenocortical system in avoidance learning. Psychol. Rep. 1: 417-420, 1955. 2. Brown, J. S., J. W. Meryman and F. N. Marzocco. Soundinduced startle response as a function of time since shock. J. comp. physiol. Psychol. 49: 190-194, 1956. 3. Davis, M. Effects of interstimulus interval length and variability on startle-response habituation in the rat. J. comp. physiol. Psychol. 72: 177-192, 1970. 4. Davis, M. and A. R. Wagner. Startle responsiveness after habituation to different intensities on tone. Psyehonom. Sci. 12: 337-338, 1968. 5. Fleshier, M. Adequate acoustic stimulus for startle reaction in the rat. J. comp. physiol. Psyehol. 60: 200-207, 1965. 6. Groves, P. M. and R. F. Thompson. Habituation: A dual process theory. Psychol. Rev. 77: 419-450, 1970. 7. Hoffman, H. S. and J. L. Searle. Acoustic variables in the modification of startle reaction in the rat. J. comp. physiol. Psychol. 60: 53-58, 1965. 8. Horlington, M. A method for measuring acoustic startle response latency and magnitude in rats: Detection of a single stimulus effect using latency measurements. Physiol. Behav. 3: 839--844, 1968.

9. Johnson, J. E. T. Influence of pituitary-adrenal hormones on habituation and conditioned avoidance behavior. Unpublished doctoral dissertation, Stanford University, 1970. 10. Levine, S. Stress and behavior. Scient. Am. 224: 26-32, 1971. 11. Moncloa, F., F. G. Peron and R. I. Dorfman. The fluorimetric determination of corticosterone in rat adrenal tissue and plasma: Effect of administering ACTH subcutaneously. Endocrinology 65: 717-724, 1959. 12. Moyer, K. E. Effect of adrenalectomy on the startle response in the rat. J. genet. Psychol. 109: 327-329, 1966. 13. Moyer, K. E. and B. N. Bunnell. Effect of exogenous adrenalin on the startle response in the rat. J. genet. Psychol. 95: 255-259, 1959. 14. Moyer, K. E. and B. N. Bunnell. Effect of adrenal demedullation on the startle response of the rat. J. genet. Psychol. 67: 341-344, 1960. 15. Peron, F. G. and R. I. Dorfman. A method for evaluation of adrenocorticotropic hormone suppressing action of corticoids. Endocrinology 64:431-436, 1959. 16. Selye, H. The Stress of Life. New York: McGraw-Hill, 1957.