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Reflex modulation due to supplementary stimulation
BEHAVIORAL BIOLOGY 22, 375--387 (1978)
Reflex Modulation due to Supplementary Stimulation ~ CLAIRE ADVOKAT *'2 AND PETER L . CARLTON~"
*Rutgers University and tOepartment of Psychiatry, College of Medicine and Dentistry of New Jersey-Rutgers Medical School, New Brunswick, New Jersey 08903 A series of experiments examined the effect of supplementary acoustic stimulation on the acoustic startle reflex of the rat. Separate groups of animals received low intensity pulses of white noise (prepulses) at one of several intervals (10, 100, 500, or 2500 msec) prior to startle-eliciting pulses of white noise. At interpulse intervals of 100 and 500 msec the startle reflex was attenuated on trials preceded by prepulses; at the 10- and 2500-msec intervals, on the other hand, the reflex was not attenuated, although the latter conclusion must be tentative because of apparatus limitations. Furthermore, at 2500 msec, amplitudes on all trials were greater than those obtained in controls that did not receive prepulses. This was the case regardless of whether supplementary stimuli preceded or followed the startle stimuli. This elevation of amplitude, unlike the attenuation, was eliminated after repetitive startle stimulation. These differential effects of supplementary stimulation suggest that two systems may mediate reflex elicitation.
It has been demonstrated that both the magnitude and latency of the startle reflex may be significantly altered as a consequence of preliminary stimulation. The direction and extent of such modification depend upon both the intensive and temporal characteristics of such antecedent stimulation. Hoffman and his colleagues (Hoffman and Fleshier, 1963; Hoffman and Searle, 1965, 1968; Hoffman and Wible, 1969) have shown that the reflex may be either attenuated or facilitated depending upon the relationship between startle stimuli and the prevailing physical environment. In particular, reflex amplitude may be attenuated if a supplementary acoustic stimulus precedes a startle stimulus by a lead time of approximately 20 to 2000 msec. This attenuation has been termed prepulse inhibition. Furthermore, such attenuation is not restricted to stimulus onset but may be 1 This report is based on portions of a dissertation submitted by the first author to Rutgers University in partial fulfillment of the requirements for the Ph.D. The research was partially supported by General Research Support funds granted by College of Medicine and Dentistry of New Jersey-Rutgers Medical School to Dr. Peter L. Carlton. 2 Requests for reprints should be sent to Dr. Claire Advokat, Division of Neurobiology and Behavior, Department of Physiology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York City, New York, 10032. 375 0091-6773/78/0223-0375502.00/0 Copyright@ 1978by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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produced by both stimulus offset or changes in the frequency of an otherwise continuous tone (Stitt et al., 1973; Stitt et al., 1974). Prepulse inhibition is not limited either to the acoustic modality or to the startle reflex of the rat: Comparable results have been obtained with visual stimuli (Buckland et al., 1969; Ison and Hammond, 1971; Stitt, 1975; see also Hilgard, 1933); the effect has been demonstrated in several species, including rats (Hoffman and Fleshier, 1963), rabbits (Ison and Leonard, 1971), pigeons (Stitt, 1975), and humans (Graham, 1975; Graham et al., 1975; Hilgard, 1933; Krauter et al., 1973). Prepulse inhibition represents one means by which the startle reflex may be profoundly modified. It is also the case, however, that the reflex is attenuated as a consequence of repetitive startle stimulation per se, i. e., in the absence of extra stimulation. This form of behavioral decrement has been termed habituation (Davis, 1970; Davis and Wagner, 1968, 1969). Both forms of response decrement, prepulse inhibition and habituation, represent instances of reflex modulation produced by antecedent stimulation. Yet, the extent to which these phenomena interact is unknown. This fact warrants consideration, since analyses of prepulse inhibition require repetitive presentation of startle stimuli. The recent work of Graham and her colleagues (Graham, 1975; Graham et al., 1975) provides an example of such methodological influences in analyses of reflex responsiveness. These investigators reported both attenuation and enhancement of the human eyeblink response to startle stimuli as a consequence of preliminary acoustic stimulation. However, such enhancement was dependent upon the experimental procedure; enhancement was obtained only when each subject was tested with all interpulse intervals ("within" subjects design). When the interpulse interval remained constant ("between" subjects design) amplitudes were not elevated. In view of these considerations, the present studies were designed to assess the interaction, if any, of repeated startle stimuli (producing habituation) and supplementary stimuli (producing prepulse inhibition).
GENERAL METHODS Subjects
The subjects were naive, male albino Charles River rats obtained from Charles River Distributors, Wilmington, Massachusetts. Upon arrival at the laboratory the subjects were housed individually, provided with constant illumination and permitted ad lib. access to food and water. At the time of testing the subjects weighted at least 215 and not more than 500 g; the majority were approximately 250 to 350 g.
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Apparatus The experimental chamber was housed in an Industrial Acoustic Chamber (IAC 10771), the inside dimensions of which were 79.5 x 60.0 x 59.5 cm. The experimental "startle chamber" consisted of a 21.0 x 8.5 x 8.5-cm perforated Plexiglas cage with a grid floor that was mounted rigidly to a 32.5 x 26.0-cm Plexiglas platform. The platform was, in turn, fixed by four heavy-duty springs to a 48.0 x 26.0-cm wooden board. This assembly was secured to the floor of the IAC. T h e startle response was monitored as the voltage output from a phonograph cartridge (Astatic N4-2, Model 16) mounted on the side of the cage; this voltage was recorded by a printing voltmeter. The cartidge had a 4.5-cm rod weighted at the end inserted where the needle would normally be. This arrangement, although insensitive to gross bodily movements made by the rat, was sensitive to the abrupt, ballistic movements produced by the startle stimulus. Movement of this rigidly mounted system could not be detected by gross observation. A 60.0-cm Altec midrange driver and horn (Model 27A) was used to deliver the startle stimuli and prepulses. A constant background noise was provided by a BRS-Foringer audio generator (Model AU-902) coupled to an 8.0-ohm speaker mounted above the experimental chamber. The delivery and timing of stimuli were controlled by standard Tektronix programmming equipment housed in a separate room. Startle stimuli were provided by a Grason-Stadler noise generator (Model 901B) coupled to a Grason-Stadler electronic switch (Model 829). Prepulses were generated by a second Grason-Stadler noise generator. Stimulus intensity (in decibels with respect to 0.0002 dyn/cm 2) was calibrated before each daily use. For this purpose the stimulus was turned on continuously and its intensity calibrated with a General Radio octave band noise analyzer (Model 1558-BP). The microphone for the analyzer was placed at the geometric center of the animal chamber. The startle stimulus consisted of a 120 + 1 db, 10-msec burst of white noise (rise time = 10/zsec). The prepulse was a 90 + 1 db, 10-msec burst of white noise. The duration of these stimuli was calibrated on an oscilloscope. The constant background noise was 72 _+ 2 db.
EXPERIMENT
1
All subjects were randomly assigned to one of four groups (N = 6/group). Each rat was placed in the startle chamber for an initial 20-min foreperiod. At the end of this period each subject received 40 startle stimuli, 20 of which were preceded by prepulses. The startle and prepulse trials were presented in a counterbalanced order as follows: S - P S - P S -
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S - P S - S - S - P S . The first trial, therefore, was never preceded by a prepulse. This sequence was presented five times for a total of 40 trials. Each of the four groups received a different interpulse interval on PS trials. For one group (Group 10) the interpulse interval (IPI) was 10 msec; for the second group (Group 100) it was 100 msec; for the third group (Group 500) it was 500 msec; and for the fourth group (Group 2500) it was 2500 msec. At the end of the initial 40 trials, all subjects received an additional 60 trials consisting only of startle pulses (called "habituation trials' ,3 hereafter). This series was followed by another 40 trials in the same counterbalanced sequence as the first series. The interval between all stimulus presentations, S or PS, was 30 sec. Results The results of Experiment 1 are summarized in Fig. 1. The median amplitude is plotted for five trial blocks for Group 10 (Fig. 1A), Group 100 (Fig. 1B), Group 500 (Fig. 1C), and Group 2500 (Fig. 1D). Amplitude for S trials is indicated by filled circles, for PS trials by open circles, and for the first trials by filled squares. The first feature of Fig. 1 is the presence or absence of prepulse inhibition. It is clear that, during Blocks 1-4, prepulse inhibition did not occur in either Group 10 or Group 2500, whereas there was a reliable attenuation of amplitude on PS relative to S trials in both Groups 100 and 500. These group differences are compared directly in the two parts of Fig. 2, in which the median amplitude of all 20 PS trials (open circles) and all 20 S trials (filled circles) are shown for each of the four groups. Prehabituation trials are shown on the left side and posthabituation trials on the right. The arrows indicate those groups in which all six subjects had lower median amplitudes on PS relative to S trials ( P < 0.05 by Wilcoxin's T test). This occurred in Groups 100 and 500, both before and after the interposed habituation trials. However, Fig. 2 also shows a third and new aspect of the effect of interpulse interval: The response amplitudes of both Groups 500 and 2500 appear to be elevated on both S and PS trials. Statistical comparison of reflex magnitude supports this observation. Before habituation (Fig. 2A), the groups differed significantly on both S trials (P < 0.04 by KruskalWallis two-tailed test) and PS trials (P < 0.02). After the interposed startle trials (Fig. 2B), the g_roups did not differ in magnitude on S trials (P > 0.05) but did differ on PS trials (P < 0.04). This result reflects the maintenance of In this context, "habituation" refers only to the response decrement engendered by repeated stimulus presentation rather than to any hypothetical process that may be presumed to underlie that decrement.
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inhibition in Groups 100 and 500; it also provides additional confirmation of the persistence of prepulse inhibition following habituation. The effect of the 500-msec interpulse interval on S trials is especially interesting. Prior to interposed habituation the startle amplitude (filled circle in Fig. 2A) was greater than those of Group 100 (P < 0.05 by two-tailed Mann-Whitney U test) and Group 10 (P < 0.02); after this treatment, there were no differences among these groups on startle-only trials. This result indicates that the attenuation produced by a 500-msec POST-HABITUATION
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FIG. 2. Median startle amplitudes on startle trials (filled circles) and prepulse trials (open circles) as a function of interpulse interval (log scale). Amplitudes on prehabituation trials are shown in A and amplitudes on posthabituation trials are shown in B. Arrows indicate those intervals at which all subjects had lower startle amplitudes on prepulse trials than on startle trials. The stimulus configuration on prepulse trials is shown in the inset.
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interpulse interval prior to repetitive stimulation is not simply a result of elevated startle amplitudes during these trials. That is, prepulse inhibition was obtained both before (when startle-only amplitudes were high) and after (when startle-only amplitudes were low) interposed habituation. Finally, the effect of interpulse interval on startle-only trials was not limited to the prehabituation test trials; differences were maintained during the initial trials of habituation, especially in Group 2500 (see Fig. 1). To determine the extent to which these differences were maintained, the groups were compared on successive thirds of the habituation trials (the center fraction of the points in each part in Fig. 1). For each animal, startle scores were summed over each one-third of these trials; i.e., Trial Blocks 5-8, 9-12, and 13-16. Statistical comparisons indicated that on Trial Blocks 5-8 Group 2500 was different from Group 10 (P < 0.05 by two-tailed Mann-Whitney U test) and from Group 100 (P < 0.03) but not from Group 500; this latter group differed from none of the others. The differences between Group 2500 and Groups 10 and 100 were not maintained during the latter two-thirds of the trials.
Discussion Experiment 1 demonstrated that the phenomenon of prepulse inhibition can be obtained within a single group of subjects at interpulse intervals that are comparable to those used in studies in which all interpulse intervals are presented to every subject (Hoffman and Searle, 1965). Furthermore, Experiment 1 also indicated that prepulse inhibition is not eliminated by extended exposure to startle stimulation: Those intervals that were effective before this manipulation were also effective after it. In this respect, prepulse inhibition is similar to the "nonhabituating refractory relationship" obtained for pairs of startle pulses (Wilson and Groves, 1973). The results of Experiment 1 also indicate an additional effect of interpulse interval; an increase in baselevel amplitude at long interpulse intervals. This amplitude difference was obtained when prepulse inhibition was either present (Group 500) or absent (Group 2500). Furthermore, it should be noted that these amplitude differences are conservative estimates. This is the case because reflex magnitude was defined by the limits of the voltmeter that registered the startle reflex. The limit of this voltmeter was such that a "ceiling" score of 120 was imposed on all responses. Because Group 2500 contained a large number of these maximum scores, it can be assumed that the actual startle amplitude was often greater and that the group differences obtained in this and subsequent studies are conservative ones. Furthermore, this limitation precluded comparison of S vs PS trials for Group 2500. Thus, it was not possible to determine whether prepulse inhibition might not have been obtained at this interpulse interval.
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Be that as it may, Experiment 1 did demonstrate that the effect of prepulses on baselevel amplitude is not confined to the immediate time period during which such prepulses are presented. Rather, responsivity may be significantly altered even after such extra stimulation has ceased. In Experiment 1, group differences were maintained for at least the first 20 (of a total of 60) trials in the absence of any preliminary stimulation. This fact attests to the profound influence of minor fluctuations of the physical environment on reflex activity. However, after extended exposure to startle stimulation, i.e., after habituation has taken place, there were no differences in baselevel startle amplitude. It is not clear, however, whether a 10- or a 100-msec interpulse interval attenuates all responses or whether a 2500-msec interpulse interval augments all responses relative to those amplitudes that would have occurred in the absence of prepulses. Experiment 2 examined this question by comparing the extremes of interpulse intervals (10 and 2500 msec) with a third condition in which startle stimuli were presented in the total absence of prepulses. EXPERIMENT 2
Subjects were randomly assigned to one of three groups (N = 6/group). After an initial 20-min foreperiod in the startle chamber, all subjects received 40 trials at a fixed 30-sec interval. For one group (Group 0) only startle trials were presented; no prepulses were given. For the other two groups the trials consisted of a sequence of startle and prepulse trials in the same counterbalanced order used in Experiment 1. The interpulse interval for one of these two groups (Group 10) was 10 msec; for the second group (Group 2500) it was 2500 msec. Results and Discussion
The results of Experiment 2 are summarized in Fig. 3. Median amplitudes on S trials (filled circles) and PS trials (open circles) are shown for Groups 10 and 2500. The horizontal dashed line indicates the average amplitude for all trials of Group 0. This average was derived in the following manner: The 40 trials for subjects in Group 0 were separated into two categories, those that (1) occurred at the same sequential point in time as the S trials of the other two groups ( " S " trials) and those that (2) occurred at the same sequential point in time as the PS trials of the other two groups ( " P S " trials). The median amplitude of the 20 " S " and the 20 " P S " trials was obtained for each subject in Group 0; the mean of these two was then calculated for each animal. The horizontal line in Fig. 3 is the median of these individual mean scores. However, for statistical comparison, the S and PS trials of Groups 10 and 2500 were compared with the " S " and " P S " trials, respectively, of Group 0.
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INTERPULSE INTERVAL(MSEC) FIG. 3. Median startle amplitudes of Groups 0, 10, and 2500. The dashed horizontal line indicates the average startle amplitude of Group 0. Amplitudes on startle trials of Groups 10 and 2500 are indicated by filled circles and amplitudes on prepulse trials are indicated by open circles. The arrow indicates that all subjects in Group 10 had lower amplitudes on prepulse trials than on startle trials.
On the first trial, none of the groups differed; furthermore, the initial trials of Group 0 and Group 10 in this study were comparable to those of Group 10 in Experiment 1; the median amplitude of Groups 0 and 10 on the first trial was 120, and the median amplitude on the first trial for Group l0 was 118 in Experiment I. The amplitudes of Group 10 for all S trials, although somewhat lower, did not reliably differ from those of Group 0 (P > 0.05 by Mann-Whitney U test) nor did the amplitudes on PS trials (P > 0.05). However, within this group, all subjects had lower startle amplitudes on PS trials than they did on S trials (P < 0.05 by Wilcoxin's T test). This fact is noted by the arrow in Fig. 3. This latter effect is apparently not a robust one in that such inhibition was not observed under comparable conditions in Experiment 1. In contrast, the median amplitude of Group 2500 was significantly different from both that of Group 0 on " S " trials (P < 0.026) and o n " P S ' , trials (P < 0.042); the S and PS values for Group 2500 did not, however, differ from each other. It is not clear, however, whether this reflects a true lack of attenuation or the imposition of the "ceiling" effect previously noted. It appears, therefore, that the difference in Experiment 1 was due to an elevation in Group 2500 rather than to a reduction in Group 10 relative to amplitudes that would have been obtained with a startle-only condition. This result means that the obtained elevation of amplitudes is a nonspecific phenomenon in that it is not confined to PS trials as in prepulse inhibition. Furthermore, this result implies that a prepulse during Trial n (a PS trial) can elevate amplitude on Trial n + 1 (an S trial) when these trials are separated by 30 sec or more. This latter implication leads to
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the expectation that the elevation in amplitude might be obtained even when the relationship of the supplementary pulse to the startle pulse was reversed.
EXPERIMENT 3 Subjects were randomly divided into two groups (N = 6/group). After an initial 20-min foreperiod in the startle chamber, all subjects received 40 trials at a fixed 30-sec intertrial interval. On half of these trials (SP) the startle pulse was followed by a supplementary pulse in the same counterbalanced order used in previous experiments. The interval between the onset of the startle and the onset of this postpulse was 10 msec for one group (Group 10) and 2500 msec for the second (Group 2500).
Results The results of Experiment 3 are summarized in Fig. 4. The median amplitudes on S trials (filled circles) and SP trials (open circles) are shown for Groups 10 and 2500. Although the two groups did not differ on the first trial (P > 0.05 by two-tailed Mann-Whitney U test), they were significantly different on S trials (P < 0.002) and SP trials (P < 0.002). The results of Experiment 3 thus demonstrate that the elevation in amplitude due to a 2500-msec interpulse interval is independent of the sequence of the two pulses.
GENERAL DISCUSSION These experiments demonstrate two effects of supplementary stimulation on reflex magnitude in the rat. The first of these is prepulse inhibition, defined as the attenuation of reflex amplitude produced by supplementary
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stimulation presented prior to the reflex eliciting stimulus. Heretofore, as noted in the introductory section, the temporal parameters of such attenuation had been examined only on the basis of comparisons within the same subject; the present studies indicate that prepulse inhibition is not dependent upon such a procedure and that reflex attenuation may be obtained at comparable intervals in separate groups of subjects. Furthermore, such attenuation is not eliminated after a series of reflexeliciting stimuli; attenuation is obtained after amplitude has decremented as a consequence of such habituation. This result suggests that the sensitivity of the system mediating prepulse inhibition is maintained after prolonged startle stimulation in that response decrements due to startle stimulation did not impair the capacity of supplementary stimuli to attenuate the reflex. This conclusion complements earlier reports that prepulse inhibition is also maintained after extended exposure to the supplementary stimulus itself (Ison et al., 1973; Russo et al., 1975) or after prolonged intervals without stimulation (Ison et al., 1973). The second effect of supplementary stimulation on the reflex amplitude is an elevation produced by the interpolation of supplementary stimuli during the presentation of reflex-eliciting stimuli. Such an effect of discrete auditory pulses at long interpulse intervals has not previously been reported. This has probably been the case because of methodological limitations in the earlier studies. In particular, in these studies each subject received supplementary stimuli at every interpulse interval. Under such conditions reflex elevation resulting from long intervals could have been obscured by the fact that all prepulse effects were evaluated against a single baseline. This baseline may have been elevated by the inclusion of both short and long interstimulus intervals. Thus, reflex attenuation could easily be detected, whereas reflex augmentation could not. It should be borne in mind, however, that the augmented amplitudes seen at 2500 msec may have masked an attenuation that was superimposed upon that augmentation. That is, the limits of the recording device were such that, as previously noted, values greater than 120 could not be recorded; such a "ceiling effect" may have thereby obscured a truly present attenuation. The present studies also demonstrated that these two effects of supplementary stimulation differ in at least two ways. First, unlike prepulse inhibition, reflex augmentation is not retained after habituation. That is, if supplementary stimuli are reinstated after a response decrement due to habituation, reflex elevation is not obtained. Second, unlike prepulse inhibition, reflex augmentation is independent of pulse sequence; reflex magnitudes remain elevated regardless of whether the supplementary pulse is presented before or after a particular reflex-eliciting pulse. Furthermore, this influence extends to all trials,
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both those that are, as well as those that are not, preceded by prepulses. This lack of dependence on stimulus sequence bears on several interpretations of the bases of augmentation. For example, it might be postulated that supplementary stimuli have a "signaling" function that increases the "expectation" of the subsequent reflex-eliciting stimuli (Davis, 1970). As a result, the response to an "anticipated" stimulus might be different from that to an "unexpected" stimulus. Moreover, the responses induced by such signals may bear a resemblance to that class of autonomic reflexes traditionally cited as indexes of the orienting response (Sokolov, 1963). Orienting responses are elicited by novel stimuli; they presumably provide an index of the animal' s capacity to detect alterations in the surrounding physical environment. The latencies of such responses fall within the 2500-msec interval found to be effective in these studies. Finally, orienting responses decrement as a consequence of repetitive stimulation. Thus, the orienting response attendant upon supplementary stimulation might enhance the subsequent reflex. However, this interpretation would also imply that enhancement would be obtained only when the signal preceded the eliciting stimulus so that the latter stimulus could be anticipated. Experiment 3 clearly indicated that such an ordering of stimuli was not a necessary condition for enhancement. Furthermore, such interpretations cannot easily account for the maintenance of reflex amplitudes after supplementary stimuli were withdrawn. Rather, these characteristics imply an alteration in the " s t a t e " of the animal. State influences on reflex activity have long been recognized (Pavlov, 1927; Sherrington, 1906). Recently, such influences have been incorporated into a dual-process model in which response decrement is taken to be due to an inferred process of habituation (Groves and Thompson, 1970). This theoretical position maintains that every stimulus that evokes a response has two properties: It controls the response and influences the "state" of the organism. These two effects are mediated by separate systems: An " S - R pathway" is taken as the most direct route through the central nervous system from stimulus to discrete motor response and mediates habituation of the reflex itself; " s t a t e " o r " sensitization" is the general level of excitation, arousal, activation, tendency to respond, etc. of the organism. During the course of repetitive stimulation, activity in both of these systems is presumed to change so that they jointly determine the course and ultimate decrement in reflex amplitude. These considerations suggest that any account of augmentation (e.g., "signaling," orienting response) that relies on a specificity of the relation of the eliciting stimulus and the supplementary stimulus will necessarily be incomplete, whereas an account like that involving "sensitization" can better encompass the data reported here.
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I f so, the t w o p h e n o m e n a d e s c r i b e d h e r e m a y be o f v a l u e in e v a l u a t i n g dual-process theory, especially because they can be independently man i p u l a t e d . I n p a r t i c u l a r , it m a y b e that p r e p u l s e i n h i b i t i o n , b e c a u s e o f its sensitivity and dependence on very brief interpulse intervals, can provide a n i n d e p e n d e n t i n d e x of the i n t e g r i t y of the S - R s y s t e m . A l t e r n a t i v e l y , the effects o f s u p p l e m e n t a r y s t i m u l a t i o n at long i n t e r v a l s that are i n d e p e n d e n t o f p u l s e s e q u e n c e m a y p r o v i d e a n i n d e p e n d e n t i n d e x of the p r o c e s s called s e n s i t i z a t i o n .
REFERENCES Buckland, G., Buckland, J., Jamieson, C., and Ison, J. R. (1969). Inhibition of startle response to acoustic stimulation produced by visual prestimulation. J. Comp. Physiol. Psychol. 67, 493-496. Davis, M. (1970). Effects of interstimulus interval length and variability on startle-response habituation in the rat. J. Comp. Physiol. Psychol. 72, 177-192. Davis, M., and Wagner, A. R. (1968). Startle responsiveness after habituation to different intensities of tone, Psychon. Sci. 12, 337-338. Davis, M., and Wagner, A. R. (1969). Habituation of startle response under incremental sequence of stimulus intensities. J. Comp. Physiol. Psychol. 67, 486-492. Graham, F. (1975). The more or less startling effects of weak prestimulation. Psychophysiology 12, 238-248. Graham, F. K., Putnam, L. E., and Leavitt, L. A. (1975). Lead-stimulation effects on human cardiac orienting and blink reflexes. J. Exp. Psychol. Human Percep. Perf. 104, 161-169. Groves, P. M., and Thompson, R. F. (1970). Habituation: A dual-process theory. Psychol. Rev. 77, 419-450. Hilgard, E. R. (1933). Reinforcement and inhibition of eyelid reflexes. J. Gen. Psychol. 8, 85-113. Hoffman, H. S., and Fleshier, M. (1963). Startle reaction: Modification by background acoustic stimulation. Science 141, 928-930. Hoffman, H. S., and Searle, J. L. (1965). Acoustic variables in the modification of startle reaction in the rat. J. Comp. Physiol. Psychol. 60, 53-58. Hoffman, H. S., and Searle, J. L. (1968). Acoustic and temporal factors in the evocation of startle. J. Acous. Soc. Amer. 45, 269-283. Hoffman, H. S., and Wible, B. L. (1969). Temporal parameters in startle facilitation by steady background signals. J. Acous. Soc. Amer. 45, 7-12. Ison, J. R., and Hammond, G. R. (1971). Modification of the startle reflex in the rat by changes in the auditory and visual environments. J. Comp. Physiol. Psychol. 75, 435-452. Ison, J. R., Hammond, G. R., and Krauter, E. E. (1973). Effects of experience on stimulusproduced reflex inhibition in the rat. J. Comp. Physiol. Psychol. 83, 324-336. Ison, J. R., and Leonard, D. W. (1971). Effects of auditory stimulation on the amplitude of the nictitating membrane reflex of the rabbit (Oryctolagus cumiculus). J. Comp. Physiol. Psychol. 75, 157-164. Krauter, E. E., Leonard, D. W., and Ison, J. R. (1973). Inhibition of human eyeblink by brief acoustic stimulus. J. Comp. Physiol. Psychol. 84, 246-251. Pavlov, I. V. (1927). "Conditioned Reflexes." New York: Dover. Russo, J. M., Reiter, L. A., and Ison, J. R. (1975). Repetitive exposure does not attenuate the sensory impact of the habituated stimulus. J. Comp. Physiol. PsychoL 88,665-669. Sherrington, C. S. (1906). "Integrative Action of the Nervous Sysfem." New Haven: Yale University Press.
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Sokolov, Y. N. (1963). Perception and the Conditioned Reflex. London: Pergamon Press. Stitt, C. L. (1975). "Modification of the Pigeon's Startle Reaction by the Sensory Environment." Unpublished doctoral dissertation, Bryn Mawr College. Stitt, C. L., Hoffman, H. S., and Marsh, R. (1973). Modification of the rat's startle reaction by the termination of antecedent acoustic signals. J. Comp. Physiol. Psychol. 84, 207-215. Stitt, C. L., Hoffman, H. S., Marsh, R., and Boskoff, K. J. (1974). Modification of the rat's startle reaction by an antecedent change in the acoustic environment. J. Comp. Physiol. Psychol. 86, 826-836. Wilson, C. J., and Groves, P. M. (1973). Refractory period and habituation of acoustic startle response in rats. J. Comp. Physiol. Psychol. 83, 492-498.
Reflex Modulation due to Supplementary Stimulation ~ CLAIRE ADVOKAT *'2 AND PETER L . CARLTON~"
*Rutgers University and tOepartment of Psychiatry, College of Medicine and Dentistry of New Jersey-Rutgers Medical School, New Brunswick, New Jersey 08903 A series of experiments examined the effect of supplementary acoustic stimulation on the acoustic startle reflex of the rat. Separate groups of animals received low intensity pulses of white noise (prepulses) at one of several intervals (10, 100, 500, or 2500 msec) prior to startle-eliciting pulses of white noise. At interpulse intervals of 100 and 500 msec the startle reflex was attenuated on trials preceded by prepulses; at the 10- and 2500-msec intervals, on the other hand, the reflex was not attenuated, although the latter conclusion must be tentative because of apparatus limitations. Furthermore, at 2500 msec, amplitudes on all trials were greater than those obtained in controls that did not receive prepulses. This was the case regardless of whether supplementary stimuli preceded or followed the startle stimuli. This elevation of amplitude, unlike the attenuation, was eliminated after repetitive startle stimulation. These differential effects of supplementary stimulation suggest that two systems may mediate reflex elicitation.
It has been demonstrated that both the magnitude and latency of the startle reflex may be significantly altered as a consequence of preliminary stimulation. The direction and extent of such modification depend upon both the intensive and temporal characteristics of such antecedent stimulation. Hoffman and his colleagues (Hoffman and Fleshier, 1963; Hoffman and Searle, 1965, 1968; Hoffman and Wible, 1969) have shown that the reflex may be either attenuated or facilitated depending upon the relationship between startle stimuli and the prevailing physical environment. In particular, reflex amplitude may be attenuated if a supplementary acoustic stimulus precedes a startle stimulus by a lead time of approximately 20 to 2000 msec. This attenuation has been termed prepulse inhibition. Furthermore, such attenuation is not restricted to stimulus onset but may be 1 This report is based on portions of a dissertation submitted by the first author to Rutgers University in partial fulfillment of the requirements for the Ph.D. The research was partially supported by General Research Support funds granted by College of Medicine and Dentistry of New Jersey-Rutgers Medical School to Dr. Peter L. Carlton. 2 Requests for reprints should be sent to Dr. Claire Advokat, Division of Neurobiology and Behavior, Department of Physiology, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York City, New York, 10032. 375 0091-6773/78/0223-0375502.00/0 Copyright@ 1978by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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produced by both stimulus offset or changes in the frequency of an otherwise continuous tone (Stitt et al., 1973; Stitt et al., 1974). Prepulse inhibition is not limited either to the acoustic modality or to the startle reflex of the rat: Comparable results have been obtained with visual stimuli (Buckland et al., 1969; Ison and Hammond, 1971; Stitt, 1975; see also Hilgard, 1933); the effect has been demonstrated in several species, including rats (Hoffman and Fleshier, 1963), rabbits (Ison and Leonard, 1971), pigeons (Stitt, 1975), and humans (Graham, 1975; Graham et al., 1975; Hilgard, 1933; Krauter et al., 1973). Prepulse inhibition represents one means by which the startle reflex may be profoundly modified. It is also the case, however, that the reflex is attenuated as a consequence of repetitive startle stimulation per se, i. e., in the absence of extra stimulation. This form of behavioral decrement has been termed habituation (Davis, 1970; Davis and Wagner, 1968, 1969). Both forms of response decrement, prepulse inhibition and habituation, represent instances of reflex modulation produced by antecedent stimulation. Yet, the extent to which these phenomena interact is unknown. This fact warrants consideration, since analyses of prepulse inhibition require repetitive presentation of startle stimuli. The recent work of Graham and her colleagues (Graham, 1975; Graham et al., 1975) provides an example of such methodological influences in analyses of reflex responsiveness. These investigators reported both attenuation and enhancement of the human eyeblink response to startle stimuli as a consequence of preliminary acoustic stimulation. However, such enhancement was dependent upon the experimental procedure; enhancement was obtained only when each subject was tested with all interpulse intervals ("within" subjects design). When the interpulse interval remained constant ("between" subjects design) amplitudes were not elevated. In view of these considerations, the present studies were designed to assess the interaction, if any, of repeated startle stimuli (producing habituation) and supplementary stimuli (producing prepulse inhibition).
GENERAL METHODS Subjects
The subjects were naive, male albino Charles River rats obtained from Charles River Distributors, Wilmington, Massachusetts. Upon arrival at the laboratory the subjects were housed individually, provided with constant illumination and permitted ad lib. access to food and water. At the time of testing the subjects weighted at least 215 and not more than 500 g; the majority were approximately 250 to 350 g.
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Apparatus The experimental chamber was housed in an Industrial Acoustic Chamber (IAC 10771), the inside dimensions of which were 79.5 x 60.0 x 59.5 cm. The experimental "startle chamber" consisted of a 21.0 x 8.5 x 8.5-cm perforated Plexiglas cage with a grid floor that was mounted rigidly to a 32.5 x 26.0-cm Plexiglas platform. The platform was, in turn, fixed by four heavy-duty springs to a 48.0 x 26.0-cm wooden board. This assembly was secured to the floor of the IAC. T h e startle response was monitored as the voltage output from a phonograph cartridge (Astatic N4-2, Model 16) mounted on the side of the cage; this voltage was recorded by a printing voltmeter. The cartidge had a 4.5-cm rod weighted at the end inserted where the needle would normally be. This arrangement, although insensitive to gross bodily movements made by the rat, was sensitive to the abrupt, ballistic movements produced by the startle stimulus. Movement of this rigidly mounted system could not be detected by gross observation. A 60.0-cm Altec midrange driver and horn (Model 27A) was used to deliver the startle stimuli and prepulses. A constant background noise was provided by a BRS-Foringer audio generator (Model AU-902) coupled to an 8.0-ohm speaker mounted above the experimental chamber. The delivery and timing of stimuli were controlled by standard Tektronix programmming equipment housed in a separate room. Startle stimuli were provided by a Grason-Stadler noise generator (Model 901B) coupled to a Grason-Stadler electronic switch (Model 829). Prepulses were generated by a second Grason-Stadler noise generator. Stimulus intensity (in decibels with respect to 0.0002 dyn/cm 2) was calibrated before each daily use. For this purpose the stimulus was turned on continuously and its intensity calibrated with a General Radio octave band noise analyzer (Model 1558-BP). The microphone for the analyzer was placed at the geometric center of the animal chamber. The startle stimulus consisted of a 120 + 1 db, 10-msec burst of white noise (rise time = 10/zsec). The prepulse was a 90 + 1 db, 10-msec burst of white noise. The duration of these stimuli was calibrated on an oscilloscope. The constant background noise was 72 _+ 2 db.
EXPERIMENT
1
All subjects were randomly assigned to one of four groups (N = 6/group). Each rat was placed in the startle chamber for an initial 20-min foreperiod. At the end of this period each subject received 40 startle stimuli, 20 of which were preceded by prepulses. The startle and prepulse trials were presented in a counterbalanced order as follows: S - P S - P S -
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S - P S - S - S - P S . The first trial, therefore, was never preceded by a prepulse. This sequence was presented five times for a total of 40 trials. Each of the four groups received a different interpulse interval on PS trials. For one group (Group 10) the interpulse interval (IPI) was 10 msec; for the second group (Group 100) it was 100 msec; for the third group (Group 500) it was 500 msec; and for the fourth group (Group 2500) it was 2500 msec. At the end of the initial 40 trials, all subjects received an additional 60 trials consisting only of startle pulses (called "habituation trials' ,3 hereafter). This series was followed by another 40 trials in the same counterbalanced sequence as the first series. The interval between all stimulus presentations, S or PS, was 30 sec. Results The results of Experiment 1 are summarized in Fig. 1. The median amplitude is plotted for five trial blocks for Group 10 (Fig. 1A), Group 100 (Fig. 1B), Group 500 (Fig. 1C), and Group 2500 (Fig. 1D). Amplitude for S trials is indicated by filled circles, for PS trials by open circles, and for the first trials by filled squares. The first feature of Fig. 1 is the presence or absence of prepulse inhibition. It is clear that, during Blocks 1-4, prepulse inhibition did not occur in either Group 10 or Group 2500, whereas there was a reliable attenuation of amplitude on PS relative to S trials in both Groups 100 and 500. These group differences are compared directly in the two parts of Fig. 2, in which the median amplitude of all 20 PS trials (open circles) and all 20 S trials (filled circles) are shown for each of the four groups. Prehabituation trials are shown on the left side and posthabituation trials on the right. The arrows indicate those groups in which all six subjects had lower median amplitudes on PS relative to S trials ( P < 0.05 by Wilcoxin's T test). This occurred in Groups 100 and 500, both before and after the interposed habituation trials. However, Fig. 2 also shows a third and new aspect of the effect of interpulse interval: The response amplitudes of both Groups 500 and 2500 appear to be elevated on both S and PS trials. Statistical comparison of reflex magnitude supports this observation. Before habituation (Fig. 2A), the groups differed significantly on both S trials (P < 0.04 by KruskalWallis two-tailed test) and PS trials (P < 0.02). After the interposed startle trials (Fig. 2B), the g_roups did not differ in magnitude on S trials (P > 0.05) but did differ on PS trials (P < 0.04). This result reflects the maintenance of In this context, "habituation" refers only to the response decrement engendered by repeated stimulus presentation rather than to any hypothetical process that may be presumed to underlie that decrement.
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inhibition in Groups 100 and 500; it also provides additional confirmation of the persistence of prepulse inhibition following habituation. The effect of the 500-msec interpulse interval on S trials is especially interesting. Prior to interposed habituation the startle amplitude (filled circle in Fig. 2A) was greater than those of Group 100 (P < 0.05 by two-tailed Mann-Whitney U test) and Group 10 (P < 0.02); after this treatment, there were no differences among these groups on startle-only trials. This result indicates that the attenuation produced by a 500-msec POST-HABITUATION
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FIG. 2. Median startle amplitudes on startle trials (filled circles) and prepulse trials (open circles) as a function of interpulse interval (log scale). Amplitudes on prehabituation trials are shown in A and amplitudes on posthabituation trials are shown in B. Arrows indicate those intervals at which all subjects had lower startle amplitudes on prepulse trials than on startle trials. The stimulus configuration on prepulse trials is shown in the inset.
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interpulse interval prior to repetitive stimulation is not simply a result of elevated startle amplitudes during these trials. That is, prepulse inhibition was obtained both before (when startle-only amplitudes were high) and after (when startle-only amplitudes were low) interposed habituation. Finally, the effect of interpulse interval on startle-only trials was not limited to the prehabituation test trials; differences were maintained during the initial trials of habituation, especially in Group 2500 (see Fig. 1). To determine the extent to which these differences were maintained, the groups were compared on successive thirds of the habituation trials (the center fraction of the points in each part in Fig. 1). For each animal, startle scores were summed over each one-third of these trials; i.e., Trial Blocks 5-8, 9-12, and 13-16. Statistical comparisons indicated that on Trial Blocks 5-8 Group 2500 was different from Group 10 (P < 0.05 by two-tailed Mann-Whitney U test) and from Group 100 (P < 0.03) but not from Group 500; this latter group differed from none of the others. The differences between Group 2500 and Groups 10 and 100 were not maintained during the latter two-thirds of the trials.
Discussion Experiment 1 demonstrated that the phenomenon of prepulse inhibition can be obtained within a single group of subjects at interpulse intervals that are comparable to those used in studies in which all interpulse intervals are presented to every subject (Hoffman and Searle, 1965). Furthermore, Experiment 1 also indicated that prepulse inhibition is not eliminated by extended exposure to startle stimulation: Those intervals that were effective before this manipulation were also effective after it. In this respect, prepulse inhibition is similar to the "nonhabituating refractory relationship" obtained for pairs of startle pulses (Wilson and Groves, 1973). The results of Experiment 1 also indicate an additional effect of interpulse interval; an increase in baselevel amplitude at long interpulse intervals. This amplitude difference was obtained when prepulse inhibition was either present (Group 500) or absent (Group 2500). Furthermore, it should be noted that these amplitude differences are conservative estimates. This is the case because reflex magnitude was defined by the limits of the voltmeter that registered the startle reflex. The limit of this voltmeter was such that a "ceiling" score of 120 was imposed on all responses. Because Group 2500 contained a large number of these maximum scores, it can be assumed that the actual startle amplitude was often greater and that the group differences obtained in this and subsequent studies are conservative ones. Furthermore, this limitation precluded comparison of S vs PS trials for Group 2500. Thus, it was not possible to determine whether prepulse inhibition might not have been obtained at this interpulse interval.
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Be that as it may, Experiment 1 did demonstrate that the effect of prepulses on baselevel amplitude is not confined to the immediate time period during which such prepulses are presented. Rather, responsivity may be significantly altered even after such extra stimulation has ceased. In Experiment 1, group differences were maintained for at least the first 20 (of a total of 60) trials in the absence of any preliminary stimulation. This fact attests to the profound influence of minor fluctuations of the physical environment on reflex activity. However, after extended exposure to startle stimulation, i.e., after habituation has taken place, there were no differences in baselevel startle amplitude. It is not clear, however, whether a 10- or a 100-msec interpulse interval attenuates all responses or whether a 2500-msec interpulse interval augments all responses relative to those amplitudes that would have occurred in the absence of prepulses. Experiment 2 examined this question by comparing the extremes of interpulse intervals (10 and 2500 msec) with a third condition in which startle stimuli were presented in the total absence of prepulses. EXPERIMENT 2
Subjects were randomly assigned to one of three groups (N = 6/group). After an initial 20-min foreperiod in the startle chamber, all subjects received 40 trials at a fixed 30-sec interval. For one group (Group 0) only startle trials were presented; no prepulses were given. For the other two groups the trials consisted of a sequence of startle and prepulse trials in the same counterbalanced order used in Experiment 1. The interpulse interval for one of these two groups (Group 10) was 10 msec; for the second group (Group 2500) it was 2500 msec. Results and Discussion
The results of Experiment 2 are summarized in Fig. 3. Median amplitudes on S trials (filled circles) and PS trials (open circles) are shown for Groups 10 and 2500. The horizontal dashed line indicates the average amplitude for all trials of Group 0. This average was derived in the following manner: The 40 trials for subjects in Group 0 were separated into two categories, those that (1) occurred at the same sequential point in time as the S trials of the other two groups ( " S " trials) and those that (2) occurred at the same sequential point in time as the PS trials of the other two groups ( " P S " trials). The median amplitude of the 20 " S " and the 20 " P S " trials was obtained for each subject in Group 0; the mean of these two was then calculated for each animal. The horizontal line in Fig. 3 is the median of these individual mean scores. However, for statistical comparison, the S and PS trials of Groups 10 and 2500 were compared with the " S " and " P S " trials, respectively, of Group 0.
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On the first trial, none of the groups differed; furthermore, the initial trials of Group 0 and Group 10 in this study were comparable to those of Group 10 in Experiment 1; the median amplitude of Groups 0 and 10 on the first trial was 120, and the median amplitude on the first trial for Group l0 was 118 in Experiment I. The amplitudes of Group 10 for all S trials, although somewhat lower, did not reliably differ from those of Group 0 (P > 0.05 by Mann-Whitney U test) nor did the amplitudes on PS trials (P > 0.05). However, within this group, all subjects had lower startle amplitudes on PS trials than they did on S trials (P < 0.05 by Wilcoxin's T test). This fact is noted by the arrow in Fig. 3. This latter effect is apparently not a robust one in that such inhibition was not observed under comparable conditions in Experiment 1. In contrast, the median amplitude of Group 2500 was significantly different from both that of Group 0 on " S " trials (P < 0.026) and o n " P S ' , trials (P < 0.042); the S and PS values for Group 2500 did not, however, differ from each other. It is not clear, however, whether this reflects a true lack of attenuation or the imposition of the "ceiling" effect previously noted. It appears, therefore, that the difference in Experiment 1 was due to an elevation in Group 2500 rather than to a reduction in Group 10 relative to amplitudes that would have been obtained with a startle-only condition. This result means that the obtained elevation of amplitudes is a nonspecific phenomenon in that it is not confined to PS trials as in prepulse inhibition. Furthermore, this result implies that a prepulse during Trial n (a PS trial) can elevate amplitude on Trial n + 1 (an S trial) when these trials are separated by 30 sec or more. This latter implication leads to
REFLEX MODULATION
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the expectation that the elevation in amplitude might be obtained even when the relationship of the supplementary pulse to the startle pulse was reversed.
EXPERIMENT 3 Subjects were randomly divided into two groups (N = 6/group). After an initial 20-min foreperiod in the startle chamber, all subjects received 40 trials at a fixed 30-sec intertrial interval. On half of these trials (SP) the startle pulse was followed by a supplementary pulse in the same counterbalanced order used in previous experiments. The interval between the onset of the startle and the onset of this postpulse was 10 msec for one group (Group 10) and 2500 msec for the second (Group 2500).
Results The results of Experiment 3 are summarized in Fig. 4. The median amplitudes on S trials (filled circles) and SP trials (open circles) are shown for Groups 10 and 2500. Although the two groups did not differ on the first trial (P > 0.05 by two-tailed Mann-Whitney U test), they were significantly different on S trials (P < 0.002) and SP trials (P < 0.002). The results of Experiment 3 thus demonstrate that the elevation in amplitude due to a 2500-msec interpulse interval is independent of the sequence of the two pulses.
GENERAL DISCUSSION These experiments demonstrate two effects of supplementary stimulation on reflex magnitude in the rat. The first of these is prepulse inhibition, defined as the attenuation of reflex amplitude produced by supplementary
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stimulation presented prior to the reflex eliciting stimulus. Heretofore, as noted in the introductory section, the temporal parameters of such attenuation had been examined only on the basis of comparisons within the same subject; the present studies indicate that prepulse inhibition is not dependent upon such a procedure and that reflex attenuation may be obtained at comparable intervals in separate groups of subjects. Furthermore, such attenuation is not eliminated after a series of reflexeliciting stimuli; attenuation is obtained after amplitude has decremented as a consequence of such habituation. This result suggests that the sensitivity of the system mediating prepulse inhibition is maintained after prolonged startle stimulation in that response decrements due to startle stimulation did not impair the capacity of supplementary stimuli to attenuate the reflex. This conclusion complements earlier reports that prepulse inhibition is also maintained after extended exposure to the supplementary stimulus itself (Ison et al., 1973; Russo et al., 1975) or after prolonged intervals without stimulation (Ison et al., 1973). The second effect of supplementary stimulation on the reflex amplitude is an elevation produced by the interpolation of supplementary stimuli during the presentation of reflex-eliciting stimuli. Such an effect of discrete auditory pulses at long interpulse intervals has not previously been reported. This has probably been the case because of methodological limitations in the earlier studies. In particular, in these studies each subject received supplementary stimuli at every interpulse interval. Under such conditions reflex elevation resulting from long intervals could have been obscured by the fact that all prepulse effects were evaluated against a single baseline. This baseline may have been elevated by the inclusion of both short and long interstimulus intervals. Thus, reflex attenuation could easily be detected, whereas reflex augmentation could not. It should be borne in mind, however, that the augmented amplitudes seen at 2500 msec may have masked an attenuation that was superimposed upon that augmentation. That is, the limits of the recording device were such that, as previously noted, values greater than 120 could not be recorded; such a "ceiling effect" may have thereby obscured a truly present attenuation. The present studies also demonstrated that these two effects of supplementary stimulation differ in at least two ways. First, unlike prepulse inhibition, reflex augmentation is not retained after habituation. That is, if supplementary stimuli are reinstated after a response decrement due to habituation, reflex elevation is not obtained. Second, unlike prepulse inhibition, reflex augmentation is independent of pulse sequence; reflex magnitudes remain elevated regardless of whether the supplementary pulse is presented before or after a particular reflex-eliciting pulse. Furthermore, this influence extends to all trials,
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both those that are, as well as those that are not, preceded by prepulses. This lack of dependence on stimulus sequence bears on several interpretations of the bases of augmentation. For example, it might be postulated that supplementary stimuli have a "signaling" function that increases the "expectation" of the subsequent reflex-eliciting stimuli (Davis, 1970). As a result, the response to an "anticipated" stimulus might be different from that to an "unexpected" stimulus. Moreover, the responses induced by such signals may bear a resemblance to that class of autonomic reflexes traditionally cited as indexes of the orienting response (Sokolov, 1963). Orienting responses are elicited by novel stimuli; they presumably provide an index of the animal' s capacity to detect alterations in the surrounding physical environment. The latencies of such responses fall within the 2500-msec interval found to be effective in these studies. Finally, orienting responses decrement as a consequence of repetitive stimulation. Thus, the orienting response attendant upon supplementary stimulation might enhance the subsequent reflex. However, this interpretation would also imply that enhancement would be obtained only when the signal preceded the eliciting stimulus so that the latter stimulus could be anticipated. Experiment 3 clearly indicated that such an ordering of stimuli was not a necessary condition for enhancement. Furthermore, such interpretations cannot easily account for the maintenance of reflex amplitudes after supplementary stimuli were withdrawn. Rather, these characteristics imply an alteration in the " s t a t e " of the animal. State influences on reflex activity have long been recognized (Pavlov, 1927; Sherrington, 1906). Recently, such influences have been incorporated into a dual-process model in which response decrement is taken to be due to an inferred process of habituation (Groves and Thompson, 1970). This theoretical position maintains that every stimulus that evokes a response has two properties: It controls the response and influences the "state" of the organism. These two effects are mediated by separate systems: An " S - R pathway" is taken as the most direct route through the central nervous system from stimulus to discrete motor response and mediates habituation of the reflex itself; " s t a t e " o r " sensitization" is the general level of excitation, arousal, activation, tendency to respond, etc. of the organism. During the course of repetitive stimulation, activity in both of these systems is presumed to change so that they jointly determine the course and ultimate decrement in reflex amplitude. These considerations suggest that any account of augmentation (e.g., "signaling," orienting response) that relies on a specificity of the relation of the eliciting stimulus and the supplementary stimulus will necessarily be incomplete, whereas an account like that involving "sensitization" can better encompass the data reported here.
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I f so, the t w o p h e n o m e n a d e s c r i b e d h e r e m a y be o f v a l u e in e v a l u a t i n g dual-process theory, especially because they can be independently man i p u l a t e d . I n p a r t i c u l a r , it m a y b e that p r e p u l s e i n h i b i t i o n , b e c a u s e o f its sensitivity and dependence on very brief interpulse intervals, can provide a n i n d e p e n d e n t i n d e x of the i n t e g r i t y of the S - R s y s t e m . A l t e r n a t i v e l y , the effects o f s u p p l e m e n t a r y s t i m u l a t i o n at long i n t e r v a l s that are i n d e p e n d e n t o f p u l s e s e q u e n c e m a y p r o v i d e a n i n d e p e n d e n t i n d e x of the p r o c e s s called s e n s i t i z a t i o n .
REFERENCES Buckland, G., Buckland, J., Jamieson, C., and Ison, J. R. (1969). Inhibition of startle response to acoustic stimulation produced by visual prestimulation. J. Comp. Physiol. Psychol. 67, 493-496. Davis, M. (1970). Effects of interstimulus interval length and variability on startle-response habituation in the rat. J. Comp. Physiol. Psychol. 72, 177-192. Davis, M., and Wagner, A. R. (1968). Startle responsiveness after habituation to different intensities of tone, Psychon. Sci. 12, 337-338. Davis, M., and Wagner, A. R. (1969). Habituation of startle response under incremental sequence of stimulus intensities. J. Comp. Physiol. Psychol. 67, 486-492. Graham, F. (1975). The more or less startling effects of weak prestimulation. Psychophysiology 12, 238-248. Graham, F. K., Putnam, L. E., and Leavitt, L. A. (1975). Lead-stimulation effects on human cardiac orienting and blink reflexes. J. Exp. Psychol. Human Percep. Perf. 104, 161-169. Groves, P. M., and Thompson, R. F. (1970). Habituation: A dual-process theory. Psychol. Rev. 77, 419-450. Hilgard, E. R. (1933). Reinforcement and inhibition of eyelid reflexes. J. Gen. Psychol. 8, 85-113. Hoffman, H. S., and Fleshier, M. (1963). Startle reaction: Modification by background acoustic stimulation. Science 141, 928-930. Hoffman, H. S., and Searle, J. L. (1965). Acoustic variables in the modification of startle reaction in the rat. J. Comp. Physiol. Psychol. 60, 53-58. Hoffman, H. S., and Searle, J. L. (1968). Acoustic and temporal factors in the evocation of startle. J. Acous. Soc. Amer. 45, 269-283. Hoffman, H. S., and Wible, B. L. (1969). Temporal parameters in startle facilitation by steady background signals. J. Acous. Soc. Amer. 45, 7-12. Ison, J. R., and Hammond, G. R. (1971). Modification of the startle reflex in the rat by changes in the auditory and visual environments. J. Comp. Physiol. Psychol. 75, 435-452. Ison, J. R., Hammond, G. R., and Krauter, E. E. (1973). Effects of experience on stimulusproduced reflex inhibition in the rat. J. Comp. Physiol. Psychol. 83, 324-336. Ison, J. R., and Leonard, D. W. (1971). Effects of auditory stimulation on the amplitude of the nictitating membrane reflex of the rabbit (Oryctolagus cumiculus). J. Comp. Physiol. Psychol. 75, 157-164. Krauter, E. E., Leonard, D. W., and Ison, J. R. (1973). Inhibition of human eyeblink by brief acoustic stimulus. J. Comp. Physiol. Psychol. 84, 246-251. Pavlov, I. V. (1927). "Conditioned Reflexes." New York: Dover. Russo, J. M., Reiter, L. A., and Ison, J. R. (1975). Repetitive exposure does not attenuate the sensory impact of the habituated stimulus. J. Comp. Physiol. PsychoL 88,665-669. Sherrington, C. S. (1906). "Integrative Action of the Nervous Sysfem." New Haven: Yale University Press.
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Sokolov, Y. N. (1963). Perception and the Conditioned Reflex. London: Pergamon Press. Stitt, C. L. (1975). "Modification of the Pigeon's Startle Reaction by the Sensory Environment." Unpublished doctoral dissertation, Bryn Mawr College. Stitt, C. L., Hoffman, H. S., and Marsh, R. (1973). Modification of the rat's startle reaction by the termination of antecedent acoustic signals. J. Comp. Physiol. Psychol. 84, 207-215. Stitt, C. L., Hoffman, H. S., Marsh, R., and Boskoff, K. J. (1974). Modification of the rat's startle reaction by an antecedent change in the acoustic environment. J. Comp. Physiol. Psychol. 86, 826-836. Wilson, C. J., and Groves, P. M. (1973). Refractory period and habituation of acoustic startle response in rats. J. Comp. Physiol. Psychol. 83, 492-498.