Journal of Experimental Psychology: Animal Behavior Processes 1978, Vol. 4, No. 2, 104-119

Habituation of the Forelimb-Withdrawal Response in Neonatal Rats Donald J. Stehouwer and Byron A. Campbell Princeton University The purpose of the present research was to examine the ontogeny of habituation in the neonatal rat, using the forelimb-withdrawal response. Thresholds and latencies of the response, changing patterns of responding to shock stimuli, and habituation of the response were studied in rats 3 to IS days of age. It was found that although response thresholds do not change during this period of development, response latencies decrease and amplitudes increase. Three- and 6-day-old pups remain active much longer following shock than do 10- and IS-day-olds. When compared for habituation to different frequencies of stimulation, 3-day-old rat pups were found to be much more susceptible to habituation at low frequencies than 15-day-olds. Insertion of a simple intense shock in the habituation series produced marked dishabituation in the 3-day-olds but little or none in the 15-day-olds. This pattern of results was obtained regardless of the locus of the dishabituating shock. Analysis of response latencies showed that although 15-day-olds responded 2 to 3 times more quickly than 3-day-olds, both age groups responded more rapidly to a strong shock than to a weak one. During habituation, response amplitudes of both 3- and 15-day-old pups declined with no change in latency. Age-related differences in habituation were shown to be independent of differences in reactivity to shock and more likely due to the emergence of response sensitization.

Habituation and sensitization are considered by most psychologists and neurobiologists to be among the most primitive forms of learning as well as the substrate from which many higher learning processes evolved. Habituation has been observed in representatives of the oldest animal phyla (see Wyers, Peeke, & Herz, 1973), which show few other forms of learning or plasticity. Moreover, habituation persists in higher mammals as a neural and behavioral phenomenon apparently little changed by evolution. Habituation has been postulated to appear early in the developmental sequence because of its inherent simplicity (Thompson, 1972), but This research was supported by Grant MH01562-19 from the National Institute of Health. Requests for reprints should be sent to Byron A. Campbell, Department of Psychology, Princeton University, Princeton, New Jersey 08540.

in spite of its fundamental importance, little work has been done to examine habituation in infraprimate mammalian neonates. In the human infant, habituation has been studied quite extensively and has been widely used as a tool to study perceptual and cognitive development (e.g., Bornstein, Kessen, & Weiskopf, 1976; Cohen, 1976; Jeffrey, 1976). Study of the ontogeny of habituation in animals has been complicated by the use of diffuse behaviors, such as exploration of an open field (e.g., Bronstein, Neiman, Wolkoff, & Levine, 1974) or adaptation to a testing environment (e.g., Bronstein & Dworkin, 1974; Feigley, Parsons, Hamilton, & Spear, 1972). In these instances, not only is the response ambiguous, but so are t h e e li c jting stimuli, since they are . , and, ^ 11 j t_ ^i i • ^ m nad y

controlled by the subject

rather than the experimenter. Effective

Copyright 1978 by the American Psychological Association, Inc. All rights of reproduction in any form reserved.

104

HABITUATION OF FORELIMB WITHDRAWAL study of habituation requires a response that is reliably elicitable, well defined in terms of both the response itself and the stimuli eliciting it, and, optimally, one whose underlying neuronal circuitry is known. Few, if any, studies on the ontogeny of mammalian habituation meet these simple requirements. The studies that come closest to meeting these standards are by File and Plotkin (1974) and File and Scott (1976), who found that the number of trials required to produce habituation to an airpuff increased until Day 8 in the neonatal rat, but then showed a sudden drop of 50% on Day 9. The most difficult problem in developmental research on habituation is finding a response that can be readily elicited and recorded. The present study demonstrates that the forelimb-withdrawal response elicited by mild electric shock can serve as a useful tool for the study of habituation in the neonatal rat. The withdrawal response can be easily elicited neonatally, first appearing at about 16 days of gestation (Narayanan, Fox, & Hamburger, 1971; Vaughn & Grieshaber, 1973). In addition, this response has been widely used in neurophysiological studies of habituation in adult mammals (cf. Griffin, 1970; Groves & Thompson, 1970). The aim of the present research is to describe parametrically the ontogenetic changes that occur during the course of development. Of primary interest is the age at which habituation of the forelimbwithdrawal response first appears and how the characteristics of habituation change as central nervous system maturation proceeds. Experiment 1 The purpose of the first experiment was to measure spontaneous flexion activity and determine flexion response thresholds in the developing neonate. Although little published work is available on the responsivity of neonatal rats to electric shock, Collier (Note 1) reported that the aversion threshold for shock did not change from 5 to 20 days of age.

105

Method Subjects were 80 albino rats of Sprague-Dawley descent, born and raised in the Princeton University vivarium. Litters were culled to 8 pups at 3 days of age and left undisturbed until the time of testing. All were kept in 48 X 27 X 15-cm plastic rat cages with the dam. Purina Rat Chow and water were continuously available, and the colony room was maintained on a 16:8, day might cycle at 23-2S°C. During testing subjects were suspended in fabric harnesses, which allowed free movement of all four legs, and placed in a heated chamber. The temperature of the chamber was adjusted to thermoneutrality for the subjects as follows: 37 °C for the 3- and 6-day-old rats, 34 °C for the 10-day-olds, and 33 °C for the 15-day-olds (Conklin & Heggeness, 1971; Taylor, 1960). Temperatures were controlled to ±.S °C by a Yellow Springs thermostat and relative humidity maintained at approximately 45% by surrounding the heating element with dishes of water. Pilot studies indicated that lowering the temperature by as little as 3 °C reduced responding in the youngest subjects by more than 50%. A 3-mm diameter stainless steel tube ran the length of the subject's back and served as the indifferent electrode. Liberally applied electrode paste ensured good contact. The stimulating electrode consisted of a fine (.05 mm diameter) platinumiridium wire fastened around the carpus of the right forelimb. The wire was very soft and not aversive, allowing unrestricted movement of the limb. Schematic representations of 3- and 15-day-old subjects in the apparatus are shown in Figure 1. Forelimb-withdrawal responses were transduced by inducing a current in a coil via a magnet tied to the forepaw being stimulated. The magnet was counterbalanced to reduce the force necessary to move it to 1 g ± . 1 g. The output of this transducer was amplified by a solid-state amplifier built in the Princeton University Psychology Department electronics shop. Withdrawal responses were defined by this amplifier, which closed a relay for 31.6 ± 9.75 msec whenever a response exceeded a threshold velocity of 3.18 ± .48 cm/sec. Responses were counted on electromagnetic counters via associated programming equipment. At either 3 or 15 days, litters were evenly divided among the experimental groups, with age as a between-litters variable. Four groups of 10 animals at each age were assigned to one of the following conditions: no shock, .04 mA, .08 mA, or .16 mA shock. The 60 Hz constant-current AC shocks were 35 msec in duration and were delivered at 45 sec intervals. The dependent variable was the proportion of shocks that were effective in eliciting a withdrawal response. To minimize "spontaneous" responses due to general activity, only responses occurring within 1 sec following the stimulus were counted. Response latencies were not recorded in this experiment.

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DONALD J. STEHOUWER AND BYRON A. CAMPBELL

Figure 1. Schematic representation of 3- (right) and 15-day-old (left) rats suspended in habituation apparatus. The shock source is connected via alligator clips to an indifferent electrode on the subject's back and to a stimulating electrode around the forepaw. A fine thread connects the stimulated forepaw to the motion transducer, whose output is amplified by a solid-state amplifier (not shown).

Results and Discussion The results of this experiment, shown in Figure 2, indicate that 3-day-old pups are more active in the test situation than 15day-olds, regardless of shock intensity. The probability that a shock will elicit a response is directly related to stimulus intensity, an effect that is independent of age. An analysis of variance supports these conclusions, since age, F(l, 72) = 7.556, p < .01, and intensity, F(3, 72) = 25.601, p < .001, were significant, but their interaction, F(3, 72) = .529, p > .05, was not. These data suggest that the major developmental change is a decrease in general activity rather than a change in shock sensitivity, since the same agedependent differences are apparent in the no-shock groups. The response threshold was defined as the shock intensity that elicited a level of

responding midway between spontaneous levels and 100% responding (Campbell & Masterson, 1969). In the present study, spontaneous responses occurred at a rate of 24% in the 3-day-olds and 8% in the 15-day-olds. Thus, threshold levels of responding were calculated to be 62% in the 3-day-olds and 54% in the 15-day-olds, which, by interpolation, would be elicited by a .12 mA shock in both cases. Experiment 2 Experiment 1 established that the nociceptive response threshold does not differ in 3- and 15-day-old rats. The second experiment was conducted to determine whether the aftereffects of shock changed during ontogenesis, as well as to extend the findings of the first experiment to additional age groups and to a wider range of shock intensities.

HABITUATION OF FORELIMB WITHDRAWAL

107

Method Twenty litters of 8 pups were drawn from the same population as those in Experiment 1. Each litter was evenly divided among treatment groups at either 3, 6, 10, or 15 days of age, resulting in 10 subjects per group. The apparatus and procedure were unchanged from Experiment 1, except that the shock intensities were .04 mA, .16 mA, .64 mA, and 2.56 mA. In addition to recording the number of shocks eliciting a response, we also recorded the number of responses occurring during the first 10 consecutive 3-sec periods following each shock. Also recorded was the total number of responses that occurred during the entire 45 sec interstimulus interval (ISI) in order to detect possible habituation or sensitization effects. Ten shocks were delivered, but due to a programming error, response data for the 10th shock was lost.

Results and Discussion Presented in Figure 3 is the probability of responding at each age as a function of shock intensity. As in Experiment 1, the functions are similar for animals of different ages, with the younger pups being slightly more active than the older subjects at lower shock intensities. At the two highest intensities every shock elicited a response regardless of age. These data replicate the findings of Experiment 1 for the 3- and 100 3 DAYS 15 DAYS

O 80 x 60

20

0 -

I

1

0.00 0.04 0.08 0.16 SHOCK INTENSITY (mA)

Figure 2. Mean percentage of elicited-by-shock stimulation in 3- and 15-day-old rats as a function of shock intensity.

100

80

60,

.40,

tt

3 DAYS O—O 6 DAYS *—A 10 DAYS 15 DAYS

20

0.04

0.16

0.64

2.56

SHOCK INTENSITY (mA)

Figure 3. Mean percentage leg flexions in 3-, 6-, 10-, and 15-day-old rats as a function of shock intensity.

15-day-olds and extend the results to include 6- and 10-day-old pups. Figure 4 shows the mean total number of responses occurring to each shock over the entire 45 sec ISI for each shock intensity. Examination of the figure reveals that the 3- and 6-day-olds were more responsive than the 10- and 15-day-olds, especially at the higher intensities. An analysis of variance on these data indicated that age, F(3, 144) = 20.720, p < .001; shock intensity, F(3, 144) = 20.449, p < .001; and their interaction, F(9, 144) = 3.959, p < .001, were highly significant, but that the effect of shock repetition, F(8, 1152) = 1.061, p > .05, was not. The time course of responding during the postshock interval is also of considerable interest and is shown in Figure 5. Here it is evident that the younger rats not only respond much more vigorously to shock but continue to respond for a longer period of time following shock. A significant Age X Time interaction, F(21, 1296) = 14.935, p < .001, confirms the observation. In summary, the results of the first two experiments show that the threshold of responding to electric shock does not change between 3 and 15 days of age, but that 3and 6-day-old rats respond more vigorously to shock and for a longer period of time following shock than 10- and 15-day-olds. Observation of the subjects revealed that in the two youngest age groups, the initial

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DONALD J. STEHOUWER AND BYRON A. CAMPBELL

Experiment 3

30 .

o

8

co cc £ co

20

O X

10

I 0.04

I

I

I

O.I6 0.64 2.56 INTENSITY (mA)

Figure 4. Mean number of leg flexions occurring during the entire 45 sec interstimulus interval in 3-, 6-, 10-, and 15-day-old rats as a function of shock intensity.

withdrawal response was followed by repeated forelimb flexion and by squirming and movement of the other limbs in a pattern similar to locomotor activity characteristic of this developmental period. Similar generalized responses to localized stimuli in fetal and neonatal rats have been previously reported (Narayanan et a!., 1971; Stelzner, 1971). The 10- and especially 15-day-olds did not show this behavior, presumably because of maturation of (inhibitory) integrating mechanisms that modulate or control diffuse neuronal activity. At these ages, shocks elicited discrete withdrawal responses, which were occasionally followed by attempts to scratch the stimulated area with the ipsilateral hindlimb. These observations and interpretations concur with those of Stelzner (1971), who also reported repeated flexion and prolonged activity in neonatal rats following noxious cutaneous stimulation up to approximately 8 days of age, and File and Scott (1976), who found a sudden reduction in elicited head-turns on Day 9.

The previous experiments demonstrate the feasibility of using the forelimb withdrawal response in developmental research. As noted, the response is relatively easy to record, and the stimulus threshold for withdrawal is invariant across the ages studied. Our next step was to determine whether repeated stimulation produced habituation of the response. Three critical characteristics of habituation, as identified by Thompson and Spencer (1966), were examined in the present experiment. First, does occurrence of the withdrawal response decline as a negative exponential function of stimulus presentation? Second, is the rate and amount of response decline directly proportional to frequency of stimulation? Third, does presentation of an intense stimulus following habituation result in reappearance of the original response (dishabituation) ? A further purpose of this experiment was to study the effects of age on these characteristics of habituation. 3 DAYS

6 DAYS

• 0.04 mA "0.16 mA • 0.64 mA ° 2.56 mA

co obi 3

9

3

9

X UJ

15 21 27 10 DAYS

3

9

15 21 27 15 DAYS

a 10 LJ

-I 8

§6 LJ

15 21 27 3 9 15 21 27 TIME AFTER SHOCK IN SECONDS

Figure 5. Mean number of responses elicited in 3-, 6-, 10-, and 15-day-old rats as a function of shock intensity and time following shock.

HABITUATION OF FORELIMB WITHDRAWAL Method

3-DAY OLDS

loop Twenty-four litters of 8 pups were drawn from the same population as those in the two previous experiments. Each litter was evenly divided among the treatment groups at either 3, 6, 10, or 15 days of age, resulting in 12 subjects per group. The apparatus and procedure were identical to those described in Experiments 1 and 2, with the following exceptions: 160 shock stimuli of .18 mA intensity were delivered at either 1.0, 2.0, 4,0, or 8.0 sec intervals. The 161st shock was a 2.0 mA dishabituating shock, followed by 40 more stimuli of the original intensity and frequency. The .18 mA electric shock used in this and subsequent experiments was selected as the habituating stimulus on the basis of pilot work that indicated that this intensity produced a high, stable baseline of initial responding and rapid habituation. Recorded was the proportion of shocks that elicited a response within 1.0 sec of shock onset.1

Results The results of Experiment 3 are presented in Figure 6. Most striking is the changing effect of ISI during development. At 3 days of age, ISIs of 1 and 8 sec produce equal rates of response decrement. As the animals grow older, they habituate progressively less to infrequently presented stimuli, until at 15 days of age only slight habituation occurs at the longest ISI. An analysis of variance confirmed these observations, as the overall Age X ISI X Blocks effect, F(13S, 2640) = 1.255, p < .05, and the ISI X Blocks effect in 15-day-olds, F(45, 2640) = 15.141, p < .001, were significant, but in 3-day-olds, neither the ISI, F(3, 176) = 1.274, p > .05, nor the ISI X Blocks effect, ^(45, 2640) = 1.^12, p > .05, were significant. Equally striking is the decrease in dishabituation produced by the intense 2 mA shock stimulus as maturation proceeded. At 3 days of age the shock produced marked dishabituation, but at 15 days little or no dishabituation was observed. An analysis of variance performed on the block preceding and the block immediately following dishabituation yielded a significant Age X Blocks interaction, ^(3, 176) = 25.694, p < .001, to support this conclusion.

109 6-DAY OLDS

• 1.0 sec

4

2.0 sec »

• 4.0 sec ° 8.0 sec

1 4 8 12 16 20 15-DAY OLDS

1 4

8

12

16 20

8

12

16

20

BLOCKS OF 10 SHOCKS

Figure 6. Habituation of the forelimb-withdrawal response to repeated shock in 3-, 6-, 10-, and 15-dayold rats with 1.0, 2.0, 4.0, and 8.0 sec interstimulus intervals. Arrows in the top right corners indicate insertion of a single intense (dishabituation) stimulus into the habituation series.

Discussion In general the results of this research are in close agreement with the defining features of habituation listed by Thompson and Spencer (1966). The percentage of shocks eliciting the withdrawal response declines exponentially with repeated stimu1 Since the recording equipment registered only movements and not static limb position, a reduction in responding could result from either tonic flexion or extension. The former would more likely reflect sensitization to the stimulus, the latter, habituation. In order to determine whether response decrements reflected habituation or sensitization, eight rat pups at 3 and 15 days of age received 200 stimuli at either 1.0 sec or 8.0 sec intervals. During the last 100 stimulus presentations these subjects were independently scored for tonic flexion or extension by the first author and one of four other observers. It was found that the forelimb of every subject was tonically extended (habituated) at asymptote, although occasional struggling resulted in limb flexion prior to the shock and a subsequent failure to record a response to that shock. However, no observer reported these incidents to occur at a rate exceeding 6% of the observations, regardless of experimental condition. Therefore, the decrease in responding to shock reflected an increased tendency to leave the leg extended.

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DONALD J. STEHOUWER AND BYRON A. CAMPBELL 1.0 SEC 181

8.0 SEC ISI

Figure 7. Polygraph records of a typical habituation session in 3- and IS-day-old rats, with shock stimuli presented at 1.0 or 8.0 sec intervals. Each upward deflection in the top trace represents a recorded relay closure. Downward deflections in the bottom trace reflect response amplitude. Each deflection in the center trace represents a stimulus presentation, the downward deflection in the middle of the session marking the delivery of a strong dishabituating shock.

lation, the amount of habituation is proportional to frequency of stimulation, and strong shock produces marked dishabituation. Two of the three characteristics studied changed markedly during development. First, at 3 days of age the pups habituated rapidly to all frequencies of stimulation, but later in development pronounced habituation occurred only at the higher frequencies. These results suggest that 3day-old pups habituate more easily than older pups and that the interstimulus interval would have to be increased well beyond 8.0 sec to attenuate habituation in these young animals. Second, the magnitude of dishabituation produced by strong shock declined with age. This result parallels the ontogenetic decline in interstimulus responding found in the second experiment, suggesting that the greater dishabituation observed in the younger pups may be due to their greater reactivity to strong shock. Experiment 4 The third experiment leaves unanswered a number of fundamental questions about habituation of the flexor-withdrawal re-

sponse during development. For example, does response amplitude decrease as response frequency declines? What is the latency of the response, and does it change as a function of age and habituation? Do responses immediately following dishabituation differ from those occurring early in the habituation sessions? Are response latencies to strong and weak shocks the same? And, of central importance to the present study, is the all-or-none relay closure an accurate reflection of changes in leg flexion? The following experiment was conducted to answer these questions. Method Subjects were 3- and 15-day-old animals drawn from the same population of rats as in the other experiments. The apparatus was unchanged from the first three experiments, except for the following modifications. A Grass polygraph (Model 7C) was used to record both relay closures (the measure used in all other experiments) and the amplified output of the magnetic motion transducer. The procedure was identical to that of Experiment 3, with the following exceptions: 100 .18 mA shock stimuli were delivered at either 1.0 or 8.0 sec intervals. The 101st shock was a 2.0 mA shock, and was followed by 100 additional .18 mA shocks. The behavior of four pups in each age and frequency condition was recorded at high speed (SO mm/sec)

HABITUATION OF FORELIMB WITHDRAWAL

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Table 1 Median Latency of Response (in msec) Trials Age

Interval

1-10

51-60

Dishabituation

3 days

1.0 sec 8.0 sec 1.0 sec 8.0 sec

90 50 20 30

100 55 25 12.5

22.5 15 5 5

15 days

in order to measure response latencies. Since it was not feasible to record the entire session, and since few responses occurred late in habituation, only responses to the first 10 stimuli, the 51st to 60th stimuli, the dishabituating stimulus, and the 5 stimuli immediately following dishabituation were monitored. In addition, records for an entire session were obtained from four pups in each age and treatment condition at low paper speeds (50 mm/ min in the 1.0 sec ISI groups; 5 mm/min in the 8.0 sec ISI groups) to show the changes in response amplitude that occur during habituation and following dishabituation.

Results Representative low-speed records (Figure 7) show that during habituation there is a gradual waning of response amplitude. Furthermore, there is close correspondence between the response decline measured directly from the transducer and that measured as a decline in the probability of a relay closure. Not surprisingly, response amplitudes were greater in the 15day-olds than in the 3-day-olds. Response latencies from shock onset for those trials on which a response was recorded were measured to the nearest 5 msec from high-speed records and are presented in Table 1. This table shows that latencies decrease with development but do not change during the course of habituation. In both 3- and 15-day-old animals, the response latencies to the intense dishabituating shocks were much shorter than to the mild shocks. In the case of the 15-day-olds, the response latencies to the dishabituating stimulus were too short to be measured accurately at the paper speed used, but all were 5 msec or less. Response latencies to the first 5 weak stimuli following dishabituation did not

101-105 95 60 20 20

differ from those occurring prior to dishabituation. Since responses to the mild shocks in the sampled periods did not differ in latency, these data were combined to generate the frequency distribution of latencies shown in Figure 8. From Figure 8 and Table 1 , it is clear that latencies were much longer in the 3-day-olds than in the 15day-olds, and that for the 3-day-olds, latencies were longer when stimulated at 1.0 sec intervals than at 8.0 sec intervals. Analyses of variance confirmed this, since age, F(l, 270) = 131.979, p < .001; ISI, /?(!, 270) = 11.571, p < .001; and their interaction, F(l, 270) = 31.028, p < .001, were all significant. Analysis of the interaction' showed that 15-day-olds responded with shorter latencies than 3-dayolds regardless of ISI, Fs(l, 270) > 16.882, ps < .001, and that ISI had an effect on latencies of 3-day-olds, F(i, 270) = 36.507, p < .001, but not on the 15-day-olds, F(l, 270) = 2.620, p > .05. It is also apparent in Figure 8 that the shorter interval between stimuli increased latency variability in the 3-day-olds. Discussion This experiment shows that habituation of the flexor-withdrawal response in neonatal rats parallels a decline in the amplitude of the response and that latency to respond does not change during the course of habituation. When the response latencies to weak shock stimuli are compared with those elicited by the intense dishabituating stimulus, it is obvious that the former are much slower. A not unexpected finding is the observation that 15-day-old rats have

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DONALD J. STEHOUWER AND BYRON A. CAMPBELL l.O sec ISI

8.0 sec ISI

100-

old

100-

o £15 days „ °- old *>

Thompson and Spencer (1966) and Kandel (1976). If dishabituation in the present experiments is specific to the response pathway stimulated, then stimulation of a different site should be ineffective. However, if dishabituation reflects an increase in generalized arousal, the site of stimulation should be of little importance. In this experiment, subjects were habituated as previously, given a strong shock to the contralateral hindlimb, and then tested for dishabituation. Method

RESPONSE LATENCY (msec)

Figure 8. Frequency histograms of response latencies of 3- and 15-day-old rats habituated to shocks occurring at either 1.0 or 8.0 sec intervals.

response latencies 2 to 3 times shorter than 3-day-olds, which is quite likely due to increased myelination of axons in the response pathway (Jacobson, 1970) or maturation of some other aspect of neural function. The greater variability of latencies in the 3-day-old, 1.0 sec ISI condition may be due to superimposition of elicited responses on a more variable background of intertrial responding (Experiments 2 and 3, this study). When high levels of intertrial responding are present, the shock stimulus is about as likely to occur during the extension phase as in the flexion phase. Coincidence of extension and shock onset may be incompatible with flexion and thereby lengthen response latency. Conversely, stimulation during flexion may lead to spuriously short latencies. Similarly, response amplitudes may be either reduced or augmented.

A litter of eight 3-day-old rats and a litter of eight 15-day-old rats, maintained as previously, were used in this experiment. The apparatus was changed to allow delivery of a 2.0 mA dishabituating shock to the contralateral hindlimb through a wire electrode identical to that used for delivering the habituating stimulus. All other procedural details are identical to Experiment 3, except that all subjects were habituated to stimuli occurring at 1.0 sec intervals, and the dishabituating stimulus was presented after ISO stimuli.

Results Comparison of Figures 6 and 9 shows that stimulation of the contralateral limb is just as effective in producing dishabituation as stimulation of the original site.

Experiment 5 In the preceding experiments the dishabituating stimulus was presented to the same locus as the habituating stimulus. It is therefore possible that the resulting restoration of responding is due either to specific facilitation of the withdrawal response pathway or to an increase in generalized arousal, as postulated by

1

5 10 15 BLOCKS OF 10 SHOCKS

19

Figure 9. Dishabituation of the forelimb-withdrawal response in 3- and 15-day-old rats following a single intense stimulus delivered to the contralateral hindlimb.

HABITUATION OF FORELIMB WITHDRAWAL

As in Experiment 3, the 3-day-old subjects showed a large response increment, while 15-day-olds showed virtually none. These conclusions are confirmed by an analysis of variance, which yielded a significant Age X Dishabituation interaction, F(\, 14) = 14.029, p < .01, and subsequent t tests showing a significant response increase in 3-day-olds, t(T) = 5.289, p < .01, but not in 15-day-olds, J(7) = 1.468, p > .05.

113

to pups of different ages result in markedly different declines in frequency of responding. For example, when a shock stimulus is presented every 4 sec, the 3-day-old pups show a sharp decline in responding, while 15-day-old pups show little change. This does not necessarily mean, however, that the 15-day-old pups habituate less to the shock stimulation than the 3-day-old pups. As Davis and Wagner (1968) have elegantly demonstrated, apparent differences in habituation may be confounded Discussion with the response-eliciting properties of These results show that dishabituation the stimulus. In other words, a lesser does not depend on direct stimulation of response decline using a strong stimulus the habituated site, suggesting that the could be due either to reduced habituation strong shock produces dishabituation or to the greater exciting value of a strong through generalized arousal rather than stimulus. Using auditory startle stimuli of through specific response facilitation. As different intensities, Davis and Wagner noted, greater dishabituation was produced (1968) showed that weak stimuli produce in 3- than in 15-day-old pups, a finding again a greater decrement in responding than consistent with the observation that shock strong stimuli but result in less habituation has a greater and more pervasive effect when the subjects are subsequently tested on stimuli of the same intensity. This on 3-day-old than on 15-day-old rats. same general design was used to compare development of habituation in rats of Experiment 6 different ages. The preceding series of studies have shown that both reactivity to supra- Method threshold shock and amount of habituation Subjects from 32 litters (N = 256) were mainto repetitive shock stimulation (at low in a manner identical to the previous experifrequencies) decline with age in the de- tained ments, and were tested at either 3, 6, 10, or 15 days veloping rat. This coincidence of findings of age. suggests that reducing the intensity of The apparatus and procedure were the same as stimulation applied to younger animals, Experiment 3, with the following changes: At each thereby making the stimulation more age 16 animals were habituated at either .09 or .18 mA intensity, and with either a 2.0 or 4.0 sec similar in subjective intensity to the shock ISI. Each group of animals received 100 shocks received by the older subjects, might during habituation, was divided in half, and 30 sec produce less habituation in the two later tested for habituation at either the high or low youngest age groups. Such a result, how- intensity. This design is summarized in Table 2. The test consisted of 10 shocks delivered at the same ever, is extremely unlikely in view of the frequency to which the subject was originally many studies showing a greater decline in habituated. As before, Utters were divided evenly responding at low stimulus intensities among treatment groups, with age as a between(Thompson & Spencer, 1966). The first litters variable. phase of Experiment 6 compares habituation to repetitive shock stimulation at two Results intensities and two frequencies in the The results of the initial habituation developing rats. A question of more penetrating interest phase of this experiment are presented in concerns the relative degree of habituation Figure 10. The 2.0 and 4.0 sec ISI groups produced in those instances where the same habituated to .18 mA intensity shock show frequency and intensity of shock applied virtually the same developmental pattern

DONALD J. STEHOUWER AND BYRON A. CAMPBELL

114

Table 2 Experimental Design for Experiment 6 Age (days) 3, 6, 10, 15 Interstimulus interval (sec) 2.0 4.0 Training intensity (mA) .09 .18 .09 .18 Testing intensity (mA) .09 .18 .09 .18 .09 .18 .09 .18

of habituation as the corresponding groups in Experiment 3. Again, probability of responding increases at longer ISI as the animals grow older, suggesting that habituation to repetitive shock stimuli decreases as a function of age. This observation was verified by a significant Age X ISI X Trials interaction, F(27, 2160) = 1.815, p < .05. Reducing intensity of the stimulus to .09 mA had the effect of reducing the level of responding and yielded a more rapid response decrement. Furthermore, at this shock level the older animals showed a decline in responding comparable to the younger rats at the 4.0 sec ISI. Analysis of variance of these data confirmed that intensity, F(l, 240) = 22.988, p < .001, and Intensity X Blocks, F(9, 2160) = 3.932, p < .001, effects were significant, but the Age X ISI X Blocks effect for the .09 mA group was not, F(2T, 2160) = .977, p > .05.

The effect of varying stimulus intensity during habituation on subsequent test performance can be seen in Figure 11, which shows mean percent leg flexions for the 10 test trials. As described, half of the subjects in each training shock intensity were retested at that intensity and half at the other intensity. The results shown in Figure 11 are the combined (averaged) frequencies of responding for both shock intensities. As expected, more habituation was seen in the high-intensity groups than the low-intensity groups. Frequency of shock stimulation had little effect during this second habituation test, except perhaps in the 15-day-olds. Analysis of variance yielded a significant effect of habituationshock intensity, F(l, 224) = 20.45, p < .001, and an Age X Habituation-Shock Intensity interaction, F(3, 224) = 2.74, p < .05. However, the Age X Habituation-

TPC-T '2.0 sec ISI, 0,09mA • 2.0sec ISI, 0.18mA TDAIM "Z.OseclSI TRAIN O40sec |SI lt;>l «4.0sec ISI, 0.09mA = 4.0 sec IS. 0.18mA 3 DAYS

60

6 DAYS

A A • D

2.0 sec ISI, 4.0 2.0 4.0

0.09mA 0.09 0.18 0.18

40

UJ

20

0' 1 5

IOT I

5

IOT

1 5

IOT

5

IOT

BLOCKS OF 10 SHOCKS

Figure JO. Effects of stimulus intensity and frequency on habituation of the forelimb-flexion response in 3-, 6-, 10-, and 15-day-old rats during acquisition and on a subsequent test for habituation.

6

10 AGE (DAYS)

15

Figure 11. Mean percent flexions elicited during the habituation test as a function of stimulus intensity and frequency during habituation, collapsed across test intensity.

HABITUATION OF FORELIMB WITHDRAWAL

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Shock Intensity X ISI interaction failed to the smaller decline in frequency of respondattain significance, F(3, 224) < 1.0. The ing seen in 10- and 15-day-old pups com15-day-olds appeared to violate this general pared to 3- and 6-day-olds seen in Experipattern, but statistical support was mar- ment 3 and the initial portion of this ginal. Therefore, to determine the reliability experiment may reflect the emergence of of this result, two more groups of 12 another process. Within the context of IS-day-old pups were used in an exact habituation theory, this process is most replication of the 4.0 sec ISI, .18 and .09 likely that of sensitization (Groves & mA conditions, tested at .09 mA. These Thompson, 1970). The possible role of groups were a major source of the ap- sensitization in maintaining responding in parently deviant data point in Figure 11. the older age groups is explored in the This replication showed that 15-day-old following experiment. pups, like the younger pups, were more completely habituated after strong stimuExperiment 7 lation than after weak stimulation on the 10 test trials, /(22) = 2.33, p < .05. Thus In their dual process theory, Groves and it appears that habituation is more pro- Thompson (1970) proposed that two innounced in pups of all ages, including dependent processes, habituation and sensi15-day-olds, at higher intensities, even tization, interact to yield a particular though the response decrement during the behavioral output when an animal is habituation series is less. Moreover, the exposed to repetitive, inescapable stimulaamount of habituation observed with this tion. According to this analysis, the ageprocedure does not vary appreciably with related decline in responding to repetitive age. Therefore, the smaller decline in stimulation seen in Experiments 3 and 6 frequency of responding in 15-day-olds of this report could reflect either a decrease does not reflect increased resistance to in habituation to repetitive stimulation or habituation but suggests instead that there the emergence of sensitization during may be another factor that maintains a development. However, as noted in the high level of responding in these animals last experiment, habituation does not during the initial habituation series. appear to vary as a function of age when measured under the conditions proposed by Davis and Wagner (1968). This suggests Discussion that some other process, such as sensitizaThe first phase of this experiment tion, is emerging during development to allowed us to examine the possibility that modify (or mask) the manifestation of the greater habituation to shock observed habituation. Additional evidence for this possibility in the younger pups was due to the more intense behavioral reactivity elicited by can be seen in Experiment 3, where 10-dayshock in those animals. The results of this old rats show an initial decline in respondexperiment do not support this view, since ing, followed by an increase. This pattern reducing shock intensity and the con- of responding, a decrease followed by an comitant perseverative behavioral excita- increase, is characteristic of the concurrent bility did not produce greater habituation. development of habituation and sensitizaIn the second phase of this experiment, tion under some conditions. Moreover, in we found that high-intensity shock pro- many instances, several hundred trials are duced greater habituation than low-inten- required for sensitization to reach a sity shock when habituation was measured maximum, according to Groves, Lee, and as frequency of responding to a standard Thompson (1969). With these considerations in mind, the shock intensity. Moreover, when measured in this fashion, habituation of the forelimb- purpose of the following experiment was withdrawal reflex does not appear to vary to study the effects of repetitive shock appreciably as a function of age. Therefore, stimulation on the forelimb-withdrawal

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DONALD J. STEHOUWER AND BYRON A. CAMPBELL • O A A

3 DAYS 6 DAYS 10 DAYS 15 DAYS

o x

o o: uj Q.

0 I 5 10 BLOCKS OF 50 SHOCKS

Figure 12. Percentage of shock stimuli eliciting a forelimb-withdrawal response in 3-, 6-, 10-, and 12-day-old rats.

response over an extended habituation session consisting of 500 shock-stimulus presentations.

so that after 300 shocks, they are responding at a rate greater than or equal to first-block levels. These differences are supported by an analysis of variance in which age, F(3, 190) = 49.312, p < .001; blocks, F(9, 1710) = 16.021, p < .001; and the Age X Blocks interaction, F(27, 1710) = 4.894, p < .001, were all significant. The increment in responding following the initial decline in the 10- and 15-day-olds was also found to be significant. An analysis of variance performed in Trial Blocks 2 through 6 yielded a significant Age X Blocks interaction, F(12, 760) = 1.841, p < .05, and a significant effect of blocks in the 10- and 15-day-old rats, Fs(4, 760) > 3.593, ps < .01, but not in the 3- and 6-day-old pups, Fs(4, 760) < .525, ps > .05. Discussion

The data obtained in this experiment suggest that shock-induced sensitization of the forelimb-withdrawal response emerges between 6 and 10 days of age in the developing rat. At ages up to and including Method 6 days, repeated shock stimulation results Subjects for this experiment were 194 rat pups in a rapid decline in frequency of responding from 36 litters, drawn from the same population to repeated stimulation. Over the course as previously. The number of pups tested at each age was as follows: 3-day-olds, 39 pups; 6-day-olds, of the next 4 days of development, this SO pups; 10-day-olds, 56 pups; 15-day-olds, 49 pups. pattern of responding changes as seen in The apparatus was unchanged, except for the 10- Figure 11. In both 10- and 15-day-old and 15-day-old pups the indifferent electrode was rats, a brief initial decline is followed by a changed from the metal tube and electrode paste slight but significant increase in frequency used previously to a 9 mm wound clip applied to the base of the back. During training, pups received SOO of responding. This pattern of results, .18 mA shocks to the forepaw at 4.0 sec intervals, coupled with the finding that amount of with responses (relay closures) recorded as before. habituation does not change appreciably with age (Experiment 6) leads us to speculate that sensitization resulting from Results repeated shock stimulation is the mechaAcquisition data, presented in Figure 12 nism that prevents the decline of respondas percent of responses per block of 50 ing in 10- and 15-day-old animals. The initial decline in frequency of shocks, show again that 3- and 6-day-old pups undergo a much greater response responding in 10- and 15-day-old rats is decrement than do 10- and 15-day-olds hypothesized to reflect habituation and the when the ISI is 4.0 sec. The younger subsequent increase to result from sensitianimals declined quickly to a low level of zation of the response. This sequence and responding and remained at that level for its time course are similar to the functions the remaining trials. Responding in 10- and generated by Groves et al. (1969) in a 15-day-old rats, on the other hand, de- parametric investigation of habituation and clined from the first to second block, but sensitization. Further support for this view then increased over the next five blocks has been reported by Stehouwer (1977),

HABITUATION OF FORELIMB WITHDRAWAL who found that 10- and 15-day-old rats given four ISO-shock habituation trials showed higher levels of responding 24 hr later on a habituation test series. This increase was interpreted as evidence for long-term retention of sensitization. At the same time 3- and 6-day-old rats showed no evidence of sensitization or retention of sensitization over the 24-hr period. General Discussion The major contribution of this research is the establishment of habituation of the flexor-withdrawal response as a suitable preparation for studying the development of behavioral plasticity in the neonatal rat. The withdrawal response can be reliably elicited by a specified electrical stimulus, and habituation of this response varies systematically and predictably with variations in intensity and interstimulus intervals. It offers the further advantage of early maturation and is simple in relation to other types of responses studied ontogenetically (e.g. Bronstein & Dworkin, 1974; Bronstein et al., 1974; Feigley et al., 1972; File & Plotkin, 1974; Williams Hamilton, & Carlton, 1975). These experiments demonstrated that the limb-withdrawal response exhibits at least four of the characteristics that define habituation (Thompson & Spencer, 1966). Namely, the response declines with repeated stimulation; the amount of the decrement is directly related to stimulus frequency; a strong shock produces dishabituation; and the within-session decrement is directly related to the stimulus intensity. Developmentally, the present study found no changes in the flexor reflex threshold for electrical stimulation between 3 and 15 days of age. During the same period of development, however, the aftereffects of shock become more abbreviated, and latency of the response decreased. When pups were habituated to varying frequencies of stimulation, it was found that responding declined less in older pups. Concomitantly, the effectiveness of a

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strong shock in producing dishabituation decreased. When pups were given an extended habituation session, it was found that 10- and 15-day-old pups first showed a decline in response frequency to the shock stimulus, followed by an increase in responding. The decrease was thought to reflect habituation and increase the sensitization. The 3- and 6-day-old groups showed only a decline in responding. These data were interpreted as suggesting that the process of sensitization emerges somewhere between 6 and 10 days of age in the developing rat. This study complements others concerned with the early behavioral development of the rat. Like Stelzner and his associates (Stelzner, 1971; Stelzner, Ershler, & Weber, 1975; Weber & Stelzner, 1977) we found that during the first postnatal week, noxious stimuli elicited persistent and generalized responding. Early in the second week of development, this mode of responding was replaced by brief, discrete responses. These behavioral changes are correlated, according to Stelzner (1971), with the development of the spinal cord ultrastructure. For example, the size and number of synaptic contacts increases dramatically during the first 3 weeks of life. Furthermore, most of the synapses he observed in newborn rats were distally located on dendrites, the more proximal synapses developing later. Since inhibitory contacts are generally located nearer the soma than excitatory synapses (Eccles, 1973), this evidence suggests that the development of behavioral inhibition results from increased neural inhibition. Bodian (1968) has also correlated synaptic development with the transition from generalized responding to localized reflexes in the fetal macaque and similarly concluded that this transition resulted from the functional onset of long, intersegmental connections. Again, ultrastructural evidence suggests that the synaptic connections responsible for the behavioral change were of the inhibitory type. There is a paucity of studies on the development of the various supraspinal projections to the spinal cord of the rat,

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DONALD J. STEHOUWER AND BYRON A. CAMPBELL

but some are obviously present at birth, since the newborn pup responds to tactile stimulation of the face with movements extending the length of the trunk. The corticospinal tract, however, does not make functional connections in the cord until 12 to 15 days of age (Donatelle, 1977; Hicks & D'Amato, 1975), at which time they manifest themselves in the fine adjustment of limbs, the emergence of tactile placing (Hicks & D'Amato, 1975), and the development of forelimb behaviors such as object manipulation (Barren, 1934). In view of the results reported in this paper, it is of interest to note that Barren (1934) concluded that the corticospinal tract is mainly concerned with the regulation of forelimb flexor movements. Thus, it is quite clear that nervous control of movement improves greatly in the first 3 weeks and that this improvement reflects both spinal maturation and the development of supraspinal connections to the cord. Our results also show that the disappearance of perseverative activity following a single shock parallels the emergence of sensitization. It is not inconceivable, therefore, that inhibition of neonatal perseverative responding and the emergence of sensitization are mediated by maturation of the same neural systems. Reference Note 1. Collier, A. C. The ontogeny of response to shock in the neonatal rat pup and its effect on suckling. Paper presented at the 46th meeting of the Eastern Psychological Association, New York, April 1975.

References Barron, D. H. The results of unilateral pyramidal section in the rat. Journal of Comparative Neurology, 1934, 60, 45-56. Bodian, D. Development of fine structure of spinal cord in monkey fetuses. II: Prereflex period to period of long intersegmental reflexes. Journal of Comparative Neurology, 1968, 133, 113-166. Bornstein, M. H., Kessen, W., & Weiskopf, S. The categories of hue in infancy. Science, 1976, 191, 201-202. Bronstein, P. M., & Dworkin, R. Replication: The persistent locomotion of immature rats. Bulletin of the Psychonomic Society, 1974, 4, 124-126.

Bronstein, P. M., Neiman, H., Wolkoff, F. D., & Levine, M. J. The development of habituation in the rat. Animal Learning and Behavior, 1974, 2, 92-96. Campbell, B. A., & Masterson, F. A. Psychophysics of punishment. In B. A. Campbell & R. M. Church (Eds.), Punishment and aversive behavior. New York: Appleton-Century-Crofts, 1969. Cohen, L. B. Habituation of human infant attention. In T. J. Tighe & R. N. Leaton (Eds.), Habituation: Perspectives from child development, animal behavior and neurophysiology. Hillsdale, N. J.: Erlbaum, 1976. Conklin, P., & Heggeness, F. W. Maturation of temperature homeostasis in the rat. American Journal of Physiology, 1971, 220, 333-336. Davis, M., & Wagner, A. R. Startle responsiveness after habituation to different intensities of tone. Psychonomic Science, 1968, 12, 337-338. Donatelle, J. M. Growth of the corticospinal tract and the development of placing reactions in the postnatal rat. Journal of Comparative Neurology, 1977, 175, 207-232. Eccles, J. C. The understanding of the brain. New York: McGraw-Hill, 1973. Feigley, D. A., Parsons, P. J., Hamilton, L. W., & Spear, N. E. The development of habituation to novel environments in the rat. Journal of Comparative and Physiological Psychology, 1972, 79, 443-452. File, S. E., & Plotkin, H. C. Habituation in the neonatal rat. Developmental Psychobiology, 1974, 7, 121-127. File, S. E., & Scott, E. M. Acquisition and retention of habituation in the preweanling rat. Developmental Psychobiology, 1976, 9, 97-107. Griffin, J. P. Neurophysiological studies into habituation. In G. Horn & R. A. Hinde (Eds.), Short-term changes in neural activity and behavior. London: Cambridge University Press, 1970. Groves, P. M., Lee, D., & Thompson, R. F. Effects of stimulus frequency and intensity on habituation and sensitization in acute spinal cat. Physiology and Behavior, 1969, 4, 383-388. Groves, P. M., & Thompson, R. F. Habituation: A dual-process theory. Psychological Review, 1970, 77, 419-450. Hicks, S. P., & D'Amato, C. J. Motor-sensory cortex-corticospinal system and developing locomotion and placing in rats. American Journal of Anatomy, 1975, 143, 1-42. Jacobson, M. Developmental neurobiology. New York: Holt, Rinehart & Winston, 1970. Jeffrey, W. E. Habituation as a mechanism for perceptual development. In T. J. Tighe & R. N. Leaton (Eds.), Habituation: Perspectives from child development, animal behavior and neurophysiology. Hillsdale, N. J.: Erlbaum, 1976. Kandel, E. R. Cellular basis of behavior: An introduction to behavioral neurobiology. San Francisco: Freeman, 1976. Narayanan, C. H., Fox, M. W., & Hamburger, V. Prenatal development of spontaneous and evoked

HABITUATION OF FORELIMB WITHDRAWAL activity in the rat (rattus norvegicus albinus). Behavior, 1971, 40, 100-134. Stehouwer, D. J. Habituation of the limb-withdrawal response in the developing rat. Unpublished dissertation, Princeton University, 1977. Stelzner, D. J. The normal postnatal development of synaptic end-feet in the lumbrosacral spinal cord and of responses in the hindlimb of the albino rat. Experimental Neurology, 1971, 31, 337-357. Stelzner, D. J., Ershler, W. B., & Weber, E. O. Effects of spinal transection in neonatal and weanling rats: Survival of function. Experimental Neurology, 1975, 46, 156-177. Taylor, P. M. Oxygen consumption in newborn rats. Journal of Physiology, 1960, 154, 153-168. Thompson, R. F., & Spencer, W. A. Habituation: A model phenomenon for the study of neuronal substrates of behavior. Psychological Review, 1966, 73, 16-43. Thompson, W. R. Storage mechanisms in early

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experience. Minnesota Symposium on Child Psychology, 1972, 6, 97-127. Vaughn, J. E., & Grieshaber, J. A. A morphological investigation of an early reflex pathway in developing rat spinal cord. Journal of Comparative Neurology, 1973, 148, 177-209. Weber, E. D., & Stelzner, D. J. Behavioral effects of spinal cord transection in the developing rat. Brain Research, 1977, 125, 241-255. Williams, J. M., Hamilton, L. W., & Carlton, P. L. Ontogenetic dissociation of two classes of habituation. Journal of Comparative and Physiological Psychology, 1975, 89, 733-737. Wyers, E. J., Peeke, H. V. S., & Herz, M. J. Behavioral habituation in invertebrates. In H. V. S. Peeke & M. J. Herz (Eds.), Habituation (Vol. 1: Behavioral Studies}. New York: Academic Press, 1973.

Received November 22, 1976 Revision received December IS, 1977 •