- Email: [email protected]
Development of fear-related heart rate responses in neonatal rabbits
~.1"~ ~'7~.'7,~ ~ ,~.~
Journal of the
ELSEVIER
Autonomic Nervous System Journal of the Autonomic Nervous System 50 (1994) 231-238
Development of fear-related heart rate responses in neonatal rabbits Laura Sebastiani, Domenico Salamone, Pasquale Silvestri, Alfredo Simoni, Brunello Ghelarducci * Dipartimento di Fisiologia e Biochimica, Universit?t di Pisa, Via S. Zeno 29/31, 56127 Pisa, Italy
Received 21 January 1994; revision received 16 March 1994; accepted 30 March 1994
Abstract
Classical simple conditioning of heart rate (HR) was studied in rabbits between the 1st and 18th neonatal day. An auditory stimulus (1000 Hz, 5 s) served as the conditioned stimulus (CS), and a train of electric impulses (100 Hz, 500 ms, 1-1.5 mA) was used as the unconditioned stimulus (US). HR responses developed during orientation session (CS-alone) as well during acquisition (CS-US paired) were analyzed and compared to those developed by young adult rabbits (3-month-old). In all neonatal animals tested, baseline HR measured during an adaptation session preceding conditioning, was similar though significantly higher than that measured in adult rabbits (Newman-Keuls P < 0.05). Before the 10th neonatal day, the animals did not show either somatomotor or H R orienting responses to the CS-alone presentations. Consequently, since orienting reactions play a necessary role in the formation and manifestation of conditioned reflexes, 1 to 10-day-old infant rabbits were not submitted to the acquisition session. All the other neonatal groups, while showing orienting behaviours similar to those exhibited by adults (head and pinna movement), presented different patterns of H R orienting responses (no response, bradycardia, tachycardia, bradycardia/tachycardia etc.). As for the acquisition session, the first bradycardic response, similar to that developed by adult rabbits, was found in 18-day-old rabbits. However, also in this neonatal group the amplitude of the conditioned response was significantly smaller when compared to that exhibited by young adults (Newman-Keuls P < 0.01). In addition, in some of the 10-day-old neonates, H R appeared very unstable and dropped to very low values (as low as 146 b e a t s / m i n ) early during conditioning, apparently as a consequence of CS-US association. As for the unconditioned response, no differences were found between adult rabbits and the neonatal animals older than 12 days. In contrast, most of the 10-day-old rabbits showed either bradycardia or no response to the unconditioned stimulus. Considering the ability of mammalian infants to learn somatomotor conditioned responses at early stages of maturation, conditioning of H R responses occurs late during ontogeny. Since this incapacity to show H R conditioned responses before the 18th postnatal day cannot be ascribed to their inability to show phasic H R changes nor to a failure in detecting the auditory stimulus, these results suggest the possibility that H R conditioned responses may be mediated by neural structures developing later during maturation. Keywords: Heart rate response; Aversive conditioning; Rabbit; Neonate
* Corresponding author. Tel." (39-50) 554074; Fax: (39-50) 552183. 0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(94)00090-7
232
L. Sebastiani et ak /Journal of the Autonomic Nervous System 50 (1994) 231-238
I. Introduction
It is well known that neonatal mammals are able to perform relatively complex associations [4,14,18,22] in spite of the immaturity of their sensory and motor systems. However, the emergence of classically conditioned motor and heartrate responses follows the sequential development of the sensory mechanisms (olfactory, tactile, auditory, visual) involved as conditioned stimuli [5,6,11,22,28,29]. Aside from the complete maturation of the appropriate sensory and motor systems, a conditioned response also requires the complete development of the central nervous structures involved in the association process [6]. A striking observation from these previous studies is that autonomic conditioned responses develop considerably later in ontogeny when compared with motor conditioning, and that this delay is evident for all the sensory modalities used as conditioned stimulus (CS) [6,7,9,23,24]. In rats, for example, while motor conditioning to an olfactory stimulus emerges within the first days of life, conditioned HR responses using the same olfactory modality are not present until the 15th postnatal day [24]. Though rabbits have been widely used in behavioral and neurophysiological studies concerning fear-related responses and their neural substrates [16,20,25,27,30], at present little is known about the maturation of fear-related heart rate orienting and conditioned responses in infant rabbits. In contrast, much is known of the development of somatomotor responses (pinna orientation, head movement etc.) which are components of the orienting response to an acoustic novel stimulus in adult rabbits [31]. The main purpose of this study is to determine the age at which both orienting and classically conditioned HR responses to an auditory stimulus emerge in infant rabbits. This study represents an essential step in determining the influence exerted on the development of the HR conditioned responses by those neural structures which are known to modulate this response in the adult.
A preliminary report of these data was presented at the Meeting of the Physiological Society, King's College, London, December 1993 [12].
2. Materials and methods
2.1. Animals and experimental groups Forty-eight New Zealand white neonatal rabbits, bred and reared in the animal colony of our Department were used for the study. At the start of the conditioning procedure rabbits were divided in 5 groups of different ages: (1-9)-day-old (n = 8); 10-day-old (n --- 8); 12-day-old (n = 8); 15-day-old (n = 8) and 18-day-old (n = 8). For the entire duration of the experiment they were housed with their mothers and with other rabbits of the same age and had free access to water and food. An additional group of eight adult rabbits (3month-old) weighing 2.5-3.0 kg were used. These animals belonged to the same breed as the neonatal groups. All neonates and adult animals were submitted to a simple conditioning protocol [27]
2.2. Apparatus and conditioning procedure All neonatal animals were tested while restrained in a full-body sling attached to the ceiling of a sound-attenuating chamber (50 x 45 × 40 cm), diffusely illuminated by a 20-W lamp. The chamber's ventilation system also provided the background noise (60 dB). As for adult rabbits they were placed inside the sound-attenuating chamber, held in a plexiglass restrainer (Tecniplast), which limited both body and head movements. Before behavioral testing, in all animals fine stainless steel wire loops were inserted subcutaneously at the precordial level to allow for ECG recording. Two similar wire loops chronically inserted in the border of the right pinna served as stimulating electrodes for delivering the unconditioned stimulus (US), a 500-ms train of 0.5-ms pulses at 100 Hz, with an intensity of
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
1-1.5 mA provided by a constant current stimulator. The conditioned stimulus (CS) consisted of a continuous 1000-Hz tone (5 s, 90 dB) and was presented to the rabbit through a loudspeaker mounted on the ceiling of the chamber about 25 cm above the head of the animal. A Commodore 64 computer was programmed to control the characteristics and the presentation sequence of the stimuli (CS and US). Before starting the conditioning procedure all the animals were handled for at least a 1-hour session. Successively, each animal received a 1hour adaptation session on each of 4 consecutive days, in order to adapt to the test enviroment. For the group of animals tested before the 5th day of life this procedure was shortened. Following this procedure, all animals were given 10 min of adaptation during which heart rate (HR) was sampled at 1-min intervals. Next, 15 consecutives unreinforced 1000-Hz tones (CS-alone) were presented at pseudorandom intertrial intervals (5565 s; mean = 60 s). This was to habituate the heart rate orienting response to a novel stimulus commonly observed in adult rabbits. Simple conditioning started immediately thereafter and consisted of 45 trials in which CS was paired to US at a 60 s ITI. The offset of CS coincided with US onset.
2.3. Heart rate analysis On-line acquisition of ECG, as well as subsequent off-line measurement and analysis of H R responses were performed by means of a computer system (Microsys Electronics 386). HR changes to each CS-alone and CS-US paired presentation were calculated as the difference between the average H R (beats/min) during the 5-s CS and that during the 5-s pre-CS baseline period. For successive analysis differences were grouped in blocks of 5 trials. The HR unconditioned response to pinna shock was calculated by comparing the 5-s period following US offset to the baseline H R measured in the 5-s period preceding US onset. A repeated measures analysis of variance (ANOVA) was performed on the difference scores. Post-hoc Newman-Keuls comparisons as
233
well as two-tailed paired t-test between means were performed when appropriate. Differences were considered significant at P < 0.05.
3. Results
3.1. Adaptation In all the neonatal animals tested, baseline HR measured at the end of the adaptation session was similar (means ± SD: 297.08 + 38.83, (19)-day-old; 320.46+24.86, 10-day-old; 291.51 ± 48.64, 12-day-old; 302.16 + 29.58, 15-day-old; 321.98 + 28.86, 18-day-old). However, in all groups, baseline HR was higher than that measured in adult rabbits (249.73 ± 27.41). Indeed, ANOVA performed on these data yelded a significant group effect (F(5,42) = 5.394, P < 0.001) and post-hoc Newman-Keuls test revealed that all neonatal groups significantly differed from adult animals (10 and 18-day-old P < 0.01; (1-9),12,15day-old P < 0.05).
3.2. Orientation We considered that a H R orienting response consisted of any change in H R (tachycardia or bradycardia) significantly different from baseline, shown in response to the first CS-alone presentation. In adult rabbits this response, usually a bradycardia that habituated over a few trials, was accompanied by typical somatomotor orienting reponses (pinna orientation, sudden head and body movements, pupillary dilatation). However, a great variability in the magnitude and duration of this response was seen in normal adult rabbits.
Adults All adult animals showed H R and behavioral orienting responses similar to those found in previous studies [15,17,25,26,27]. In particular, the heart rate response to the CS-alone presentation consisted of an initial deceleration which soon habituated over the 15 trials. In all the animals tested the highest HR responses occurred during the first block of 5 CS-alone presentations ( - 14.3 + 5.4 beats/min: mean + SE).
234
L. Sebastiani et aL / Journal of the Autonomic Nervous System 50 (1994) 231-238
Neonatal animals During habituation all the animals, with the exception of the (1-9)-day-old neonates, showed marked somatomotor orienting responses at the onset of the novel stimulus (tone) which were similar to those described for the adult rabbit. However, at difference with adult rabbits in which these responses are typically accompanied by a bradycardia, neonatal rabbits exhibited different patterns of H R changes not only for the different age groups but also within a single group. Statistical analysis performed on these responses did not yield any significant result thus preventing any conclusion on the existence of a preferred pattern related to a particular age group. However, when the prevailing pattern of response was considered in each group, as described below and summarized in Table 1, it was possible to note a systematic change of the prevailing pattern of response in relation to age. (1-9)-day-old rabbits. None of these animals showed any somatomotor orienting response (sudden movements of the head a n d / o r the body, orientation of the pinna, etc.) or any change in HR in response to CS. Therefore, they were not submitted to the conditioning testing. Indeed, since the orienting reaction plays an essential role in the formation and manifestation of the conditioned reflex, its appearance was considered a necessary prerequisite before proceeding to the conditioning session. lO-day-old rabbits. As indicated in Table 1, most of the neonates (n = 5) did not show any H R orienting response, while of the remaining three animals one exhibited a bradycardic re-
sponse comparable to that found in adult rabbits, another an initial tachycardia followed by bradycardia, and the last one a small tachycardic response. 12 and 15-day-old rabbits. Both groups of neonates showed very different patterns of H R orienting responses at the onset of the tone. As shown in Table 1, half of the 12-day-old animals (n = 4 ) showed no responses to the CS-alone presentation, while half of the 15-day-old neonates exhibited bradycardia and only three of this group of animals did not show any H R orienting response. 18-day-old rabbits. In most of the animals (n = 6) behavioral responses were accompanied by bradycardia. Out of six animals only one did not show any H R response and another one exhibited a small tachycardia at the onset of the CS (Table 1).
3.3. Acquisition Baseline H R during conditioning. The mean pre-CS H R values for all the groups submitted to the acquisition session were analyzed during conditioning. No changes in baseline H R were observed as a consequence of training. In fact, with the exception of three 10-day-old rabbits whose H R appeared very unstable and dropped to very low values (down to 146 b e a t s / m i n ) during conditioning, for all the remaining groups baseline H R calculated during the last block of acquisition trials did not differ significantly from that measured at the end of the adaptation session (paired t-test). In addition, A N O V A performed on base-
Table 1 Somatomotor and heart rate responses exhibited by neonatal rabbits between the 1st and 18th postnatal day. The number of neonates showing a specific orienting response is expressed as percentage of the total number of animals belongingto the same age group Somatomotor 1-9 days 10 days 12 days 15 days 18 days and heart rate (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) orienting responses Pinna orientation none 100% 100% 100% 100% Head movement none 100% 100% 100% 100% No heart rate changes 100% 62.5% 50.0% 37.5% 12.5% Slight tachycardia or biphasic response none 25.0% 37.5% 12.5% 12.5% Bradycardia none 12.5% 12.5% 50.0% 75.0%
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
trials a n d t h e n showing a g r a d u a l a d a p t a t i o n tow a r d s b a s e l i n e values.
CS-US paired 5
Q) c
c~ c~
0
E 0 L ~--
--5
r, r~ 0
--10
,
n~ "1C 0
--15
E -20
I 5
i 10
i 15
i 20
i 25
0a 3
235
315
i 40
45
5 Trials Blocks
Fig. 1. Mean HR changes from pre-CS baseline recorded during the CS-US paired presentations in rabbit of different postnatal ages (e 10 days, v 12 days, • 15days, [] 18 days and • adult). Data points represent means for five trials blocks. line v a l u e s c a l c u l a t e d across t h e 45 acquisition trials d i d n o t yield any significant trial effect, thus i n d i c a t i n g t h a t in all t h e e x p e r i m e n t a l groups, b a s e l i n e H R was stable t h r o u g h o u t t h e c o n d i t i o n ing session. Heart rate conditioning. Fig. 1 shows t h e H R r e s p o n s e s e x h i b i t e d by t h e five e x p e r i m e n t a l g r o u p s d u r i n g t h e acquisition session.
Adult A d u l t a n i m a l s s h o w e d a typical c o n d i t i o n e d b r a d y c a r d i a c o m p a r a b l e to t h a t p r e v i o u s l y d e s c r i b e d by o t h e r a u t h o r s [15,17,25-27]. I n p a r t i c u lar, b r a d y c a r d i a d e v e l o p e d within t h e first 15 trials, r e a c h i n g its p e a k d u r i n g t h e 3rd b l o c k o f 5
Neonatal animals lO-day-old. A s m e n t i o n e d above, in 3 o f t h e a n i m a l s s t u d i e d , b a s e l i n e H R was very u n s t a b l e d u r i n g c o n d i t i o n i n g . Such f l u c t u a t i o n s r e s u l t e d in a s e p a r a t i o n o f d a t a f r o m t h e s e a n i m a l s which was n o t i n c l u d e d in s u m m a r y d a t a . A s shown in T a b l e 2, t h e s e 10-day-old n e o n a t e s s h o w e d e i t h e r t a c h y c a r d i a o r b r a d y c a r d i a . H o w e v e r , in c o n t r a s t to n o r m a l a d u l t r a b b i t s in which b i g g e r H R c h a n g e s a r e typically shown a r o u n d t h e third b l o c k o f trials, t h e a m p l i t u d e o f the r e s p o n s e s e x h i b i t e d by t h e s e t h r e e a n i m a l s was l a r g e r in t h e first b l o c k of C S - U S p a i r e d p r e s e n t a t i o n s , a n d successively d e c r e a s e d , or even r e v e r s e d in sign d u r i n g t h e c o n d i t i o n i n g session. A s for the rem a i n i n g animals, t h e y d i d n o t d e v e l o p any condit i o n e d H R c h a n g e as a c o n s e q u e n c e o f C S - U S a s s o c i a t i o n (see Fig. 1). 12 and 15-day-old. D u r i n g acquisition sessions r a b b i t s d i d not d e v e l o p any c o n d i t i o n e d c h a n g e in H R as a c o n s e q u e n c e o f C S - U S pairing. 18-day-old. A typical p a t t e r n o f c o n d i t i o n e d b r a d y c a r d i a a p p e a r s for t h e first t i m e in this age group. T h e a v e r a g e r e s p o n s e o f all t h e a n i m a l s is shown in Fig. 1. In t h r e e o f t h e s e r a b b i t s t h e t i m e course, as well as t h e a m p l i t u d e o f t h e r e s p o n s e , was comp a r a b l e to t h a t typical o f a d u l t animals. In contrast, t h e a m p l i t u d e o f t h e b r a d y c a r d i c r e s p o n s e e x h i b i t e d by t h e o t h e r t h r e e n e o n a t e s was s m a l l e r t h a n t h a t of t h e adults. O u t o f eight animals, only two d i d n o t d e v e l o p c o n d i t i o n e d b r a d y c a r d i a . A d e t a i l e d p i c t u r e o f t h e d i f f e r e n t r e s p o n s e types is shown in Fig. 2.
Table 2 Comparison between the heart rate (HR) responses developed, during the acquisition session (B1 to B9), by three 10-day-old neonates with unstable baseline HR and those exhibited by adult animals. Data represent mean HR changes from pre-CS baseline recorded during the CS-US paired presentations. Each number represents the mean value of five trials blocks Name
B1
B2
B3
B4
B5
B6
B7
B8
B9
nl0b nl0c nl0f Adult
+ 22.8 + 11.4 -50.8 - 9.5
+ 3.2 +4.9 -20.9 - 12.6
+ 6.6 +8.9 - 10.6 - 14.9
- 1.6 + 1.4 -7.5 - 14.1
- 13.4 -0.8 - 12 - 11.8
- 17.7 -0.7 -8 - 8.4
- 0.5 +3.5 -4.8 - 8.9
- 0.2 +2 - 10.1 - 9.3
- 0.5 +0.9 +0.6 - 6.0
236
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238 CS-US
tent movements of the animals following the electric shock.
paired
A
B
m o 221
0
4. Discussion
E © ~_
-5
cJ~ c (3 zz o ~f 3=
-10
-15
c (3 ~3
E
-20 0
I
l
i
5
10
15
210
5 Trials
I
I
t
t
J
25
30
35
40
45
Blocks
Fig. 2. Detailed representation of mean HR changes from pre-CS baseline recorded during the CS-US paired presentations in 18-day-old neonates. The curves depict conditioned responses comparable (zx n = 3) and smaller ( • n = 3) than those exhibited by adults ( • n = 8). Two animals showed no response (~ n = 2).
Statistical analysis
A repeated analysis of variance performed on data from all the 5 experimental groups yielded highly significant G r o u p and Trial effects ( F (4,32) = 13.658, P < 0.0001 and F(8,256) = 3.474, P < 0.001, respectively) and a significant GroupsxTrials interaction (F(32,256) = 1.478, P = 0.05). In addition, post-hoc Newman-Keuls test revealed that values obtained from all neonatal groups significantly differed from those obtained in adults ( P < 0.01). H R response to US. In adult animals as well as in all the neonatal groups older than 10 days, the H R response to the US was a tachycardia. In contrast, most of the 10-day-old rabbits exhibited either bradycardia or no changes in HR. However, for all the neonates and especially for 10day-old infants, it was difficult to have reliable measures of the unconditioned response (UR) across the whole acquisition session due to persis-
The major finding of the present research is that classically conditioned bradycardia elicited in rabbits by pairing a tone (CS) to an electric shock (US) does not emerge until the 18th postnatal day. This result is quite surprising, since sensory systems (auditory and nociceptive) necessary for conditioned bradycardia are mature before this age [21]. Moreover, conditioned somatomotor defence reflexes in response to an acoustic stimulus are known to be present, in the rabbit, between the 10th and 13th postnatal day [31]. These data indicate that rabbits are able to perform complex associations between stimuli at early stages of development and that the failure of neonates to show H R C R cannot be ascribed to a general inability of neonatal rabbits to learn. In addition, the presence of a marked bradycardia following the US observed in our experiments in some of the neonatal rabbits, suggests that the lack of H R conditioning cannot be due to an overall inability to decelerate the heart. This is in agreement with the findings reported by several authors which indicate that the baroreceptor reflex pathways are already functional at early stages of development (see, for review, Ref. 8). The present results also confirm the existence of a marked dissociation between the time of emergence of somatomotor and autonomic conditioned responses, as previously described in other species (rats, pygmy goats) by various authors [6,9,24]. Also the H R orientation reflexes exhibited a delay which parallels the dissociation observed between the somatomotor and H R conditioned responses. In fact, no signs of orientation could be found in the animals grouped between the 1st and 10th postnatal day. Moreover, the somatomotor reflexes typically shown by adult rabbits in response to an acoustic novel stimulus, were present starting from the 10th postnatal day. This is in agreement with the results of previous studies performed in the rabbit, according to which very primitive orienting reflexes to sounds (startling, standing still) appear around the 10th postnatal day and the
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
first orienting-exploring reflexes in response to an acoustic stimulus (turning of the head, movements of the ears, fixing of eyes) occur between the 12-15th postnatal day [10,31]. The first reliable signs of a HR orienting response appeared around the 10th day of life. Starting from this age all the groups of neonates tested showed very complex patterns of response (no response, bradycardia, tachycardia, biphasic responses, bradycardia followed by tachycardia) (see Table 1). In spite of this variability, it was observed that a HR decelerating response, comparable to that typical of the adult, became more evident in the older neonates (10-day°old: 12.5%; 12-day-old: 12.5%; 15-day-old: 50% and 18-dayold: 75%). As a whole, these observations not only indicate that the emergence of the typical bradycardic orienting response occurs rather late during development but, also, that the trend of this response towards bradycardia in the neonates parallels their advance to maturity. In all the neonates, baseline HR was significantly higher than that measured in adults. This result is consistent with what has been described in rats [6] and pygmy goats [9] and attributed to a higher overall level of resting sympathetic tone in very young kids. As suggested for rat pups by Hofer [13], this increase in background sympathetic tone could represent an early phase characterized by a sympathetic dominance in the development of the control of baseline HR. The presence of a high sympathetic tone during the first postnatal weeks may also explain why the magnitude of the HR conditioned bradycardia developed by 18-day-old rabbits was significantly reduced when compared to that shown by adult animals. The immaturity of the systems controlling HR is also suggested by the instability of the baseline HR shown by the 10-day-old rabbits during conditioning. While in adults and in most of the older neonatal rabbits baseline HR remained stable across the whole acquisition session, in 3 out of 8 animals of the 10-day-old group, baseline HR dropped to very low values following the first CS-US paired presentations (Table 2). In conclusion, these studies indicate that the processes mediating specific HR responses to
237
certain types of enviromental stimulation are not present at birth and require several days to mature. Since the sensorimotor systems specifically involved in the expression of the aversive responses are known to be functional early in development, this delay could be ascribed to incomplete maturation of CNS regions involved in associative processes. Among these structures, a significant role may be exerted by the cerebellar vermis which has been identified as one of the brain structures that control classically conditioned bradycardia [27,30]; in addition, its maturation is not complete at birth [1-3,19]. Future studies will be aimed at investigating the possible relation between the appearance of conditioned bradycardia and maturation of the cerebellar vermis.
Acknowledgements The excellent technical assistance of G. Bresciani, whose constant care to neonatal rabbits in the animal colony made this work possible, is gratefully acknowledged. This work has been supported by a grant from the Italian Ministry of University and Scientific Research.
References [1] Altman, J. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol., 145 (1972) 353-398. [2] Altman, J. Postnatal development of the cerebellar cortex in the rat. II. Phases of maturation of Purkinje cells and the molecular layer. J. Comp. Neurol., 145 (1972) 399-464. [3] Altman, J. Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol., 145 (1972) 465-514. [4] Amsel, A., Burdette, D.R. and Letz, R. Appetitive learning, patterned alternation, and extinction in 10-d-o rats with non-lactating suckling as reward, Nature, 262 (1976) 816-818. [5] Ronca, A.E. and Bernston, G.G. Development of classically conditioned heart rate patterns to tactile stimuli iia rats. Abstr. International Society for developmental psychobiology, Dallas, TX, 1985, p. 68.
23~
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
[6] Campbell, B.A. and Ampuero, M.X. Dissociation of autonomic and behavioral components of conditioned fear during development in the rat, Behav. Neurosci., 99 (1985) 1089-1102. [7] de Toledo, L. and Black, A.H. Heart rate: changes during conditioned suppression in rats. Science, 152 (1966) 1404-1406. [8] Downing, S.E. Baroreceptor regulation of the heart. In: Berne R.M., Sperelakis, N. and Geiger, S.R. (Eds.) Handbook of Physiology. Section 2: The cardiovascular System. Vol. I. The heart. American Physiological Society, Bethesda, Maryland, 1979, pp. 621-652. [9] Fitzgerald, R.D., Francisco, D.L., Metcalfe, J. and Lawson, M.S. Classically conditioned heart rate and respiratory-motor activity in newborn and neonatal pygmy goats, Anim. Learn. Behav., 12 (1984) 217-222. [10] Foss, I. and Flottorp, G. A comparative study of the development of hearing and vision in various species commonly used in experiments. Acta Otolaryngol. 77 (1974) 202-214. [11] Gemberling, G.A., Domjan, M. and Amsel, A. Aversive learning in 5-day-old rats: taste-toxicosis and textureshock associations, J. Comp. Physiol. Psychol., 94 (1980) 734-745. [12] Ghelarducci, B., Salamone, D., Silvestri, P., Simoni, A. and Sebastiani, L. Development of fear-related heart rate responses in neonatal rabbits. Proc. Physiol. Soc., C126 (1993) 124-125P. [13] Hofer, M.A. The role of early experience in the developmerit of autonomic regulation. In Di Cara L.V. (Ed.), Limbic and autonomic nervous systems research, New York: Plenum Press, 1974, pp. 195-221. [14] Johanson, I.B. and Hall, W.G. Appetitive learning in 1-day-old rat pups, Science, 205 (1979) 419-421. [15] Kapp, B.S., Frysinger, R.C., Gallagher, M. and Haselton, J.R. Amygdala central nucleus lesions: effect on heart rate conditioning in the rabbit, Physiol. Behav., 23 (1979) 1109-1117.
[16] Kapp, B.S., Gallagher, M., Applegate, C.D. and Frysinger, R.C. The amygdala central nucleus: contribution to conditioned cardiovascular responding during aversive Pavlovian conditioning in the rabbit, In: Woody, C.D. (Ed.) Conditioning: representation of involved neural function, Plenum Press, New York, 1982. [17] Kazis, E., Mitligann, W.L. and Powell, D.A. Autonomicsomatic relationships: blockade of heart rate and corneo-retinal potential responses, J. Comp. Physiol. Psychol., 84 (1978) 98-110. [18] Kenny, J.T. and Blass, E.M. Suckling as incentive to instrumental learning in pre-weanling rats, Science, 196 (1977) 898-899.
[19] Larsell, O. The comparative anatomy and histology of the cerebellum from monotremes through apes. Vol 2. Jansen J. (Ed.) University of Minnesota Press, Minneapolis, 1970. [20] Lavond, D.G., Lincoln, J.S., Mc Cormick, D.A. and Thompson, R.F. Effect of bilateral lesions of the dentate and interpositus cerebellar nuclei on conditioning of heart-rate and nictitating membrane/eyelid responses in the rabbit, Brain Res., 305 (1984) 323-330. [21] Rubel, E.W. Ontogeny of Structure and Function in Vertebrate Auditory System. In: Jacobson M. (Ed) Handbook of Sensory Physiology. Vol IX. Development of sensory systems. Springer, Berlin, 1978, pp. 135-235. [22] Rudy, J.W. and Cheatle, M.D. Odor-aversion learning in neonatal rats, Science, 198 (1977) 845-846. [23] Rudy, J.W., Vogdt, M.B. and Hyson, R.L. A developmental analysis of the rat's learned reactions to gustatory and auditory stimulation, In R. Kail and N.E. Spear (Eds.) Comparative perspective of memory development, Hillsdale, NJ: Erlbaum, 1983, pp. 181-208. [24] Sananes, C.B., Gaddy, J.R. and Campbell, B.A. Analysis of the maturation of the heart rate conditioned response in infant rats, using an olfactory stimulus, Abstr. International Society for developmental psychobiology, Dallas, TX, 1985, p. 72. [25] Schneiderman, N., Smith, M., Smith, A. and Gormezano, 1. Heart rate classically conditioning in rabbits, Psychon. Sci., 6 (1966) 241-242. [26] Schneiderman, N. Response system divergences in aversive classical conditioning. In: Black, A.H., Prokasky, W.C. (Eds.) Classical conditioning, Appleton-CenturyCrofts, New York, 1972. [27] Sebastiani, L., La Noce, A., Paton, J.F.R. and Ghelarducci, B. Influence of the cerebellar posterior vermis on the acquisition of the classically conditioned bradycardic response in the rabbit, Exp. Brain Res., 88 (1992) 193198. [28] Smotherman, W.P., Odor aversion learning by the rat fetus, Physiol. Behav., 29 (1982) 769-771. [29] Stickrod, G., Kimble, D.P. and Smotherman, W.P. In utero taste/odor aversion conditioning in the rat. Physiol. Behav., 28 (1982) 5-7. [30] Supple, W.F. Jr., Sebastiani, L. and Kapp, B.S. Purkinje cells responses in the cerebellar vermis during Pavlovian differential fear conditioning in rabbits. NeuroReport, 4 (1993) 975-978. [31] Volokhov, A.A. Comparative studies of the functional development of analyzer systems in animals in the process of ontogenesis. Progr. Brain Res. 22 (1968) 527-540.
Journal of the
ELSEVIER
Autonomic Nervous System Journal of the Autonomic Nervous System 50 (1994) 231-238
Development of fear-related heart rate responses in neonatal rabbits Laura Sebastiani, Domenico Salamone, Pasquale Silvestri, Alfredo Simoni, Brunello Ghelarducci * Dipartimento di Fisiologia e Biochimica, Universit?t di Pisa, Via S. Zeno 29/31, 56127 Pisa, Italy
Received 21 January 1994; revision received 16 March 1994; accepted 30 March 1994
Abstract
Classical simple conditioning of heart rate (HR) was studied in rabbits between the 1st and 18th neonatal day. An auditory stimulus (1000 Hz, 5 s) served as the conditioned stimulus (CS), and a train of electric impulses (100 Hz, 500 ms, 1-1.5 mA) was used as the unconditioned stimulus (US). HR responses developed during orientation session (CS-alone) as well during acquisition (CS-US paired) were analyzed and compared to those developed by young adult rabbits (3-month-old). In all neonatal animals tested, baseline HR measured during an adaptation session preceding conditioning, was similar though significantly higher than that measured in adult rabbits (Newman-Keuls P < 0.05). Before the 10th neonatal day, the animals did not show either somatomotor or H R orienting responses to the CS-alone presentations. Consequently, since orienting reactions play a necessary role in the formation and manifestation of conditioned reflexes, 1 to 10-day-old infant rabbits were not submitted to the acquisition session. All the other neonatal groups, while showing orienting behaviours similar to those exhibited by adults (head and pinna movement), presented different patterns of H R orienting responses (no response, bradycardia, tachycardia, bradycardia/tachycardia etc.). As for the acquisition session, the first bradycardic response, similar to that developed by adult rabbits, was found in 18-day-old rabbits. However, also in this neonatal group the amplitude of the conditioned response was significantly smaller when compared to that exhibited by young adults (Newman-Keuls P < 0.01). In addition, in some of the 10-day-old neonates, H R appeared very unstable and dropped to very low values (as low as 146 b e a t s / m i n ) early during conditioning, apparently as a consequence of CS-US association. As for the unconditioned response, no differences were found between adult rabbits and the neonatal animals older than 12 days. In contrast, most of the 10-day-old rabbits showed either bradycardia or no response to the unconditioned stimulus. Considering the ability of mammalian infants to learn somatomotor conditioned responses at early stages of maturation, conditioning of H R responses occurs late during ontogeny. Since this incapacity to show H R conditioned responses before the 18th postnatal day cannot be ascribed to their inability to show phasic H R changes nor to a failure in detecting the auditory stimulus, these results suggest the possibility that H R conditioned responses may be mediated by neural structures developing later during maturation. Keywords: Heart rate response; Aversive conditioning; Rabbit; Neonate
* Corresponding author. Tel." (39-50) 554074; Fax: (39-50) 552183. 0165-1838/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0165-1838(94)00090-7
232
L. Sebastiani et ak /Journal of the Autonomic Nervous System 50 (1994) 231-238
I. Introduction
It is well known that neonatal mammals are able to perform relatively complex associations [4,14,18,22] in spite of the immaturity of their sensory and motor systems. However, the emergence of classically conditioned motor and heartrate responses follows the sequential development of the sensory mechanisms (olfactory, tactile, auditory, visual) involved as conditioned stimuli [5,6,11,22,28,29]. Aside from the complete maturation of the appropriate sensory and motor systems, a conditioned response also requires the complete development of the central nervous structures involved in the association process [6]. A striking observation from these previous studies is that autonomic conditioned responses develop considerably later in ontogeny when compared with motor conditioning, and that this delay is evident for all the sensory modalities used as conditioned stimulus (CS) [6,7,9,23,24]. In rats, for example, while motor conditioning to an olfactory stimulus emerges within the first days of life, conditioned HR responses using the same olfactory modality are not present until the 15th postnatal day [24]. Though rabbits have been widely used in behavioral and neurophysiological studies concerning fear-related responses and their neural substrates [16,20,25,27,30], at present little is known about the maturation of fear-related heart rate orienting and conditioned responses in infant rabbits. In contrast, much is known of the development of somatomotor responses (pinna orientation, head movement etc.) which are components of the orienting response to an acoustic novel stimulus in adult rabbits [31]. The main purpose of this study is to determine the age at which both orienting and classically conditioned HR responses to an auditory stimulus emerge in infant rabbits. This study represents an essential step in determining the influence exerted on the development of the HR conditioned responses by those neural structures which are known to modulate this response in the adult.
A preliminary report of these data was presented at the Meeting of the Physiological Society, King's College, London, December 1993 [12].
2. Materials and methods
2.1. Animals and experimental groups Forty-eight New Zealand white neonatal rabbits, bred and reared in the animal colony of our Department were used for the study. At the start of the conditioning procedure rabbits were divided in 5 groups of different ages: (1-9)-day-old (n = 8); 10-day-old (n --- 8); 12-day-old (n = 8); 15-day-old (n = 8) and 18-day-old (n = 8). For the entire duration of the experiment they were housed with their mothers and with other rabbits of the same age and had free access to water and food. An additional group of eight adult rabbits (3month-old) weighing 2.5-3.0 kg were used. These animals belonged to the same breed as the neonatal groups. All neonates and adult animals were submitted to a simple conditioning protocol [27]
2.2. Apparatus and conditioning procedure All neonatal animals were tested while restrained in a full-body sling attached to the ceiling of a sound-attenuating chamber (50 x 45 × 40 cm), diffusely illuminated by a 20-W lamp. The chamber's ventilation system also provided the background noise (60 dB). As for adult rabbits they were placed inside the sound-attenuating chamber, held in a plexiglass restrainer (Tecniplast), which limited both body and head movements. Before behavioral testing, in all animals fine stainless steel wire loops were inserted subcutaneously at the precordial level to allow for ECG recording. Two similar wire loops chronically inserted in the border of the right pinna served as stimulating electrodes for delivering the unconditioned stimulus (US), a 500-ms train of 0.5-ms pulses at 100 Hz, with an intensity of
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
1-1.5 mA provided by a constant current stimulator. The conditioned stimulus (CS) consisted of a continuous 1000-Hz tone (5 s, 90 dB) and was presented to the rabbit through a loudspeaker mounted on the ceiling of the chamber about 25 cm above the head of the animal. A Commodore 64 computer was programmed to control the characteristics and the presentation sequence of the stimuli (CS and US). Before starting the conditioning procedure all the animals were handled for at least a 1-hour session. Successively, each animal received a 1hour adaptation session on each of 4 consecutive days, in order to adapt to the test enviroment. For the group of animals tested before the 5th day of life this procedure was shortened. Following this procedure, all animals were given 10 min of adaptation during which heart rate (HR) was sampled at 1-min intervals. Next, 15 consecutives unreinforced 1000-Hz tones (CS-alone) were presented at pseudorandom intertrial intervals (5565 s; mean = 60 s). This was to habituate the heart rate orienting response to a novel stimulus commonly observed in adult rabbits. Simple conditioning started immediately thereafter and consisted of 45 trials in which CS was paired to US at a 60 s ITI. The offset of CS coincided with US onset.
2.3. Heart rate analysis On-line acquisition of ECG, as well as subsequent off-line measurement and analysis of H R responses were performed by means of a computer system (Microsys Electronics 386). HR changes to each CS-alone and CS-US paired presentation were calculated as the difference between the average H R (beats/min) during the 5-s CS and that during the 5-s pre-CS baseline period. For successive analysis differences were grouped in blocks of 5 trials. The HR unconditioned response to pinna shock was calculated by comparing the 5-s period following US offset to the baseline H R measured in the 5-s period preceding US onset. A repeated measures analysis of variance (ANOVA) was performed on the difference scores. Post-hoc Newman-Keuls comparisons as
233
well as two-tailed paired t-test between means were performed when appropriate. Differences were considered significant at P < 0.05.
3. Results
3.1. Adaptation In all the neonatal animals tested, baseline HR measured at the end of the adaptation session was similar (means ± SD: 297.08 + 38.83, (19)-day-old; 320.46+24.86, 10-day-old; 291.51 ± 48.64, 12-day-old; 302.16 + 29.58, 15-day-old; 321.98 + 28.86, 18-day-old). However, in all groups, baseline HR was higher than that measured in adult rabbits (249.73 ± 27.41). Indeed, ANOVA performed on these data yelded a significant group effect (F(5,42) = 5.394, P < 0.001) and post-hoc Newman-Keuls test revealed that all neonatal groups significantly differed from adult animals (10 and 18-day-old P < 0.01; (1-9),12,15day-old P < 0.05).
3.2. Orientation We considered that a H R orienting response consisted of any change in H R (tachycardia or bradycardia) significantly different from baseline, shown in response to the first CS-alone presentation. In adult rabbits this response, usually a bradycardia that habituated over a few trials, was accompanied by typical somatomotor orienting reponses (pinna orientation, sudden head and body movements, pupillary dilatation). However, a great variability in the magnitude and duration of this response was seen in normal adult rabbits.
Adults All adult animals showed H R and behavioral orienting responses similar to those found in previous studies [15,17,25,26,27]. In particular, the heart rate response to the CS-alone presentation consisted of an initial deceleration which soon habituated over the 15 trials. In all the animals tested the highest HR responses occurred during the first block of 5 CS-alone presentations ( - 14.3 + 5.4 beats/min: mean + SE).
234
L. Sebastiani et aL / Journal of the Autonomic Nervous System 50 (1994) 231-238
Neonatal animals During habituation all the animals, with the exception of the (1-9)-day-old neonates, showed marked somatomotor orienting responses at the onset of the novel stimulus (tone) which were similar to those described for the adult rabbit. However, at difference with adult rabbits in which these responses are typically accompanied by a bradycardia, neonatal rabbits exhibited different patterns of H R changes not only for the different age groups but also within a single group. Statistical analysis performed on these responses did not yield any significant result thus preventing any conclusion on the existence of a preferred pattern related to a particular age group. However, when the prevailing pattern of response was considered in each group, as described below and summarized in Table 1, it was possible to note a systematic change of the prevailing pattern of response in relation to age. (1-9)-day-old rabbits. None of these animals showed any somatomotor orienting response (sudden movements of the head a n d / o r the body, orientation of the pinna, etc.) or any change in HR in response to CS. Therefore, they were not submitted to the conditioning testing. Indeed, since the orienting reaction plays an essential role in the formation and manifestation of the conditioned reflex, its appearance was considered a necessary prerequisite before proceeding to the conditioning session. lO-day-old rabbits. As indicated in Table 1, most of the neonates (n = 5) did not show any H R orienting response, while of the remaining three animals one exhibited a bradycardic re-
sponse comparable to that found in adult rabbits, another an initial tachycardia followed by bradycardia, and the last one a small tachycardic response. 12 and 15-day-old rabbits. Both groups of neonates showed very different patterns of H R orienting responses at the onset of the tone. As shown in Table 1, half of the 12-day-old animals (n = 4 ) showed no responses to the CS-alone presentation, while half of the 15-day-old neonates exhibited bradycardia and only three of this group of animals did not show any H R orienting response. 18-day-old rabbits. In most of the animals (n = 6) behavioral responses were accompanied by bradycardia. Out of six animals only one did not show any H R response and another one exhibited a small tachycardia at the onset of the CS (Table 1).
3.3. Acquisition Baseline H R during conditioning. The mean pre-CS H R values for all the groups submitted to the acquisition session were analyzed during conditioning. No changes in baseline H R were observed as a consequence of training. In fact, with the exception of three 10-day-old rabbits whose H R appeared very unstable and dropped to very low values (down to 146 b e a t s / m i n ) during conditioning, for all the remaining groups baseline H R calculated during the last block of acquisition trials did not differ significantly from that measured at the end of the adaptation session (paired t-test). In addition, A N O V A performed on base-
Table 1 Somatomotor and heart rate responses exhibited by neonatal rabbits between the 1st and 18th postnatal day. The number of neonates showing a specific orienting response is expressed as percentage of the total number of animals belongingto the same age group Somatomotor 1-9 days 10 days 12 days 15 days 18 days and heart rate (n = 8) (n = 8) (n = 8) (n = 8) (n = 8) orienting responses Pinna orientation none 100% 100% 100% 100% Head movement none 100% 100% 100% 100% No heart rate changes 100% 62.5% 50.0% 37.5% 12.5% Slight tachycardia or biphasic response none 25.0% 37.5% 12.5% 12.5% Bradycardia none 12.5% 12.5% 50.0% 75.0%
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
trials a n d t h e n showing a g r a d u a l a d a p t a t i o n tow a r d s b a s e l i n e values.
CS-US paired 5
Q) c
c~ c~
0
E 0 L ~--
--5
r, r~ 0
--10
,
n~ "1C 0
--15
E -20
I 5
i 10
i 15
i 20
i 25
0a 3
235
315
i 40
45
5 Trials Blocks
Fig. 1. Mean HR changes from pre-CS baseline recorded during the CS-US paired presentations in rabbit of different postnatal ages (e 10 days, v 12 days, • 15days, [] 18 days and • adult). Data points represent means for five trials blocks. line v a l u e s c a l c u l a t e d across t h e 45 acquisition trials d i d n o t yield any significant trial effect, thus i n d i c a t i n g t h a t in all t h e e x p e r i m e n t a l groups, b a s e l i n e H R was stable t h r o u g h o u t t h e c o n d i t i o n ing session. Heart rate conditioning. Fig. 1 shows t h e H R r e s p o n s e s e x h i b i t e d by t h e five e x p e r i m e n t a l g r o u p s d u r i n g t h e acquisition session.
Adult A d u l t a n i m a l s s h o w e d a typical c o n d i t i o n e d b r a d y c a r d i a c o m p a r a b l e to t h a t p r e v i o u s l y d e s c r i b e d by o t h e r a u t h o r s [15,17,25-27]. I n p a r t i c u lar, b r a d y c a r d i a d e v e l o p e d within t h e first 15 trials, r e a c h i n g its p e a k d u r i n g t h e 3rd b l o c k o f 5
Neonatal animals lO-day-old. A s m e n t i o n e d above, in 3 o f t h e a n i m a l s s t u d i e d , b a s e l i n e H R was very u n s t a b l e d u r i n g c o n d i t i o n i n g . Such f l u c t u a t i o n s r e s u l t e d in a s e p a r a t i o n o f d a t a f r o m t h e s e a n i m a l s which was n o t i n c l u d e d in s u m m a r y d a t a . A s shown in T a b l e 2, t h e s e 10-day-old n e o n a t e s s h o w e d e i t h e r t a c h y c a r d i a o r b r a d y c a r d i a . H o w e v e r , in c o n t r a s t to n o r m a l a d u l t r a b b i t s in which b i g g e r H R c h a n g e s a r e typically shown a r o u n d t h e third b l o c k o f trials, t h e a m p l i t u d e o f the r e s p o n s e s e x h i b i t e d by t h e s e t h r e e a n i m a l s was l a r g e r in t h e first b l o c k of C S - U S p a i r e d p r e s e n t a t i o n s , a n d successively d e c r e a s e d , or even r e v e r s e d in sign d u r i n g t h e c o n d i t i o n i n g session. A s for the rem a i n i n g animals, t h e y d i d n o t d e v e l o p any condit i o n e d H R c h a n g e as a c o n s e q u e n c e o f C S - U S a s s o c i a t i o n (see Fig. 1). 12 and 15-day-old. D u r i n g acquisition sessions r a b b i t s d i d not d e v e l o p any c o n d i t i o n e d c h a n g e in H R as a c o n s e q u e n c e o f C S - U S pairing. 18-day-old. A typical p a t t e r n o f c o n d i t i o n e d b r a d y c a r d i a a p p e a r s for t h e first t i m e in this age group. T h e a v e r a g e r e s p o n s e o f all t h e a n i m a l s is shown in Fig. 1. In t h r e e o f t h e s e r a b b i t s t h e t i m e course, as well as t h e a m p l i t u d e o f t h e r e s p o n s e , was comp a r a b l e to t h a t typical o f a d u l t animals. In contrast, t h e a m p l i t u d e o f t h e b r a d y c a r d i c r e s p o n s e e x h i b i t e d by t h e o t h e r t h r e e n e o n a t e s was s m a l l e r t h a n t h a t of t h e adults. O u t o f eight animals, only two d i d n o t d e v e l o p c o n d i t i o n e d b r a d y c a r d i a . A d e t a i l e d p i c t u r e o f t h e d i f f e r e n t r e s p o n s e types is shown in Fig. 2.
Table 2 Comparison between the heart rate (HR) responses developed, during the acquisition session (B1 to B9), by three 10-day-old neonates with unstable baseline HR and those exhibited by adult animals. Data represent mean HR changes from pre-CS baseline recorded during the CS-US paired presentations. Each number represents the mean value of five trials blocks Name
B1
B2
B3
B4
B5
B6
B7
B8
B9
nl0b nl0c nl0f Adult
+ 22.8 + 11.4 -50.8 - 9.5
+ 3.2 +4.9 -20.9 - 12.6
+ 6.6 +8.9 - 10.6 - 14.9
- 1.6 + 1.4 -7.5 - 14.1
- 13.4 -0.8 - 12 - 11.8
- 17.7 -0.7 -8 - 8.4
- 0.5 +3.5 -4.8 - 8.9
- 0.2 +2 - 10.1 - 9.3
- 0.5 +0.9 +0.6 - 6.0
236
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238 CS-US
tent movements of the animals following the electric shock.
paired
A
B
m o 221
0
4. Discussion
E © ~_
-5
cJ~ c (3 zz o ~f 3=
-10
-15
c (3 ~3
E
-20 0
I
l
i
5
10
15
210
5 Trials
I
I
t
t
J
25
30
35
40
45
Blocks
Fig. 2. Detailed representation of mean HR changes from pre-CS baseline recorded during the CS-US paired presentations in 18-day-old neonates. The curves depict conditioned responses comparable (zx n = 3) and smaller ( • n = 3) than those exhibited by adults ( • n = 8). Two animals showed no response (~ n = 2).
Statistical analysis
A repeated analysis of variance performed on data from all the 5 experimental groups yielded highly significant G r o u p and Trial effects ( F (4,32) = 13.658, P < 0.0001 and F(8,256) = 3.474, P < 0.001, respectively) and a significant GroupsxTrials interaction (F(32,256) = 1.478, P = 0.05). In addition, post-hoc Newman-Keuls test revealed that values obtained from all neonatal groups significantly differed from those obtained in adults ( P < 0.01). H R response to US. In adult animals as well as in all the neonatal groups older than 10 days, the H R response to the US was a tachycardia. In contrast, most of the 10-day-old rabbits exhibited either bradycardia or no changes in HR. However, for all the neonates and especially for 10day-old infants, it was difficult to have reliable measures of the unconditioned response (UR) across the whole acquisition session due to persis-
The major finding of the present research is that classically conditioned bradycardia elicited in rabbits by pairing a tone (CS) to an electric shock (US) does not emerge until the 18th postnatal day. This result is quite surprising, since sensory systems (auditory and nociceptive) necessary for conditioned bradycardia are mature before this age [21]. Moreover, conditioned somatomotor defence reflexes in response to an acoustic stimulus are known to be present, in the rabbit, between the 10th and 13th postnatal day [31]. These data indicate that rabbits are able to perform complex associations between stimuli at early stages of development and that the failure of neonates to show H R C R cannot be ascribed to a general inability of neonatal rabbits to learn. In addition, the presence of a marked bradycardia following the US observed in our experiments in some of the neonatal rabbits, suggests that the lack of H R conditioning cannot be due to an overall inability to decelerate the heart. This is in agreement with the findings reported by several authors which indicate that the baroreceptor reflex pathways are already functional at early stages of development (see, for review, Ref. 8). The present results also confirm the existence of a marked dissociation between the time of emergence of somatomotor and autonomic conditioned responses, as previously described in other species (rats, pygmy goats) by various authors [6,9,24]. Also the H R orientation reflexes exhibited a delay which parallels the dissociation observed between the somatomotor and H R conditioned responses. In fact, no signs of orientation could be found in the animals grouped between the 1st and 10th postnatal day. Moreover, the somatomotor reflexes typically shown by adult rabbits in response to an acoustic novel stimulus, were present starting from the 10th postnatal day. This is in agreement with the results of previous studies performed in the rabbit, according to which very primitive orienting reflexes to sounds (startling, standing still) appear around the 10th postnatal day and the
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
first orienting-exploring reflexes in response to an acoustic stimulus (turning of the head, movements of the ears, fixing of eyes) occur between the 12-15th postnatal day [10,31]. The first reliable signs of a HR orienting response appeared around the 10th day of life. Starting from this age all the groups of neonates tested showed very complex patterns of response (no response, bradycardia, tachycardia, biphasic responses, bradycardia followed by tachycardia) (see Table 1). In spite of this variability, it was observed that a HR decelerating response, comparable to that typical of the adult, became more evident in the older neonates (10-day°old: 12.5%; 12-day-old: 12.5%; 15-day-old: 50% and 18-dayold: 75%). As a whole, these observations not only indicate that the emergence of the typical bradycardic orienting response occurs rather late during development but, also, that the trend of this response towards bradycardia in the neonates parallels their advance to maturity. In all the neonates, baseline HR was significantly higher than that measured in adults. This result is consistent with what has been described in rats [6] and pygmy goats [9] and attributed to a higher overall level of resting sympathetic tone in very young kids. As suggested for rat pups by Hofer [13], this increase in background sympathetic tone could represent an early phase characterized by a sympathetic dominance in the development of the control of baseline HR. The presence of a high sympathetic tone during the first postnatal weeks may also explain why the magnitude of the HR conditioned bradycardia developed by 18-day-old rabbits was significantly reduced when compared to that shown by adult animals. The immaturity of the systems controlling HR is also suggested by the instability of the baseline HR shown by the 10-day-old rabbits during conditioning. While in adults and in most of the older neonatal rabbits baseline HR remained stable across the whole acquisition session, in 3 out of 8 animals of the 10-day-old group, baseline HR dropped to very low values following the first CS-US paired presentations (Table 2). In conclusion, these studies indicate that the processes mediating specific HR responses to
237
certain types of enviromental stimulation are not present at birth and require several days to mature. Since the sensorimotor systems specifically involved in the expression of the aversive responses are known to be functional early in development, this delay could be ascribed to incomplete maturation of CNS regions involved in associative processes. Among these structures, a significant role may be exerted by the cerebellar vermis which has been identified as one of the brain structures that control classically conditioned bradycardia [27,30]; in addition, its maturation is not complete at birth [1-3,19]. Future studies will be aimed at investigating the possible relation between the appearance of conditioned bradycardia and maturation of the cerebellar vermis.
Acknowledgements The excellent technical assistance of G. Bresciani, whose constant care to neonatal rabbits in the animal colony made this work possible, is gratefully acknowledged. This work has been supported by a grant from the Italian Ministry of University and Scientific Research.
References [1] Altman, J. Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol., 145 (1972) 353-398. [2] Altman, J. Postnatal development of the cerebellar cortex in the rat. II. Phases of maturation of Purkinje cells and the molecular layer. J. Comp. Neurol., 145 (1972) 399-464. [3] Altman, J. Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol., 145 (1972) 465-514. [4] Amsel, A., Burdette, D.R. and Letz, R. Appetitive learning, patterned alternation, and extinction in 10-d-o rats with non-lactating suckling as reward, Nature, 262 (1976) 816-818. [5] Ronca, A.E. and Bernston, G.G. Development of classically conditioned heart rate patterns to tactile stimuli iia rats. Abstr. International Society for developmental psychobiology, Dallas, TX, 1985, p. 68.
23~
L. Sebastiani et al. /Journal of the Autonomic Nervous System 50 (1994) 231-238
[6] Campbell, B.A. and Ampuero, M.X. Dissociation of autonomic and behavioral components of conditioned fear during development in the rat, Behav. Neurosci., 99 (1985) 1089-1102. [7] de Toledo, L. and Black, A.H. Heart rate: changes during conditioned suppression in rats. Science, 152 (1966) 1404-1406. [8] Downing, S.E. Baroreceptor regulation of the heart. In: Berne R.M., Sperelakis, N. and Geiger, S.R. (Eds.) Handbook of Physiology. Section 2: The cardiovascular System. Vol. I. The heart. American Physiological Society, Bethesda, Maryland, 1979, pp. 621-652. [9] Fitzgerald, R.D., Francisco, D.L., Metcalfe, J. and Lawson, M.S. Classically conditioned heart rate and respiratory-motor activity in newborn and neonatal pygmy goats, Anim. Learn. Behav., 12 (1984) 217-222. [10] Foss, I. and Flottorp, G. A comparative study of the development of hearing and vision in various species commonly used in experiments. Acta Otolaryngol. 77 (1974) 202-214. [11] Gemberling, G.A., Domjan, M. and Amsel, A. Aversive learning in 5-day-old rats: taste-toxicosis and textureshock associations, J. Comp. Physiol. Psychol., 94 (1980) 734-745. [12] Ghelarducci, B., Salamone, D., Silvestri, P., Simoni, A. and Sebastiani, L. Development of fear-related heart rate responses in neonatal rabbits. Proc. Physiol. Soc., C126 (1993) 124-125P. [13] Hofer, M.A. The role of early experience in the developmerit of autonomic regulation. In Di Cara L.V. (Ed.), Limbic and autonomic nervous systems research, New York: Plenum Press, 1974, pp. 195-221. [14] Johanson, I.B. and Hall, W.G. Appetitive learning in 1-day-old rat pups, Science, 205 (1979) 419-421. [15] Kapp, B.S., Frysinger, R.C., Gallagher, M. and Haselton, J.R. Amygdala central nucleus lesions: effect on heart rate conditioning in the rabbit, Physiol. Behav., 23 (1979) 1109-1117.
[16] Kapp, B.S., Gallagher, M., Applegate, C.D. and Frysinger, R.C. The amygdala central nucleus: contribution to conditioned cardiovascular responding during aversive Pavlovian conditioning in the rabbit, In: Woody, C.D. (Ed.) Conditioning: representation of involved neural function, Plenum Press, New York, 1982. [17] Kazis, E., Mitligann, W.L. and Powell, D.A. Autonomicsomatic relationships: blockade of heart rate and corneo-retinal potential responses, J. Comp. Physiol. Psychol., 84 (1978) 98-110. [18] Kenny, J.T. and Blass, E.M. Suckling as incentive to instrumental learning in pre-weanling rats, Science, 196 (1977) 898-899.
[19] Larsell, O. The comparative anatomy and histology of the cerebellum from monotremes through apes. Vol 2. Jansen J. (Ed.) University of Minnesota Press, Minneapolis, 1970. [20] Lavond, D.G., Lincoln, J.S., Mc Cormick, D.A. and Thompson, R.F. Effect of bilateral lesions of the dentate and interpositus cerebellar nuclei on conditioning of heart-rate and nictitating membrane/eyelid responses in the rabbit, Brain Res., 305 (1984) 323-330. [21] Rubel, E.W. Ontogeny of Structure and Function in Vertebrate Auditory System. In: Jacobson M. (Ed) Handbook of Sensory Physiology. Vol IX. Development of sensory systems. Springer, Berlin, 1978, pp. 135-235. [22] Rudy, J.W. and Cheatle, M.D. Odor-aversion learning in neonatal rats, Science, 198 (1977) 845-846. [23] Rudy, J.W., Vogdt, M.B. and Hyson, R.L. A developmental analysis of the rat's learned reactions to gustatory and auditory stimulation, In R. Kail and N.E. Spear (Eds.) Comparative perspective of memory development, Hillsdale, NJ: Erlbaum, 1983, pp. 181-208. [24] Sananes, C.B., Gaddy, J.R. and Campbell, B.A. Analysis of the maturation of the heart rate conditioned response in infant rats, using an olfactory stimulus, Abstr. International Society for developmental psychobiology, Dallas, TX, 1985, p. 72. [25] Schneiderman, N., Smith, M., Smith, A. and Gormezano, 1. Heart rate classically conditioning in rabbits, Psychon. Sci., 6 (1966) 241-242. [26] Schneiderman, N. Response system divergences in aversive classical conditioning. In: Black, A.H., Prokasky, W.C. (Eds.) Classical conditioning, Appleton-CenturyCrofts, New York, 1972. [27] Sebastiani, L., La Noce, A., Paton, J.F.R. and Ghelarducci, B. Influence of the cerebellar posterior vermis on the acquisition of the classically conditioned bradycardic response in the rabbit, Exp. Brain Res., 88 (1992) 193198. [28] Smotherman, W.P., Odor aversion learning by the rat fetus, Physiol. Behav., 29 (1982) 769-771. [29] Stickrod, G., Kimble, D.P. and Smotherman, W.P. In utero taste/odor aversion conditioning in the rat. Physiol. Behav., 28 (1982) 5-7. [30] Supple, W.F. Jr., Sebastiani, L. and Kapp, B.S. Purkinje cells responses in the cerebellar vermis during Pavlovian differential fear conditioning in rabbits. NeuroReport, 4 (1993) 975-978. [31] Volokhov, A.A. Comparative studies of the functional development of analyzer systems in animals in the process of ontogenesis. Progr. Brain Res. 22 (1968) 527-540.