Cerebellar vermis: essential for classically conditioned bradycardia in the rat

Brain Research, 509 (1990) 17-23 Elsevier

17

BRES 15175

Cerebellar vermis: essential for classically conditioned bradycardia in the rat William F. Supple, Jr. and Robert N. Leaton Dartmouth College, Department of Psychology, Hanover, NH 03755 (U.S.A.) (Accepted 11 July 1989)

Key words: Cerebellum; Vermis; Classical conditioning; Heart-rate conditioning; Lesion; Rat

The effects of lesions of the cerebellar vermis on the acquisition of heart-rate conditioning in rats was examined. Lesions of the vermis severely attenuated the acquisition of conditioned bradycardic responses in a simple conditioning procedure in restrained rats. Importantly, the vermal lesions did not affect resting heart-rate, unconditioned heart-rate orienting responses to a tone stimulus or unconditioned heart-rate responses to the shock unconditioned stimulus. It is concluded that the cerebellar vermis is an essential component of a heart-rate conditioned response circuit in the rat. The similarities between these effects and those following manipulations of the amygdala are discussed. INTRODUCTION

cerebellar vermis on simple, non-discriminative heartrate conditioning in rats. Heart rate was recorded during

The cerebellum has been shown to contribute to a variety of complex behavioral processes 3°. The midline cerebellum, which includes the cerebellar vermal cortex and associated fastigial nuclei, has been implicated in a variety of ' e m o t i o n a l ' or 'fear-related' behaviors 5'15.

several different phases of the conditioning procedure to determine the effects of these lesions on u n c o n d i t i o n e d and conditioned heart-rate changes. Heart rate was

Vermal lesions have b e e n reported to result in taming effects in cats and monkeys 4'22 and several fear-related behaviors are a t t e n u a t e d following vermal or fastigial lesions in rats 2"26'27. Consistent with these lesion effects is the elicitation of fear-related s o m a t o m o t o r and autonomic responses by stimulation of the midline cerebellum. Freezing has b e e n elicited in rats 3 and the acoustic startle response was potentiated by stimulation of the vermis 1. A u t o n o m i c responses, similar to those evoked in fear-eliciting situations, result from midline cerebellar stimulation, for example changes in heart rate 8, blood pressure x9 and respiration rate 24. Recently, the lateral cerebellar dentate-interpositus nuclear region ~7 and associated cortex ~2 has b e e n shown to be essential for the acquisition of a classically conditioned somatomotor response, the nictitating m e m b r a n e response in the rabbit. The correspondence between the effects of lesions and stimulation, coupled with the demonstration of a cerebellar role in conditioning processes suggests that the midline cerebellum may have a functional role in the expression of a Pavlovian fear-conditioned response like conditioned bradycardia. This experiment examined the effects of lesions of the

recorded during adaptation sessions and unreinforced tone conditioned stimulus presentations to determine if the lesion affected resting heart rate, the heart-rate orienting response or its habituation. D u r i n g acquisition training the u n c o n d i t i o n e d heart-rate response following the aversive u n c o n d i t i o n e d stimulus was also assessed. M e a s u r e m e n t of these u n c o n d i t i o n e d heart-rate responses during the various phases of training will indicate if the lesion has disrupted u n c o n d i t i o n e d features of the heart-rate response which might complicate the interpretation of any observed conditioning deficit. MATERIALS AND METHODS Twenty male Long-Evans derived rats obtained from the Dartmouth College Psychology Department breeding colony served as subjects. They were individually housed, maintained on a 14:10 h light/dark cycle and allowed ad libitum access to food and water. The animals were 90 days old at the time of surgery and testing began at least 1 month following surgery. Surgery was performed with the aid of a head holder which did not require the use of ear bars 12. Surgery was performed under clean but not aseptic conditions using sodium pentobarbital (45 mg/kg, i.p.) anesthesia. Atropine sulfate (20 mg/kg, i.p.) was injected prior to surgery to reduce mucous secretions. Ten rats received aspiration lesions of the vermis. A section of skull beginning 2 mm posterior to lambda and extending 2 mm on both sides of the midline caudally to the external occipital crest was removed. The vermis was

Correspondence: W.E Supple, Jr., Department of Psychology, John Dewey Hall, The University of Vermont, Burlington, VT 05405-0134, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B,V. (Biomedical Division)

18 aspirated under visual guidance with the aid ol a dissecting microscope. The cavity created by the aspiration was loosely packed with saline-moistened Gelfoam, the wound was closed, and the rat was injected i.m. with 150,000 units of a broad range antibiotic (Combiotic, Pfizer). Ten rats served as unoperated controls. At the conclusion of behavioral testing, the lesioned rats were given a lethal dose of sodium pentobarbital and perfused intracardially with physiological saline followed by 10% buffered formalin solution. The brains were removed and stored in formalin for at least 48 h, then blocked and embedded in gelatin. The gelatinembedded brains were stored in formalin for 48 h. Frozen sections (40 ~m) were taken throughout the extent of the lesion. Every fifth section was mounted and stained with Cresyl violet. The lesions were reconstructed by projecting images of the stained sections onto atlas plates taken from Pellegrino et al. 2°. The rats were tested restrained in an inverted U-shaped clear acrylic tube (E & M Products, Model 4, 7.6 cm diameter) with guillotine-like inserts that could be adjusted to hold the rat securely. The restraining device was enclosed within a darkened 56 x 42 x 38 cm sound-attenuating chamber (Lehigh Valley Electronics). A ventilation fan provided background noise (approx. 70 dB). The chamber contained an 11.4 cm speaker mounted on the 56 cm wall. Heart-rate was recorded from two acutely implanted subcutaneous needle electrodes connected to an amplifier (E & M Model 4-A Physiograph) which produced an ink-written electrocardiogram. The tone stimulus was generated by a Hewlett-Packard Oscillator (Model 200AB) and its intensity was measured daily with a sound-level meter. The unconditioned stimulus (UCS) was produced by a Lehigh Valley Electronics Constant Current Shocker (Model 1531). The shock electrodes consisted of two 5 mm steel disks embedded in surgical rubber tubing which was secured around the rat's tail. Each rat received one 30-min adaptation session on each of 5 consecutive days, (heart-rate electrodes and tail-shock electrode attached). Heart rate was sampled at 5-rain intervals throughout each adaptation session with the first sample taken as soon as the animal was placed into the chamber. Next each rat received 2 consecutive days of 20 unreinforced CS presentations on a fixed 90-s interstimulus interval (ISI). The CS was a 6.0-s, 92-dB, 1000-Hz tone. Three consecutive days of acquisition training followed in

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which t0 paired presentations of the CS and I_J(S, a 500-ms, 1.0-mA tail shock, were presented on a variable tS0-s ISI. The first daib trial was presented 7 rain after the rat was placed into the chamber The purpose of this delay was to begin stimulus presentations after baseline heart rate had reached asymptotic levels. Heart rate appeared to reach this point approximately 7 min into the session during the adaptation phase of the experiment. Heart rate during the adaptation sessions was computed by measuring the 10 consecutive beats occurring at the interval sample point and converting this value to beats per minute. Heart-rate change was determined by comparing the 10 beats immediately preceding CS onset to the first 10 beats of the CS during the habituation phase of the experiment. During acquisition the 10 beats immediately prior to CS onset were compared to the last 10 beats during the CS. The topography of the response during the CS was determined by dividing the 6-s CS period into separate 1.0-s intervals and comparing each interval to the 1.0-s period immediately preceding CS onset. The heart-rate response to the tail shock was determined by comparing the last 10 beats during the CS with the first l0 beats after UCS offset. Repeated measures analysis of variance (ANOVA) was performed on percentage change scores which were computed using the following formula: [(Pre-CSCS)]/(Pre-CS), Post-hoe Ncuman-Keuls comparisons between means were performed where appropriate. RESULTS

Recovery Post-operative recovery of the cerebellar vermallesioned animals was typically characterized by a brief period of ataxia which completely recovered in 4-7 days.

Histology Fig. 1 presents a reconstruction of the average extent of damage to the vermis. The damage was restricted primarily to the midline cerebellum with only slight .......

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Fig. 1. Coronal reconstructions of the cerebellar vermal lesions. The shaded area represents the average extent of the damaged tissue on atlas plates from Pellegrino et al. 2°. The coordinates represent the number of millimeters posterior to bregma.

19 between groups as neither the group effect nor the groups x trials interaction was significant, Fs < 1. Habituation of the response was retained over a 24-h interval. Comparison of trial 1 on day 1 with trial 1 of day 2 resulted in a significant trials effect, FL] ~ = 17.8, P < 0.05. There were no significant group or trials effects on day 2, all Fs < 1. Acquisition. Fig. 3 presents the mean heart-rate response for each trial across the 3 acquisition sessions. The control group developed decelerative heart-rate responses while the vermal lesioned group did not. This resulted in a significant group effect, F1,18 = 20.08, P < 0.001. The magnitude of the response increased across sessions for the control group, F2j s = 5.63, P < 0.05, but not for the lesioned group, F < 1. The mean percentage changes in heart rate were: control -2.59, -4.31 and -5.34; and vermis, 0.37, 0.43 and 0.16 for each of the 3 acquisition sessions. The response developed rapidly in the control group. While there was no significant difference between the groups on trial 1 of day 1, there was by trial 2, t~s = 2.52, P < 0.02. This rapid response development in the controls prevented the overall groups x trials interaction from reaching significance. The topography of the heart-rate response over the 6.0-s CS for each acquisition session is presented in Fig. 4. The overall A N O V A resulted in significant second x lesion, Fs,90 = 2.34, P < 0.05, and second x lesion x day, F~0j80 = 2.45, P < 0.05, interactions. These interactions reflect the development of robust bradycardic responses during the CS, especially during the latter seconds of the CS in the control group, but not in the

invasion into the paravermai region. Anterior to the primary fissure the caudalmost portion of the central lobule was damaged. The culmen, declive and tubervermis were almost completely destroyed. The lesion extended posteriorly into the rostral extent of the pyramis. The lesions extended ventrally into the underlying white matter; however, the fastigial, interpositus and dentate nuclei were intact. There was no damage to extracerebellar structures.

Heart rate Adaptation. Both groups showed a consistent pattern of heart-rate change within each of the 5 daily sessions. Heart rate was greatest on initial placement into the chamber and declined within the session. This withinsession decline resulted in significant trials effects for each group in each session, smallest F 6 ~ 5 4 = 3.86, P < 0,003. Heart rate was similar for both groups and did not change differentially over trials, as neither the group effect, F < 1, nor the groups x trials, F6,108 = 1.06, P = 0.39, or groups x days, F < 1, interactions were significant. Mean heart rates in beats per minute collapsed across days and trials were: control, 498.7 and vermal lesioned, 499.2. Habituation. Fig. 2 presents the heart-rate response to unreinforced presentations of the tone CS. The initial response was a deceleration which habituated over the initial 20 tone presentations on day 1 resulting in a significant trials effect, F19,342 = 4.61, P < 0.05. Importantly, both the magnitude of the unconditioned heart-rate response and its habituation were not different

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Heart-rate response following the tail-shock UCS. Fig. 5 presents the mean percentage change in heart rate following the 1.0-mA UCS for each trial during training. The U C R to shock was generally an acceleration. However, both groups decelerated after the initial UCS on the first paired training trial. Most importantly the heart-rate response to the UCS was not affected by the vermis lesion as neither the group effect, F1,18 = 1.65, P > 0.20, nor the groups × trials interaction, F < 1, were significant. Baseline heart rate during acquisition training. The mean pre-CS heart rate of each acquisition trial over the Acquisition

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3 days of training is presented in Fig. 6. The baseline heart rate of the vermal lesioned group remained relatively stable across training while that of the control group increased, particularly during the early trials of days 2 and 3. The group x trials interaction, F9,16 2 = 3.33, P < 0.001, was significant in the overall analysis, this effect was due to the heart rate of the controls changing significantly over trials, Fg,si --- 6.35, P < 0.001, while the vermal lesioned group did not, P > 0.15. Therefore, while baseline heart-rate was not different between the groups on day 1, as was also true in the adaptation sessions, baseline heart rate of the controls

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22 became elevated as training progressed while that of the vermal lesioned group did not. DISCUSSION This experiment is the first to show that the acquisition of simple pavlovian fear conditioned heart-rate responses is severely disrupted following large lesions of the cerebellar vermis in rats. The cerebellar vermal lesions appear to have specifically disrupted part of a conditioned response pathway as unconditioned heart-rate responses of the lesion group were not different from the control's on any measure. Initial levels of resting heartrate and the change across time during the adaptation sessions were unaffected by these lesions. Likewise the magnitude and habituation characteristics of the bradycardic heart-rate orienting response to the tone stimulus were also unaffected. These results suggest that the observed acquisition deficit was not secondary to lesioninduced auditory impairments or primary disruption of the ability to decelerate the heart since bradycardia in response to the novel tones is an excellent indicator that the rat heard the tone and can unconditionally decelerate the heart in this situation. Furthermore, these lesions did not disrupt heart-rate responses to the aversive UCS as responses following tail shock were not different from control responses. This finding suggests that the lesions did not disrupt sensory reception of the UCS which is in agreement with an earlier study finding no effect on nociceptive responses following similar vermal lesions 21. Therefore, there is a behavioral dissociation, vis-a-vis heart rate, with respect to the effects of vermal lesions, Unconditioned heart-rate responses are intact, thus indicating no primary sensory or motor impairments which could ultimately interfere with the acquisition and/or expression of the conditioned response. Conditioned heartrate responses, on the other hand, were severely disrupted by vermal lesions. These animals showed a severely attenuated conditioned bradycardic response magnitude to the CS and the topography of the response did not resemble that of the control's. Overall, these findings indicate that the cerebellar vermis should be considered a component of a neural circuit which is importantly involved in classically conditioned bradycardia in the rat. The baseline heart rate of the controls became elevated during acquisition training. This effect appeared to be a conditioned response as baseline heart rate was not elevated on the first day of training but increased across sessions. This response may have been a generalized conditioned fear response which was elicited by the contextual cues associated with the UCS. This effect on baseline heart rate is similar to that reported in several other studies involving rats ~8"2~. It is interesting that the

cerebellar vermis lesions disrupted this elevation in baseline heart rate as well as conditioned bradycardia during the CS. These results suggest that a rat with a lesion of the vermis may be unable to demonstrate any conditioned change in heart rate whether it be to the context or in response to a discrete CS associated with shock. These changes in baseline heart rate could also influence the direction and appearance of the conditioned response in the control animals. Elevated baselines may bias toward bradycardic responses. In a subsequent experiment we have shown that the conditioned response of the restrained control animal is not simply an artifact of an elevated baseline heart rate. The results of a differential conditioning study in which one tone, the CS+ paired with shock, and the CS-, a tone of a different frequency never paired with shock, showed that despite the elevation in baseline heart rate there was significantly more bradycardia in response to the CS+ compared to the CS -2s. Therefore, the conditioned heart-rate response of the intact animal is under associative control and the lesion of the vermis blocks the acquisition and/or expression of this conditioned response. Brain regions, other the cerebellar vermis, have previously been implicated in the acquisition of the conditioned bradycardic response and it is important to consider how the present data relate to these findings. Kapp and associates 14 have identified the amygdaloid central nucleus (ACE) as importantly involved in the conditioned bradycardic response in the rabbit. First, lesions of the ACE selectively disrupt conditioned but not unconditioned heart-rate responses; second, the A C E has anatomical connections with medullary cardioregulatory nuclei, suggesting pathways through which the A C E could influence heart rate; and lastly, ACE neurons show associative responses to a fear-conditioned CS 14. Manipulations of these two seemingly disparate brain regions, the amygdala and cerebellar vermis, exert strikingly similar effects on heart-rate conditioning suggesting that both may be components of a common, more extensive system involved in the acquisition and/or expression of this response. Both the A C E and vermis receive afferents from the hypothalamus 1~ and the lateral parabrachial nucleus m both of which have been implicated in cardiovascular regulation. In contrast to the notion that the ACE and vermis are components of a common system it could also be possible that each are components of parallel conditioned response circuits each contributing to response acquisition in an essential yet independent manner. For example, the cerebellar vermis could function to inhibit or suppress a sympathetic response elicited by the CS which might normally compete with primary parasympathetic drive mediated by the ACE.

23 A n o t h e r r e g i o n of t h e c e r e b e l l u m , t h e lateral h e m i -

g a g e distinct m e m o r y systems 29"31. T h a t b o t h r e s p o n s e

s p h e r e and r e l a t e d d e n t a t e - i n t e r p o s i t u s n u c l e u s , has b e e n

systems h a v e a c e r e b e l l a r c o m p o n e n t m a y suggest that

i m p l i c a t e d in t h e acquisition of t h e c o n d i t i o n e d nictitating

t h e y share c o m m o n f u n d a m e n t a l n e u r o p h y s i o l o g i c a i and

m e m b r a n e r e s p o n s e ( N M R ) in the rabbit. Small lesions

molecular

of the h e m i s p h e r i c a l c o r t e x o r i n t e r p o s i t u s nucleus p e r m a n e n t l y abolish the c o n d i t i o n e d N M R 17'32. It is

b e c o m i n g increasingly a p p a r e n t , t h e r e m a y be considerable a n a t o m i c a l 6,H, n e u r o c h e m i c a l 7, c y t o a r c h i t e c t o n i c 13

e x t r e m e l y i n t e r e s t i n g that t h e r e is a c e r e b e l l a r c o m p o n e n t

and f u n c t i o n a l d i f f e r e n c e s b e t w e e n the m i d l i n e versus the

to b o t h the classically c o n d i t i o n e d h e a r t - r a t e and nicti-

lateral c e r e b e l l u m , and t h e s e dissimilarities m a y p r o v i d e

tating m e m b r a n e

important

response.

The

suggestion

has b e e n

characteristics.

insight

into t h e

On

the

other

hand,

neurophysiological

as is

mecha-

m a d e t h a t t h e s e t w o r e s p o n s e s are r e p r e s e n t i v e e x a m p l e s

nisms which u n d e r l i e the u n i q u e b e h a v i o r a l f e a t u r e s of

o f s e p a r a t e b e h a v i o r a l p r o c e s s e s and s u b s e q u e n t l y en-

e a c h r e s p o n s e type.

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