ACTA NEUROBIOL. EXP. 1977, 37: 1979-190

EFFECTS OF CARDIAC VAGOTOMY ON HEART RATE CONDITIONED RESPONSES IN CATS

A. GIAVELLI, L. ASTORGA and G. SANTIBAITEZ-H.

Institute of Physiology, Humboldt University Berlin, GDR

Abstract. Normal cats were bilaterally vagotomized at cardiac level and submitted to a classical defensive conditioning using excitatory, in- hibitory and differential stimuli. That group was compared with a nor- mal one. Both groups showed an anticipatory bradycardia to the noxious excitatory stimulus and a late deceleration to the inhibitory stimuli. The experimental group presented more variable and labile responses. Basal and intertrial heart rate decreased in normal but not in operated anim- als. The autonomic regulation of the conditioned bradycardia reflex is discussed according to the hypothesis that both active and passive fear reactions are fundamentally mediated by sympathetic activity.

INTRODUCTION

Several authors have reported that in a classical defensive condition- ing the motor response is characterized by a progressive decrease of motility (13) and the cardiac response either by deceleration, or by initial acceleration, followed by a more sustained deceleration (12).

Some studies have been devoted to the exploration of the neural in- tegration of this visceral response. It has been demonstrated that a sti- mulation of septa1 regions produces conditionable decrements of the heart rate (1, ll), that ablation of the frontal pole cortex inhibits the anticipatory heart rate (HR) changes to noxious stimulation, while re- moval of the "gyrus genualis" produces a contrary effect (14). In 1969 Diaz et al. (3) demonstrated that cardiac sympathectomy produced a n unexpected effect, an abolition of conditioned bradycardia. These

authors assumed that conditioned bradycardia is a consequence of sym- pathetic inhibition rather than a result of vagal activation. This study is dedicated to investigate the effects of bilateral ablation of vagal innerva- tion a t cardiac level. On the basis of the former hypothesis, this lesion should not significantly alter the conditioned bradycardia.

METHODS

Subjects. Eleven naive male cats were divided in two groups: nor- mal group (NG) and vagotomized group (VG), consisting of 4 and 7 animals, respectively. All cats in both groups were given food and water ad lib. and were kept a t a constant temperature in a thermoregulated room.

Appuratus. The experimental situation was similar to that described by Diaz et al. (3). The animals were placed in a hammock in a restrictive condition. EKG was recorded on a Gilson polygraph. The conditioned stimulus (CS) and the differential stimulus (DS)'were tones of 500 cy- clels and 1,000 cyclels respectively, of 5 s duration. A combination of a 1,000 cyclels tone during 3 s and a 500 cyclels tone during 2 s const- ituted the conditioned inhibitory stimulus (CIS). The tones were generat- ed by two Phillips audiooscillators. The unconditioned stimulus (US) was a 8.0-mA square pulse discharge with a frequency of 300 cycle/s and a duration of 1 ms per pulse. The shock electrodes were placed on the right fore paw.

Procedure. The vagotomies were performed under deep Penthobar- bital anesthesia (35 mgkg i.p.). A section of the ramifications leading to the heart and originating in the descendent left vagal branch was cut off and removed. EKG was controlled before and after vagotomy. HR changes produced by electric stimulation of the vagal branch were also recorded. The same surgery was applied to the left and right thorax with a week's interval.

The animals received antibiotics and a special diet during the post- operatory periods. Training began a month after the last operation. A 7-days period of adaptation to the experimental situation preceeded the beginning of training. Basal HR and HR changes induced by the stimuli were registered.

All animals were trained daily during 70 sessions. Each session con- sisted of 12 trials: 6 with the CS, 3 with the DS and 3 with the CIS. The CS was ~einforced with an electric shock 5 s after its ending. For sta- tistical analysis (variance analysis and Student t-test), the EKG was divided in three periods: (i) 10 s previous to the beginning of each trial

(BEFORE); (ii) 10 s after the onset of the CS (DURING); (iii) 10 s after the onset of the US or the equivalent period for DS and CIS trials (AFTER).

Electrophysiological control. At the end of training the operated. animals were anesthetized with Chloralose. In order to observe whether vagotomy had been complete, both vagal branches were stimulated a t the cervical level while recording the EKG. Three animals were rejected and their data were not considered in the results.

RESULTS

Normal group

Pre-training period. Before the initiation of training, the different accoustic stimuli produced neither an increase nor a decrease of HR (x = 32 beats per 10 s). The electric shock produced a general motor activation, mydriasis, piloerection and tachycardia.

Training period. As training proceeded, the motor defensive response to shock progressively decreased. I t was also observed that, while the reactivity to shock was moderate at the beginning of each session, it increased gradually towards its end.

The most important results were the following: (i) The deceleratory response appeared already during the first half of session 1. Its evolu- tion can be observed in Fig. 1. In the initial 20 sessions the differences

- --

+ 5 1 NORMAL GROUP I

Fig. 1. Comparison of the 10 s BEFORE and DURING the application of the CS, DS and CIS in the normal group through 70 sessions. Note that the clearest res- ponse is bradycardia and the inhibitory responses do not differ from zero (0). Zero line, pre-Ss baseline; above line (+), tachycardia; below line (-), bradycar- dia. Full line, CS (tone 500 cyclels)+; dotted line, DS (tone 1,000 cycle1s)-; dashed

line, CIS (1,000 + 500 cycle1s)-.

Mean heart rates of periods BEFORE, DURING and AFTER the action of the CS, DS and CIS in normal groups. Last 20 sessions

c a t I Stimulus 1 BEFORE / DURING I AFTER

DS 2 (Differential stimulus, 1,000 cycle/s) 3 1 4 1

Variance analysis of mean heart rates for the CS, DS and CIS in period DURING in normal group. Last 20 sessions

1 2 3 4

I t = 0.7 5.8***

Source of Variance Sum of ,. variyce I ( Squares est~mate

27.96 26.57 22.41 26.46

R = 25.85

CS (Positive stimulus, 500 cyclels)

28.10 23.90 23.10 24.60

R = 24.90

1 2 3 4

25.85 23.53 19.78 25.37 -- 23.63

28.60 26.70

Between-groups Within-groups

Total

28.61 27.21 24.88 27.37

27.01

22.50 23.30 24.20

24.60

CIS (Conditioned inhibitory stimulus, 1,000+500 cycle/s)

Groups Differences Se of the Difference I 1 difference I (SE) / P

21.50 20.70 23.30

23.10

28.20 21.80 23.00 27.20

28.20 23.00 23.50 27.00

158.00 718.35

876.35

CS and DS CS and CIS DS and CIS

25.60 21.10 18.30 25.00

2 57

59

2.63 2.90 0.27

79.00 12.60

,

0.355 0.355 0.355

7.4 8.2 0.8

0.001 0.001

6.27 0.01

--

SESSIOrliS

Fig. 2. Comparison of the 10 s BEFORE and AFTER the application of the CS, DS and CIS in the normal group through the 70 sessions. Denotations as in Fig. 1.

t 5

t4r

between BEFORE and DURING were already considerable. They reach statistical significance in the last 20 sessions (P < 0.02) (Fig. 1, Table I). (ii) Although the inhibitory process developed slowly and gradually, it achieved stability during the last 20 sessions. The establishment of con- ditioned inhibition was much more difficult than that of differential in- hibition (Fig. 1, Table I). (iii) Table I1 shows no difference~ among the inhibitory stimuli but each of them differs significantly from the CS (P < 0.001). (iv) In all trials with the CS a tachycardia was observed after application of the US. On the contrary, the DS and CIS induced a bradycardia (Fig. 2). (v) During the initial 20 sessions the HR averaged 35-38 beats per 10 s. As training proceeded, it decreased to 25 beats per 10 s. This decrease is statistically significant (P < 0.05) (see Fig. 5).

- NORMAL GROUP 1

Vagotomized group

Pre-training period. Immediately after operation (decentralization) HR increased to an average of 45 beats per 10 s. During the first month of the post-operatory period it gradually decreased to normal values. Motor or exploratory activity appeared normal. Responses to accoustic or electric stimuli were similar to those of the control group.

Training period. (i) The conditioned bradycardia response previous to shock appeared already during the first session. I t was fairly stable all along the training and became statistically significant in the last 20 sessions (P < 0.05) (Fig. 3, Table 111). (ii) In the first 20 sessions a total lack of discrimination was observed, while a tendency to differ- entiate appeared only in the last 20 sessions. Table IV shows no signific- ant differences between the stimuli in the DURING period (last 20 ses-

Fig. 3. Comparison of the 10 s BEFORE and DURING the application of the CS, DS and CIS, through 70 sessions. Note that the most stable response is brady-

cardia. Denotations as in Fig. 1.

+5 * 4

2 + 3 s t 2 1

Mean heart rates of periods BEFORE, DURING and AFTER the action of the CS, DS and CIS in vagotomized group. Last 20 sessions

- VAGOTORIZED 6ROUP -

.

- I Stimulus 1 BEFORE / DURING I AFTER

. .

~ l r l o r ~ ~ l ~ r t n l j ~ t ~ l , , l , , , ~ ~ , ~ , l ~ , ~ , j 1 8 1

1 5 10 1s 2 0 ~ bl 55 60 65 70 SESSIONS

1 2 3 4

n = 30.05 I I =

CS (Positive stimulus, 500 cyclels)

1 2 3 4

31.38 24.54 31.52 32.77

DS (Differential stimulus, 1,000 cyclejs)

31.51 23.92 31.10 31.88

.f = 29.60

1 2 3 4

I t = 0.03 5.1**

CIS (Conditioned inhibitory stimulus, 1,000+ 500 cycle/s)

t = 1.3 3.5*

30.34 27.52 23.92 1 22.87 31.07 3 1.46

29.19

27.88 23.00 29.82 29.52

31.30 24.55 3 1.42 31.90

29.57 29.30

' 27.31

R = 29.79 1 29.75 1 27.55

30.84 24.82 31.31 3 1.99

Variance analysis of mean heart rates for the CS, DS and CIS in period DURING in vagotomized group. Last 20 sessions.

Source of variance

Total

Sum of df

I Variance squares estimate -- 13.77 2 6.885 156.30 57 2.742

* No statistically reliable difference.

55 1 5 10 15 20 51 55 60 65 70

SESSIONS

t 5 t 4 -

Fig. 4. Comparison of the 10 s BEFORE and AFTER the application of the CS, DS and CIS, through 70 sessions. Denotations as in Fig. 1.

- YAGOTOMIZFD GROUP

sions). (iii) Analysis of period AFTER shows tachycardia to the US (P < 0.05) and bradycardia to the DS (P < 0.05) and the CIS (P < 0.02) (Table 111, Fig. 4). (iv) Basal HR averaged 32 beats per 10 s in the first 20 sessions. Although there existed a tendency towards a decrease of this rate in the last 20 sessions, it did not reach statistical significance (see Fig. 5).

I

/\!.dA~7 k.. .' 2/ ,.... y:..:v ..: ... j,-8 \ T" ..+ \ .. . . . . .: . . ..

Comparison of both groups

Training period. The capacity to endure training appeared to be greater in the control group. The VG1s behavior grew more unstable as the training proceeded. Responses such as escape from the investigator, aggression, sustained mewing during the sessions, alteration of condition- ed responses, defecation and micturation appeared more easily and fre- quently in this group. Conditioned HR changes to the CS followed the same direction in both groups, and they are statistically significant in relation to their respective basal rates, although it can be observed that

4 - Acta Neurobiologiae Experimentalis

these differences are smaller in the VG (Tables I and 111). Totally vago- tomized subjects showed the conditioned deceleratory reaction.

If we compare the periods DURING, it can be observed that the VG established discriminatory responses with great difficulty and that they are not statistically significant in relation to the reaction to the US. A tendency towards discrimination was observed in the last 20 sessions (Fig. 1 and 3, Tables I1 and IV). Analysis of the period AFTER shows a similar direction of responses in both groups (Tables I and 111). The tachycardia response in the VG reached significance with reference to its basal HR (P < 0.05).

Figure 5 presents the most notorious difference between the two groups. Basal HR differs significantly already in the initial sessions (Table V) and inversely in the last sessions (P < 0.01) (Table VI).

Both groups showed a progressive tendency towards inmobility. This is related with the stabilization d1 conditioned bradycardia.

w -

s s 2 3 0 -

z s g 2 5 -

20

Variance analysis comparing basal heart r a t a of the normal and vagotomized groups. First 20 sessions

- NORMAL 6ROUP - --- YA6OTOMIZFD GROUP ":w - -1 b' p, L'

1 \ (1 Lr-' /I \-I \ I"\

1 I i1 /&

1

-

I I I I I , r I I I I 1 7 5 10 I5 20" 51 55 6 0 65 70

source of variance ) I df ) MS I F squares I

IT

SESSIONS

Fig. 5. Comparison of the evolution of basal heart rates i n normal and vagotomiz- ed groups. See explanation in text.

Between-groups Within-groups

Total

105.68 223.27

328.95

3 76

79

35.227 2.938

11.99 0.01

Variance analysis comparing basal heart rates of the normal and vagotomized groups. Last 20 sessions

Source of variance Sum of / squares / I MS 1 I

DISCUSSION

Between-groups Within-groups

Total

The main results of this work can be summarized as follows: (i) cardiac vagotomized cats are able to learn a conditioned bradycardic response, (ii) they are able to discriminate between positive and inhi- bitory stimuli, (iii) the inhibitory effects of DS and CIS are not complete, because a decrement of HR is observed 5 s after these stimuli have been applied, (iv) the conditioned bradycardia appears rapidly in the first session of training just as it does in normal animals (4, 15, 17).

How can we explain this conditioned bradycardia in animals whose heart has no vagal innervation? It is possible to assume that it is a con- sequence of the "law of denervation", that is, of an increased sensitivity of denervated structures to different stimuli. However, there are some facts that do not fit such an explanation: (i) the bradycardia is a learned reaction, that appears between the beginning of the CS and the onset of the US, (ii) the US induces a tachycardia response and not an unspecific bradycardia, (iii) according to Dempsey and Cooper (2) denervated cat hearts show only prejunctional supersensitivity; this phenomenon reaches its maximum during a period up to 3 days (6), (iv) our experiments begin 1 mo after denervation, (v) a denervated viscus in the hypersen- sitivity period reacts more intensely in comparison with a normal in- nervated one, (vi) the bradycardia conditioned response of the denervat- ed group is of about the same magnitude or somewhat smaller than the response of the control group (see Table 111). All these facts lead us to reject the above mentioned explanation. Degeneration activity as another possible effect of denervation is out of the question because it has been produced by postganglionic section ( 5 ) , which is not the case in our experiments.

Diaz et al. (3) postulated a tonic inhibition of the cardiac sympathe- tic activity as a causal mechanism to explain the lack of conditioned bra- dycardia in sympathectomized animals. The observation described in this work seems to support this point of view. The reciprocal functional re-

190.25 193.55

383.80

3 163.416 24.908

---- 76

79

0.01 2.546

lationship between sympathetic and parasympathetic cardiac nerves could be understood not only as a passive-active interaction, but as an active one, either excitatory or inhibitory. Many authors have stressed the reciprocal influence of both systems in the visceral functions (8, 9) and we are ready to accept that probably in the bradycardic conditioned activity this is also the case, although it is quite clear that this condition- ed bradycardia is mediated by a tonic inhibition of the sympathetic system.

The conditioned bradycardia would belong to the so called "passive fear reflexes", which are part of the "sham-death reflex" or "freezing", that seem to appear in cases of inminency or inescapability to the no- xious agent (10, p. 32). In fact, the CS induces, in normal and operated cats, besides the bradycardia, motor inhibition, inspiratory apnea and sometimes defecation and urination. On the contrary, the US produces cardiac acceleration, escape reactions, mews, etc. The tendency to in- mobility was tested in a pair of cats of the denervated group. They were left free in the cage. When the CS came, instead of trying to escape

40 - - NORMAL 6ROUP - --- VAGOTOHIZFD GROUP - .------ SYMPATHECTOUIZED GROUP

"

20 -

I 1 1 1 1 1 1 1 1 1 1 1 ,

I 5 10 15 20 51 55 6 0 65 70 SESSIONS

Fig. 6. Comparison of the evolution of basal heart rates in normal, vagotomized and Diaz's (3) sympathectomized groups.

the cats remained motionless and huddled as if waiting for the shock. Such behavior can be considered a learned helplessness reaction (16).

With regard to the effect of inhibitory stimuli, it is necessary to observe that DS or CIS are less effective in the operated group than in the normal one. Our experimental design does not allow us to distinguish whether this is a relief signal or consequence of the difficulty to preserve

the inhibition for a long time when the US is absent. Probably the nor- mal inhibitory mechanism is disturbed by the suppression of the vagus. Nevertheless this speculation is not satisfactory and does not rule out other possible mechanisms (hernodynamic or neurovisceral).

The last important1 phenomenon observed in this study was the evolution of basal HR. Ilchevich and Bidzilia (7) have suggested that the "law of denervation" does not affect HR. This fact, and the radical disturbance of the decrease in sympathectomized animals (see Fig. 6), lead us to suppose that the evolution of basal HR is a direct product of environmental conditioning. These data can be modified, because our controls of basal HR were made in the experimental cage and before the onset of the stimuli. The decrease of the HR could be part of that global reaction, based upon an inhibition of sympathetic fibers coupled with an activation of parasympathetic fibers. The two autonomic di- visions interact constantly and therefore the lack of one implies a modi- fication of the other, but it is difficult to postulate the exact form of this alteration without additional observations.

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Accepted 10 January 1977

A. GIAVELLI, and G. SANTIBAfilEZ-H., Institute of Physiology, Humboldt University, Berlin, GDR.