Autonomic conditioning in the anesthetized rabbit

Physiology and Behavior. Vol. 3, pp. 673-675. Pergamon Press, 1968. Printed in Great Britain

Autonomic Conditioning in the Anesthetized Rabbit M A R I A C. L I C O , A N E T T E H O F F M A N N

AND MIGUEL

R. C O V I A N

School of Medicine, Department of Physiology, Ribeiri~o Pr6to, S.P. Brazil (Received 28 March 1968) Lice, M. C., A. HOFFMANNAND M. R. COVlAN. Autonomicconditioning in the anesthetizedrabbit. PHYSIOL.BEHAV.3 (5), 673-675, 1968.--In 21 rabbits out of 61, under urethane anesthesia a conditioned fall of blood pressure was obtained. Electrical stimulation of the septal area was the unconditioned stimulus and the conditioned one consisted of a click train of 10/see repetition rate applied during 35 see; during the last 15 see the unconditioned stimulus was paired. The number of associations necessary to establish the conditioned response ranged from 6-27. The classical pavlovian characteristics of conditioning were present: extinction, differentation, generalization, external and internal inhibition. The electrocortical activity of the acoustic area during conditioning was variable. These results show that it is possible to obtain a neurovegetative conditioning when a low integrating structure as the limbic system is activated during attention blockage by anesthesia. They rinse a number of interesting questions such as the level of vigilance necessary to elicit some kind of conditioning and also to what extent lower levels of integration can accomplish all the steps of the conditioning. Conddioned reflex

Septal area

Limbic system

Consciousness

can be acquired and retained during the stages 2, 3 and 4 of sleep in man [7]; during barbiturate sleep either in human or animals [16, 18]; after the massive coagulation of the mesencephalic reticular formation in cats [1, 4]; in the hibernating squirrel [23]; has also been obtained conditioned awakening in the cat [19, 21]. On the other hand, as has been mentioned before, it is well to remember that in spite of alertness, autonomic conditioning is below the conscious level because there is neither perceptual discrimination nor conscious response, as also happens in subliminal conditioning. The purpose of the present paper is to study an adaptive reaction during attention blockage by anesthesia in order to elucidate to what extent a high level of vigilance is necessary to obtain a neurovegetativc conditioning when a low integrating structure as the limbic system is activated. It has been shown that electrical stimulation of the limbic septal area results in arterial hypotension in cats [2], rats [3], guinea pigs [13], toads [8]. The regularity and magnitude of the evoked arterial hypotenslon also in the rabbit led us to test its conditionability. Thus, the unconditioned response elicited by stimulation of the septal area in rabbits under urethane anesthesia was used as a reinforcer of a sensorial (acoustic) stimulus.

FOR THE ESTABLISHMENT of both somatic and autonomic conditioned reflexes, " a n alert state of the nervous system is absolutely essential", according to Pavlov [17]. I n spite of the fact that autonomic conditioning is usually established below the conscious level in the alert animal, the pavlovian concept has been taken for granted by researchers in this field. It is thought that alertness depends on the unspecific action of the mesencephalic reticular formation on the cerebral cortex, because in the absence of the reticular bombardment messages arriving to the cerebral cortex by the specific pathways are not consciously perceived even though they elicit evoked response. Consciousness has been defined as a matter of degree and level of complexity of perceptual discrimination [14]. In subjective terms we mean by consciousness the clarity of our awareness of our internal and external environment. In objective terms, consciousness seems most clearly described as "readiness to respond" but not just readiness of any portion of the nervous system to respond [11]. According to these classical concepts it is obvious that in those unconscious states such as sleep, anesthesia, anoxia, somnambulism, epilepsy, etc., the possibihty of perceptual discrimination and therefore of conditioned behavior is ruled out. However, it is of empirical knowledge that several kinds of adaptive responses of variable complexity exist which are performed during periods of unconsciousness. F o r instance, the conditioned awakening of a mother by the specific sound of her baby's voice while she is unaware of a thunder sound is well known; as well as the surprising discriminative ability of somnambules and epileptics during their periods of automatisms, to such an extent that they can integrate complex operative behaviors [15]. F r o m the experimental point of view it has been observed that conditioned responses

MATERIAL AND METHODS

Experiments were performed on 61 adult rabbits of both sexes weighing from 1.5-2.5 kg. Under light ether anesthesia urethane was given intravenously at doses 1.25-1.75 g/kg; this last value was very close to the lethal dose, and sometimes a dose of 1.80 g/kg killed the animal. During the anesthetic state the corneal reflex and slight pain sensitivity were present. 673

F

Blood pressure

674

LICO, HOFFMANN AND COVIAN

As usually most of the experiments lasted for 6-8 hr, addltlonal doses of urethane were administered when necessary. The head was fixed in a sterotaxic instrument carrying a stainless steel concentric electrode pair insulated except at the tip which was placed In the septal area according to the coordinates of the atlas of Sawyer [20]. Systemic arterial pressure was measured with a Statham P23-AA transducer connected to a catheter placed in the femoral artery. Respiration was recorded through a pneumograph attached around the chest. Changes of blood pressure, respiration and electrical cortical activity were recorded synchronously on a polygraph. In a group of rabbits the cortical activity was recorded separately on an 8-channel electroencephalograph. A tracheal cannula was inserted in each animal. Body temperature was maintained by radiant heat and measured with a telethermometer with a probe introduced in the rectum. Unidirectional rectangular pulses at 50/sec, 10 msec of pulse duration and peak current of about 0,5 m A delivered by an A E L stimulator through an isolation unit was the unconditioned stimulus applied to the septal area. The conditioned stimulus consisted of a click train of t0/sec repetition rate applied during 35 sec; during the last 15 sec the unconditioned stimulus was paired. The intensity of the acoustic stimulus did not have any influence on the blood pressure. A click train of 3/sec repetition rate was used as a differential stimulus. Each pairing of stimuli were applied with 2-4 min interval. At the end of each experiment the head was perfused with 4 per cent formaldehyde and the brain submitted to routine histological procedures to determine the position of the stimulating electrodes.

TABLE 1 ACOUSTICAL CONDITIONING OF BLOOD PRESSURE FALL IN 21 RABBITS UNDER URETHANE ANESTHESIA. IF IS INDICATED IN REINFORCEMENT THE PAIRING AT WHICH THE MAXIMAL ACQUIRED RESPONSE WAS OBTAINED ( R : RI-INFORCEMENT. A S " ACOUSTICAL CONDITIONED STIMULUS. SS" SEPFAL ELECTRICAL UNCONDITIONED STIMULUS).

Arterial Blood Pressure Rabb~t Remforcement systolic m mm Hg, ( ) IB parenthes~s No. No. dla~tohc percentage of change Just beforeR 1

15

2

14

3

41

4

13

5

29

6

26

7

11

8

27

9

31

10

8

11

15

12

32

13

14

14

25

15

19

16

7

17

11

18

22

19

11

20

14

21

7

RESULTS

In 21 rabbits out of 61 conditioning was clearly obtained. The number of associations necessary to establish the conditioned response ranged from 6-27. In Table 1 some details of the conditioning observed are shown; it is indicated the pairing at which the maximal acquired response was elicited. It can be seen that in 3 rabbits (no. 9, 17, 18) the conditioned fall of the blood pressure was greater than the unconditioned one; in 3 rabbits (no. 6, 7, 14) it was similar and in the remainder it was smaller. In Fig. 1 the characteristics of the conditioning for rabbit no. 6 is illustrated. In A the blood pressure fall is only elicited by the unconditioned stimulus together with a respiratory change marked by a sharp inspiration. In B the conditioning is established for blood pressure and respiration responses after 15 stimuli pairings. It is well to remember that respiratory conditioning has been obtained in cats with mesencephalic reticular formation destroyed [4]. The acquired response was progressively enhanced in successive reinforcements reaching a magnitude similar to the unconditioned one after 26 associations as shown in C. After obtaining the conditioning, classical characteristics of pavlovian conditioned responses were studied. Figure 2-D shows the 26th response elicited without reinforcement. In E extinction is obtained after the absence for 5 times of reinforcement and in F the recovery of the conditioned response following 5 associations. In Fig. 3 (rabbit no. 13) the substitution of the conditioned stimulus (a click train of 10/sec repetition rate) for one of 3/sec resulted in differentiation as is shown in B. When the repetition rate of the acoustical stimulus is again of 10/see the conditioned response reappeared (C). In some rabbits other peculiarities of conditioned responses such as external and internal inhibition and generalization were observed.

127 97 112 92 138 95 145 98 138 102 145 112 130 82 135 90 128 62 110 65 118 78 112 72 80 56 108 87 152 106 140 88 144 80 134 84 96 42 110 60 128 80

Durmg AS

Durmg SS

115 82 102 82 112 72 135 88 120 88 125 95 110 70 123 80 114 52 90 58 89 68 100 65 70 50 99 81 114 77 120 76 120 58 110 62 80 30 100 50 114 68

110 80 98 82 105 65 125 85 112 80 125 95 110 70 120 77 112 54 65 50 87 62 96 62 68 50 99 81 106 75 116 70 124 60 120 70 72 18 74 28 II0 60

(-- 9) (--15) (-- 9) (--11) (--19) (--24) (-- 7) (--10) (--13) (--14) (--14) (--15) (--15) (--15) ( - 9) (--11) (--I1) (--16) (--18) (--11) (--24) (--13) (--ll) (--10) (--12) (--11) (-- 8) (-- 7) (~25) (--27) (--14) (--14) (--17) (--27) (--18) (--26) (--17) (--28) (--9) (--17) (--11) (--15)

(--13)

(--17) (--12) (--11) (--24) (--31) (--14) (--13) (-19) (--21) (--14) (--15) (--15) (--15) (--11) (--14) (--12) (--13) (41) (--23) (--26) (--20) (--14) (--14) (--15) (--11) (-- 8) (-- 7) (--30) (--29) (--17) (--20) (--14) (--25) (--10) (--17) (--25) (--56) (--33) (--53) (--14) (--25)

The electrocortical activity of the acoustic area during conditioning was variable. In some cases desynchronization was noticed (Fig. 4) but in others no change was present (Fig. 5). DISCUSSION

The correct level of anesthesia required to obtain the conditioned response was one of the hard problems we faced. It seems that there is an optimal level at which the conditioning

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FIG. 1. Conditioning of the blood pressure fall due to septal stimulation in the rabbit with urethane anesthesia. A, septal uncon&tioned stimulus (US) elicited the blood pressure fall and a sharp inspiration. B, after 15 stimuh pa]rmgs the conditioning is established for blood pressure and respiration. C, the magnitude of the response to the con&tloned stimulus (CS) is equivalent to the unconditioned one after 26 assocmtlons. (Jactng page 674)

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FIG. 2 Condltlonmg m the rabbit under urethane anesthesia Extraction. D, once the condlttomng was estabhshed, the blood pressure fall was obtamed by the condttloned stimulus (CS) without reinforcement (US). E, after some apphcatlons of the conditioned sttmulus alone, extraction appeared. F, after some new associations the conditioned response reappeared.

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F I G . 3 C o n d i t i o n i n g m the rabbit u n d e r urethane anesthesia. Differentmtlon. A, the conditioning has been e s t a b h s h e d to a click train o f 10/sec repetition rate. B, there was n o response to a stnmulus o f 3/sec repetition rate. C, the conditioning is again s h o w n to a chck t r a m of 10/sec.

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F I G 4 Conditioning in rabbit anesthetized w~th urethane A, before conditioning septal stimulation ehclted the blood pressure fall and E E G desynchromzat]on m the cortical acoustic area. After some associations (B) the blood pressure fall was condmoned. The condJtJoned stimulus desynchromzed the EEG and determined a fall which paralleled that obtained by the unconditioned st~mttlus.

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FIG. 5. Conditioning of the blood pressure fall in rabbzt under urethane anesthesm. A, before condnt~onmg septal stnmulat~on ehc]ted no change m the cortncal acoustncal area. B, there was not change also when condnt~onmg was estabhshed.

AUTONOMIC CONDITIONING

675

is elicited, this being an intermediate stage between a deep and a light anesthesia. In this regard it is well to quote Galambos and Morgan [5] " . . . relatwely small amounts of anesthetics unfalhngly reduce animals and men to a state where no learning whatever is possible. One might naively expect that between the stages of complete anesthesia and none at all a level would be reached where the learning process was, say, only half impaired. Such a stage has, however, never been defined". In our experience it was not possible to condition rabbits anesthetized with a dose of urethane higher than 1,75 g/kg and the optimal state could not be detected, because no external characteristic sign was found. We can only define ~t as the state in which conditioning was obtained. Emphasis has been placed on the role played by subcortical structures in the process of conditioning, namely, the thalamus, hypothalamus and mesencephalic reticular formation. It would appear that the septal area is another subcortical structure of importance in conditioning. These results show that in an unconscious state there exists a certain degree of sensorial discrimination which makes possible the conditioning of the blood pressure fall. The ~nstability of this conditioning resembles that obtained in decorticate and descerebrate animals. It has been reported that in cats under acute sodium epival anesthesia conditioning

was unobtainable but some of them gave conditioned responses upon recovering from anesthesia, showing that during unconsciousness a trace was marked in the nervous system by the training trials [22]. More closely connected with our work is the conditioning of a defensive reflex found in rabbits under urethane [6, 12]. Evidence of learning was found in cats trained during sleep unconsciousness by way of a trace instrumental avoidance routine [10]. Conditioning in a subanesthetic state with sodium pentobarbital has been observed in rats [9]. Our finding is another example of conditioning obtained during unconsciousness which raises a number of interesting questions such as the activity of the reticular formation in this conditioning and the possibility that lower levels of integration, e.g. limbic structures, can accomplish all the process. It is also possible that because of certain neural peculiarities (large limbic system and small cortical development) the rabbit is special and a suitable animal for this unconscious conditioning and urethane is an anesthetic able to release the necessary pathways. On the other hand what is the meaning of this kind of learning and at what level is the information analyzed? These and other questions involving neurophysiological and even psychological implications require clarification.

REFERENCES

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