Can halothane anaesthesia have any aversive effects on the rat?

Physiology & Behavior, Vol. 22, pp. 25-29. PergamonPress and Brain ResearchPubl., 1979. Printed in the U.S.A.

Can Halothane Anaesthesia Have Any Aversive Effects on the Rat? GERARD SCHMALTZ Laboratoire de Psychophysiologie, Universit$ de Lille 1, B P 36, 59650 Villeneuve d ' A s c q , France

( R e c e i v e d 3 M a r c h 1978) SCHMALTZ, G. Can halothane anaesthesia have any aversive effects on the rat? PHYSIOL. BEHAV. 2,2(1) 25-29, 1979.--Rats (3x2 groups) were put in a Y maze and submitted to one of the f o ~ 3 Wetresm~ts OPT): 0, 3, I0 uncontrollable presboeks (PS). Each PT, administered to 2 groups, was immediately foUowed either by Imiothlmemutesthetiz/ng (groups M,,_,, M:,_., M,,_,0 or sham anaesthetizing (groups M,, M.~,M,o). One day later, the 6 Woups underwent active avoidance conditioning in the Y maze. The overall escape performanee Ofgroup M~did not differ from that Ofjroup M,,. By contrast, a marked impairment was observed in the M,,, group, which demonstrates both the effectiveness and the good memorisation of the most aversive PT (10 PS). The acquisition of group M,,_,, was as much Lqq3airedas that oflgoup M.,. Consequently, we can conclude that halothane has no amnesic effects. The performances of groups Mo-, and M~_, were inferior to those of groups M,, and M:,, respectively. These last results are discussed in terms of aversive effects of halothane. Rat

Preshocks and haiothane anaesthesia

Aversive effects

Avoidance conditioning

SCHMALTZ, G. Une anesthisie au fluothane a-t-elle des effets aversifs chez le rat? PHYSIOL. BEHAV. 22(1) 25-29, 1979.--Des rats (3x2 groupes) ref~vem, dans un labyrimhe en Y, I'un des 3 pr~traitements (FI') suivants: 0, 3, 10 checs ¢gectriques incontrblables (CI). Chaque IT, administr~ h 2 groupes de sujets, est imm~iatement suivi, soit par une anesth~sie au fluothane (groupes t}A, 3-A, It}A), soit par une anesth~sie simukbe(groupes O-AS, 3-AS, It}AS). Unjour plus tard, les 6 gronpes effectuent un conditionnement d'~vitement actif dans le labyrinthe en Y. La performance O~.hsppement) du Woupe 3--AS ne di/~h~ pes de celle du groupe t}AS. Par contre, u u nette perlturbatkm s'observe ~ le groupe 10.-AS, ce qoi d6montre it la fois l'efl]caci~ et ht bonne n~morimt/on du PT le plus aversif (I0 CI). L'aPlmmlistqle du groupe It}A est aussi perturb6 clue ceiui du groupe 10AS. Nous pouvons en conclure qne le fluothane n'a pes d'effets amn6siques. Les performances des groupes 0--A et 3-A sont, respectivement, inf6rieures h ceIles des groupes 0-AS et 3-AS. Ces derniers r~sultats sont interpr~t~s en termes d'effets aversifs du fluothane. Rat

Chocs 61ectriques et anesth~sie au fluothane

Effets aversifs

H A L O T H A N E seems to produce fewer adverse reactions in the cardio-vascular and respiratory systems than other anaesthetics [8]. It is administered by inhalation and acts very rapidly but also wears off rapidly. In Wistar strain rats, for example, narcosis occurs in less than a minute, minimizing strusgling, and yet after 2 hr under anaesthetic, the animaps behaviour returns to normal within a few minutes [2]. The administration of this treatment thus appears not to be traumatic for the animals. Partly because of these advantages, halothane has been used to interrupt the consolidation phase of memory formation [2, 4, 6, 7]. The essential evidence which permits one to draw this conclusion is the presence of a temporal gradient of amnesia, that is, the shorter the time interval between learning and treatment, the greater the impairment [2, 4, 13]. For details of the experimental results obtained on rats see the excellent reviews by Bloch [3] and Deweer [5], both concerned with the consolidation of memory. In addition to the anmesic effects of halothane, and despite its apparent inoffensiveness, it is nevertheless possible that this anaesthetic also has aversive properties. This hy-

Conditionnement d'C~vitement

pothesis has already received some confirmation, and the work of Verrier-Oisquet et al. [11], Alexinsky and Chaponthier [1] on rats and Zirgkin et al. [13] on chicks has indicted that iwlo~ane may have not only ~ but also aversive effects. The object of the present experiment is to investigate the possible aversive effects of an ludotlmae anaesthesia on the rat and not to distinguish between aversive and amnesic effects. We start from the result of a previous study [9] which has shown that aversive uncontrollable shocks administered to rats as a pretreatment (preshocks) may interfere with the later acquisition of an avoidance task. In the present study, all the rats first received preshocks in a Y maze. Then, half of the animals was immediately anaesthetized with halothane. The possible effects of halothane will be more easily detectable when there is no delay between preshocks and anaesthesht. One day after these pretreatments, all the animals were given an avoidance learning session in the Y maze. If the anaesthesia has aversive properties, then it should increase the impairment produced by the preshocks alone so that the performance of the preshocked-anaesthetized rats should be inferior to that of

C o p y r i g h t '~ 1979 Brain R e s e a r c h Publications Inc.~0031-9384/79/010025-05502.00/0

26

SCHMALTZ

the preshocked ones. In short, one should be able to detect the aversive effects of halothane anaesthesia, if any, by examining the performance of the animals during the avoidance conditioning session. METHOD Animals The animals were 85 male Sprague-Dawley rats weighing between 285-315 g at the start of the experiment. All the animals were housed individually, with ad lib food and water available in their home cages. They were handled once each morning during the week before the beginning of the experiment. Apparatus The apparatus used was a Y-nmze with an adjoining start box. All the walls were wooden. The 3 arms of the maze formed 120" angles, and each arm was 43 cm long, 12 cm wide and 30 cm high. The centrld alley was soperated from the start box by a ~ door, while each of the lateral arms had a slidlnll door 30 cm away from the ~ end. These doors ~ the e ~ t e r to isolate ~ animal in the goal box. The floor of the wmze, including the start box, consisted of parallel brass rods 3 nun in dian~ter lying at 10 m m intervals. This grid was connected to a constant current stimulator. The entire apparatus was covered with Perspex lids. Procedure The 85 animals were divided at random into 8 groups (N= 10, except for one Stoup with N=IS). For each animal, the experiment involved 2 sessions: pretreatment, then avoidance conditioning. Session 1: Pretreatment Non-anaesthetized groups. In 3 of the groups (M., M:j and M,o) the animals were placed individually in the maze where they remained for 30 win. They could not enter the start box, as door was kept closed. During this half hour the rats in these groups received, respectively, 0, 3 or 10 aversive preshocks (1,5 mA in pulsed train~--0.1 sec on; 0,4 sac oft). These l ~ k s (PS) were inesc,e p ~ and u n ~ e d , the onset, duration and intensity being controlled by the experimenter. The fact that the PS were uncontrollable considerably increased the aversiveness of the situation [10]. Immediately after the 30 rain spent in the maze, the aninmls were given sham anaesthesia (see below) then returned to their home cages. This sham a n a e s t h u was also given to rats in a control group Co, which were never placed in the maze, but simply renmined in their home cages. Anaesthetized groups. Groups C._j,, M._., M:,_,, and M,o-. received exactly the same pretreamumts (PS) as their counterparts described above, except that a FFdotex Mark 2 delivered a mixture of equal parts o f e l r and oxygen (flow 2 l/rain) with a 1,5% concentration of halothaae. The mixture passed through a Ptexigias box (25 cm×25 cm×25 cm), escaping through 4 small h ~ e s in the lid. Each rat was placed individually in this box for 60 rain. The average time to achieve narcosis was 40 sex. After spending an hour under the influence of anaesthetic, the unconscious animal was removed and placed in its home cage where it recovered fully

within 10 to 15 min. After waking there was no obvious sign of impairment in gross behaviour. The sham anaesthesia was performed in the same box, but there was no halothane in the air-oxygen mixture, and the time spent in the box was only 40 sec. Session 2: Avoidance Conditioning The active avoidance conditioning involved 15 trials in the Y-maze and took place one day after the first session. The same procedure, as previously described [9], was used for all the rats. The rat was placed in the start box at the beginning of the session. After 1 rain, the door was opened, allowing access to the maze alleys. Five sec later, the rat received footshock with the same characteristics as the pretreatment shock, except that now it could be controlled by the animal. The shock stopped when the animal, after reaching the end of one arm (first choice), turned round and went to the end of the other arm beyond the sliding door (second choice), This sliding door was then closed and the animal was left in the goal box for 30 sex before being placed in the start box for the I rain intertrial period. During the first trial it was thus necessary for the rat to cover the entire maze before escaping the aversive stimulus (escape response). During the following trials, the goal box (second choice on the first trial) was no longer electrified. The rat could now either make an escape response by entering the goal box after the shock had begun, or make a conditioned avoidance response (CAR) by entering the goal box directly--i.e., without making any spatial discrimination error--before the onset of the shock. Some animals, especially in the early stages of learning, did not escape the shock. In this case the experimenter intervened. During the first trial if, after a 20 sex stimulation period, the rat was still in the start box, it was pushed into the central alley and door was closed. If after 90 sex the animal had not made the escape response it was pushed into a goal box for 10 sex. Therefore shock could not last longer than I00 sex on any trial. The location of the goal box depended on the previous behaviour of the animal. The goal box was either the unexplored arm, or an arm was chosen at random if the rat had not explored either arm. During the remaining trials, the procedure was the same except that the goal box was already determined. RESULTS Behavioural Observations During Pretreatment While the rats were given these 3 uncontrollable PS, their most frequent response was flight. In some a n i ~ (8/20), freezing appeared on the third PS. Among those animals which were submitted to 10 uncontrollable PS, the same pattern was observed during the early shocks but freezing then increase in frequency. After the fifth PS, the animals froze consistently in response to the aversive stimulus. Avoidance Conditioning in Non-anaesthetized Groups During the active avoidance conditioning we recorded the duration of stimulation on each trial (range: 0-100 sec)for each rat and also the total duration of stimulation summed over all 15 trials. This enabled us to calculate, respectively, the mean duration of stimulation (MDS) per trial for a given group and the mean total duration of stimulation (MTDS) for the group. A Preshock×Anaesthesia analysis of variance

AVERSIVE EFFECTS OF HALOTHANE

27

demonstrated a statistically significant preshock effect, F(2,59)= 14.47, p<0.001, anaesthesia effect, F(!,59)= 16.65, p<0.001, and a significant interaction between these two effects, F(2,59)=3.25, p<0.05. The MDSs and MTDSs record chiefly the escape responses of the animals w h e t h e r " spontaneous" or "forced". We also calculated the number of CARs for each rat, but this parameter has not been taken into account essentially because it seemed relatively insensitive to the various pretreatments. We have used a two-tailed t test for all the statistical comparisons and a significance level of 0.05. Table 1 shows both the numerical values and the dispersion of the MTDSs in addition to the results of inter-group comparisons.

I000

-

==

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]

No Anaesthesia

]

Halothane Anaesthesia

800-

p.

~ ~,oo. Z n I

~ 400-

TABLE 1 O V E R A L L P E R F O R M A N C E I)URING A V O I D A N C E CONDITIONING

Groups

N

Mean Total Duration of Stimulation (see) ± SEM

P*

CO C0_ H

10 10

214.6 ± 49.3 219.2 ± 59.4

NS

M0 MO_H

l0 15

211.4 ± 36.3 453.2 ± 69.5

<0.02

M3 M3_ H

10 l0

310.3 ± 36.7 660.5 ± 53.3

<0.001

MI0 MIO_ H

I0 l0

633.9 ± 83.4 764.2 ± 77.3

NS

0

z ,¢

Lu

200.

MF

Co CO-H

MoMo-H

J M3 M3.H MlO MIO.H

FIG. 1. Results of avoidance conditioning. The bars represent the Mean Total Duration of Stimulation (MTDS) calculated by averaging the total duration of stimulation for all the rats in a given group. (*) The difference in MTDSs between M¢-Mo. and between M3-M.~.. was significant (p<0.02 and p<0.001, respectively).

*NS = nonsignificant (p>0.05; two-tailed t-test) The comparison of the overall performance of groups Ms and M,,, respectively (Fig. 1), with that of group Mo, shows the effects produced by administration of 3 and 10 uncontrollable PS on later avoidance learning. No significant difference appeared between Mo and M:; (t=l.91, p>0.05). The most aversive pretreatment (to group M , J greatly impaired the escape component (t =4.64, p<0.001). The MTDS of groups M:3 and M,) differed significantly (t =3.55, p <0.01). The performances of groups M. and Co were similar 0=0.05, p>0.10). Judging from the overall performance (MTDS) of group M3, one might conclude that the administration of 3 uncontrollable PS during pretreatment had no effect on subsequent escape responses (Fig. 1). However, the acquisition curves of M. and M.~ (Fig. 3) show that all the animals in group M:~ had to be pushed to the goal box on the first trial (MDS= 100 see), which was not at all the case for group M0 (MDS=23.6 see). On the second trial the means again differed significantly (t =2.76, p<0.02), but the curves became similar from the third trial onward.

Avoidance Conditioning in Anaesthetized Groups The overall performance of the various anaesthetized groups is shown in Fig. 1. A comparison of the MTDS of groups M._. and M:~_. indicated a significant difference (t =2.16, p <0.05). The same result appeared when the groups M._. and M,~_. were compared (t=2.93, p<0.01). By contrast, groups M:;_, and M,._, did not differ (t = 1.10, p >0.10). The escape performance of animals in group M._, signifi-

cantly differed from that of subjects in group C._. (t=2.38, p<0.05). A comparison of the performance of anaesthetized groups with their non-anaesthetized equivalents (Table 1 and Fig. 1) showed signLrw.ant differences in the escape component between groups M,, and M,_, (t=2.66, p<0.02) and also between groups M.~ and M:~-. (t =5.41, p<0.001). The performances of groups M,, and M,._. and of groups C,r-Co-. did not differ significantly (t = 1.14 and 0.05, respectively, p >0.10). Fig. 2 shows the learning curves of groups M. and M._,. Note that although the 2 performances do not start from the same level on the first trial, their temporal evolution follows the same general pattern. From the acquisition curves of groups M,~ and M3-, (Fig. 3) one can see that no animal in either group "spontaneously" escaped to the goal box on the first trial. Through the next few trials, the performance of group M:3 improved considerably. However, there was a persistent tendency in group M:~_, to freeze, so that the curves of MDS became nearly similar only at the end of the session. The learning curves of groups M,~ and M~o-. are not represented here for 2 reasons: first, both show an identical temporal pattern; second, both do not fundamentally differ from the learning curve of group M:~-Has shown on Fig. 3. DISCUSSION

The performance of the non-anaesthetized groups demonstrates the effects of un¢ontrol~ble PS on later acquisition of avoidance conditioning. As we have already noted in a previous paper [9], the predominant type of motor responso--flight or freezing--during the administration of

28

SCHMALTZ lO0.

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~ 60-

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TRIALS

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FIG. 2. Acquisition curves for 8roups Mo (No PS.--no anaesthesia) and M... (No PS+ ~ ) . Note a similar Maend ~ evolution. (*) ~ diffe~__,~,~s(p<0.05) ~ between the two curves for Trials 2, 5, 6, 7, 10, 15;

FIG. 3. Acquisition curves for groups M. (No PS--no anaesthesia), M:~(3 PS--no anaesthesia) and M:~..(3 PS+anaesthesia). (*) A trial by trial comparison of the curves for M:~and M~., revealed significant differences (p at least <0.05) for Trials 2, 3, 4, 5, 6, 10and 12).

the uncontrollable PS determines the quality of subsequent task performance to a large extent. Free exploration of the maze had no effect on the subsequent performance of the animals (no latent learning). The ~stration of 3 uncontrollable PS affected only the first 2 c o a d i t i o n ~ trials. The animals froze in ~ to shock durins the first trial am/did not attempt to escape "spontaneously." Pttshil~ produces a s ~ t behavioural "disinhibition" [10] and from the seczad trial onward, flisht replaced freeziag more and more often, thus ~ to a rapid and marked impmvetmmt in perfornumce.

fact that pushing the rats greatly improves their performances, we have found a marked deterioration of the escape component (increased freezing) in the group anaesthetized after being submitted to 3 uncontrollable PS, as compared with the equivalent non-anaesthetized animals. It looks as if anaesthetizing greatly increased the aversiveness of the situation. The association of these 2 pretreatments (3 PS +anaesthasia) was apparently highly aversive since it led to an escape performance whk:h was beth more impaired than that observed in group 0 P S + a n a e ~ and as much altered as the one seen in the animals which had received 10 uncontrollable PS during pretreatment. The performance of animals anaesthetized after receiving 10 uncontrollable PS was as poor as that of the nonmmesthatized corresponding group. So, an amaesic effect of anaesthesia can he excluded in this e x p e r b a e ~ . The most parsimonious explanation, while not necessarily the most accurate one, is that halothane made no ~ whatever. However, we may also hypothetize that the PS and the anaesthesia were still both aversive and that the apparent similarity of performances was most likely due to a ceiling effect introduced by the experimental procedme. Pusiting the animals when they failed to escape the shock tended to equalize the scores, and the experimenter had to intervene most frequently during early trials, that is, on those trials most likely to be affected by the pretreatment experience. The impeired perfonaence of a a a e ~ animals cannot be due to a systemic disturbance produced by haioth_ane, for the following reasons. First. the animals" behaviour foiIowin6 p r e t m m m ~ t seemed normal w b e t ~ r or not they had received ummatroilable PS before anaesthesia. In addition, there was a 24 hr period between the 2 sessions, so that if there was a general malaise it must have been relatively long lasting. Finally, and very si8nificantly, the anaesthetized rats

The avoidance ~

process was disrn_~_-.d in

those animals which ~ y received I0 u ~ PS. The memory of this more aversive p r e t ~ t consistent freezing, fast in the start box, then in the central alley of the maze. This respmse, which t ~ m i s ~ throuOout the early trials despite the intervention of the e x e t e r explains the poor escape perfommace. The behavioorai observations made d u r i q the present study were entirely consistent with those reported in an earlier paper [9], which leads us to conchade that, under our experimental ecmditions, freezi~, as opposed to flJJht occurs in ~ s e to an increasingly aversive situation. We consider this behavioeral shift as evidence of increaaiq conditioned fear. Halothane anaesthesia delivered itrmwdimaiy after free exploration of the maze does interfere wi~ the ~ cornpoaent of subsequent maditioaing. This ~ strom~y supports the assumption that haiothane may have aversive effects. Under these experimental conditions, we have shown (rtmnuscript in pmlmmtion) that pushing the ~ is very effective in reducing freezing. This real "help" seems to be akin to the ~ n o n described as " ' ! ~ " in Selisman's theory of learned helplessness [lO]. l~spite the

AVERSIVE EFFECTS OF HALOTHANE

29

who could not associate the anaesthesia with the experience of the maze since they remained in their cages during pretreatment (control group), did not show any impairment of performance. This last result rules out a possible nonassociative debilitating effect of halothane on the subsequent performance. We can conclude that halothane has no amnesic effect in our experiment. However, the experimental method is not appropriate to demonstrate a general lack of amnesic effect of this treatment. All the results and especially the poor escape performance of groups Mo-. and M:H,, favor an interpretation in terms of aversive effects of halothane which may interact with those of previous PS. So, we suggest that an halothane anaesthesia, while producing no overt signs of sickness, may sometimes have strong aversive effects which, in the present study, interfere with the escape component of the avoidance learning by increasing the level of conditioned fear. This suggestion is in keeping with the results of Verrier-Gisquet e t a / . [11], Alexinsky and Chapouthier [1], and the work of Vogel and Nathan [12] who, using a conditioned aversion paradigm, have shown

that various hypnotic drugs other than halothane can have punishing effects. if the conclusions of the present paper could be generalized to other experimental conditions, then it would require a reexamination and possibly an alternative interpretation of the results obtained in some previous studies where halothane was regarded solely as an amnesic agent. As already mentioned, the presence of a temporal gradient of amnesia is generally taken as strong evidence for the amnesic effect. However, one must realize that this gradient would also be predicted ff the "amnesic" treatment were simply a negative reinforcer, since the habit strength depends on the temporal contiguity between the response and the reinforcer. This last point emphasizes the need for a properly designed experiment in order to sort out a possible temporal gradient of aversion from a gradient of amnesia following the administration of halothane. ACKNOWLEDGEMENTS

The author gratefully acknowledges the assistance of Mr. P. Petit in helping to improve the English form.

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8. Luschei, E. S. and J. J. Mehaffey. Small animal anaesthesia with halothane. J. appl. Phy.viol. 22: 595-597, 1967. 9. Schmaltz, G. and D. Cl~ment-Forestier. Effets de chocs $1ectriques non contingents sur un apprentissage d'~vitement subs~.quent chez le rat, dens une situation de labyrinthe. C.r. S~anc. Soc. Biol. 170 (3): 620-626, 1976. 10. Seligman, M. E. P. Helplessness. San Francisco: W. H. Freeman & Co., 1975. 11. Verrier-Gisquet, P., T. Alexinsky and B. Dewcer. Les effets amn~siques et aversifs de la narcose au fluothanc au cours de I'extinction. J. Physiol. 71: 348A, 1975. 12. Vogel, J, R, and B. A. Nathan. Learned taste aversions induced by hypnotic drugs. Pharmac. Biochem. Behav. 3: 189-194, 1975. 13. Zinkin, S., J. P. Lecanuet and B. Deweer. Retroactive and proactive effects of anaesthesia on following in chicks. Physiol. Behav. 16: 185-189, 1976.