Psychopharmacologia (Berl.) 42, 147-152 (1975) 9 by Springer-Verlag 1975
The Effects of Nicotine on Two-Way Avoidance Conditioning in Bi-Directionally Selected Strains of Rats* JAN C. FLEMING and P. L. BROADHURST University of Birmingham, England Received July 29, 1974; Final Version January 13, 1975 Abstract. Rats of both sexes from the genetically selected Roman High Avoidance (RHA), the Roman Low Avoidance (RLA) and the Roman Control (RCA) strains were given one 30-min session of two-way escapeavoidance conditioning (30 trials) in a shuttle box with a buzzer as the conditioned stimulus and shock as the unconditioned stimulus in a factorial design involving three time intervals (0, 15 and 30 rain) following one subcutaneous injection of saline or of nicotine in five doses (0.05, 0.1, 0.2, 0.4, or 0.8 mg/kg of body weight). Six measures relating to performance were analysed in whole or part. While the
number of avoidance responses showed the expected strain differences, no effect of dose or delay interval could be detected. Avoidance and escape latencies and intertrial activity showed some effects of these variables, especially in interaction with sex and strain. Dose determined pre-sessional activity, but its effect was strain dependent. The failure to confirm previous findingsis discussedin the context of other instances in the literature, and the results of an operant experiment confirmingthe continuing sensitivityof the Roman strains to the effects of nicotine are reported.
Key words: Nicotine - Selection - Avoidance Conditioning - Rat - Latency - Freezing.
Reviews suggest that the effects of nicotine on learning in rats has generally been facilitatory (Bovet, 1965; McGaugh, 1973). But there are indications that its extent is strain dependent and limited by temporal factors such as the delay time interval after administration, especially in pre-test treatments. Thus, the Maudsley strains of rats, bidirectionally selected for extremes of emotional elimination (Broadhurst, 1960, 1962), showed differential response to 0.Smg/kg nicotine tartrate in frequency though not duration of rearing, the emotionally non-reactive strain (MNR) increasing (Garg, 1969b, 1969c), but in a study in which small doses were administered prior to operant responding, the number of rats from the two strains was probably too small to show differential effects (Morrison, 1969). Post-trial administration had no effect on scores in swimming an underwater Y-maze (Wraight et al., 1967), though some decrease in errors of the MNR strain in the Hebb-Williams maze are * Thisstudy, ofwhichapreliminaryaccounthasalready been given (Fleming and Broadhurst, 1970), formed the basis of a thesis submitted by the first author for the degree of MScof the University of Birmingham. The second author is responsible for the present report.
variously reported by Garg and Holland (1967, 1968, 1969). The Roman strains, developed by Bignami and Bovet (1965) and Bignami (1965) for differences in speed of acquisition of two-way avoidance conditioning, have also been studied. Garg (1968, 1969b) reported a similar increase in frequency only of rearing in the high avoidance (RHA) strain. Keenan and Johnson (1972) found a reduction in rearing in the Roman control strain (RCA), not used by Garg. Bovet-Nitti (1966) showed females of both the two selected strains responded to 0.2 mg/kg of nicotine sulphate given prior to a visual discrimination task with a significant improvement of performance in one experiment. But in the conditioned avoidance task for which these strains had originally been selected, Bignami et al. (1965) showed that 0.2 mg/kg of nicotine improved the performance of the low avoidance strain. In contrast is Garg's report (1969a) that 0.8 mg/kg given immediately after a series of single trials in the shuttle box improved performance in neither strain, though it appeared to cause a significant decrease in both strains' scores in the Hebb-Witliams maze (Garg, 1969a; Garg and Holland, 1967).
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These discrepant findings suggested that a systematic study of the effects of nicotine at several time delay intervals in animals of all three R o m a n strains, high, low and control a v o i d a n c e - R H A , R L A and R C A respectively-prior to performance in two-way avoidance conditioning would be of interest. The doses were chosen to span the range from the rather large dose of 0.8 mg/kg typically used by Garg to one of the same order of size (0.05) as that said to approximate the "smoking" dose in man (Silverman, 1971). The delay intervals also ranged over the post-treatment periods typically employed in the literature. A 60-rain period of drug action was investigated with double coverage of the important period 15-45 rain post-administration.
Method Experimental Design. Equal numbers drawn from both sexes within the three Roman strains of rats were used in an avoidance conditioning session of 30 trials lasting30min. The delay intervals were 0, 15, and 30 min and for each interval two rats of each sex and strain were assigned to each of five doses of nicotine, the saline-injected control group being treated as the sixth. The doses used were 0, 0.05, 0.10, 0.20, 0.40, and 0.80 mg/kg of nicotine hydrogen tartrate. Thus with 6 dose levels, 3 delay intervals, 2 sexes and 3 strains the design is a 6 • 3 • 2 • 3 factorial with two replications, requiring a total of 216 subjects who were randomly assigned to treatments within strain and sex, except that no litter mates of like sex received the same treatment, to avoid confounding litter similarity with treatment. Subjects. The albino strains of rats used were founded by Bignami and Bovet, and the development in Rome by bidirectional selective breeding for avoidance response acquisition of the first five generations is described elsewhere (Bignami and Bovet, 1965; Bignami, 1965). After the fifth generation of selection ($5) was tested, the two strains were moved to Birmingham (Broadhurst and Bignami, 1965), followed later by some Wistar rats from the original breeder. These animals were randomly bred in parallel with the sixth generation ($6) of RHA and RLA animals to found the control strain (RCA) which has subsequently been maintained by random breeding, to enable a check to be kept on environmental, including apparatus, constancy. Since S5, selection has been on the basis of the number of avoidances in 25 trials of escape-avoidance conditioning, delivered in one session with a buzzer as the conditioned stimulus (CS) in a Miller-Mowrer shuttle-box. Husbandry and testing are standardized. Fig. 1 indicates the development of the three strains. The RHA and RLA rats used were from $13 whereas the RCA's were from $12. While testing and rearing procedures were otherwise the same as those used in the ongoing selection experiment, these animals, especially bred for this experiment, were tested between 72 and 80 days of age, earlier than usual in the selection experiment because of suggestions of a greater facilitatory effect of nicotine on escape-avoidance conditioning (Robustelli, 1966) and maze learning (Linuchev and Michelson, 1965) in younger rats. The average weights were RHA males 253.2g, females
171.7, RCA males 300.3, females 200.7, and RLA males 230.1, females 165.4.
Apparatus. The apparatus consisted of three Miller-Mowrer shuttle boxes, as described by Levine and Broadhurst (1963), operated simultaneously. The unconditioned stimulus (UCS) was an electric shock of 50 Hz a.c. set at 0.25 mA delivered through a scrambler to the grid floor. The conditioned stimulus (CS) was a buzzer with a fundamental frequency of 100 Hz and measurement, using a Dawe Sound Level Meter, Type 1400F, of the CS in each box with the ventilation blower operating gave sound levels of 88, 92 and 95 db (ref. 0.0002 dynes/sq, cm) for the three boxes.
Procedure. The CS buzzer duration was limited to 18.0 sec, with the UCS (shock) duration 10.0 sec, and a CS-UCS interval of 8.0 sec. Both CS and UCS could be terminated by a rat crossing into the other compartment. Thus a conditioned avoidance response was any crossing with latency less than 8.0 sec, and an escape response was any crossing with a latency between 8.0 and 18.0 sec. If a subject failed to cross within 18.0 sec a "no response" was recorded. Intertrial intervals averaged 60 sec, varying pseudo-randomly between 40 and 80 sec: crossings during them were not punished. These are the parameters employed in the genetic selection of these strains, except that the present subjects were given 30 trials lasting 30 rain instead of 25, in order to achieve an hour's coverage with the delay intervals of 0, 15, and 30rain. Test order was partly determined by age. Treatments were allocated randomly within age and sex, and groups of three having the same delay interval were formed, also determining the allocation to the three boxes. The doses used were 0, 0.05, 0.10, 0.20, 0.40, and 0.80 mg/kg of nicotine hydrogen tartrate, with 0 the designation for normal saline. The nicotine, obtained commercially, was dissolved in 0.9 % physiological (normal) saline in concentrations of 0.025, 0.02, 0.10, 0.20, and 0.40 mg/ml. Injection volume was therefore 2 ml/kg of body weight throughout. Injections were made subcutaneously in the right flank using 1 ml tuberculin syringes and size 20 hypodermic needles. Subjects assigned to the 15- or 30-rain intervals were replaced in their home cages and not disturbed again until the time for testing. In the shuttle boxes, the first CS was delivered after a 2-min habituation period during which the number of crossings was recorded. Trial 1 of the conditioning session was the trial on which a rat first received the shock. The measures obtained were (a) number of avoidance responses, (b) avoidance latencies, (c) escape latencies, (d) number of crossings during the habituation period, (e) number of intertrial crossings, and (f) number of trials with no response. All testing was done between 0830 and 1800hr G.M.T., and in all 256 animals were tested, 88 RCA, 89 RHA, and 79 RLA, of which data for two were rejected because of experimenter error and 38 for apparatus failure. While rejections were spread randomly over treatments, they constituted a not negligible percentage (15.6) of those tested. This conservative approach to possible artefacts caused by apparatus failure was, however, deemed prudent. Results The maximum number of avoidance responses possible was 29, since trial 1 by definition could not result
J. C. Fleming and P. L. Broadhurst: Nicotine and Avoidance Conditioning in Selected Strains of Rats 8G
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(abscissa)and of delay interval (DI) in minutes after administration (receding abscissa). Each point on the surface represents the mean for eight RHA and RCA rats
in such a response. The anticipated strain differences were striking, with means of 19.0 + (SD) 3.9 for the R H A strain, 11.0 _+ 6.9 for the R C A and 1.6 _+ 3.3 for the RLA, or 65.5 %, 37.9 % and 5.5 % respectively which agree well with the percentages for the rest of the animals of the corresponding generations shown in Fig. 1. Animals of variance ( A N O V A ) showed the main effect for strain was significant beyond the 0.1% level, confirming the difference between the means above, but no effect of sex or dose or delay interval was detected. Fig. 2 presents, in part, the data on which this analysis was based, from which it will be clear that few consistent variations are apparent for either dose or delay interval. This impression was confirmed by extensive trend analyses reported by Fleming (1968) together with the details of this and subsequent ANOVA's. The mean avoidance latency of the R H A ' s of 2.8 + 0.8 sec was shorter than that for the R C A rats of 3.8 + 1.2. But the A N O V A revealed no significant main effect for dose or dose interval, though R H A males had shorter latencies than R H A females (2.5 + 0.8 sec vs. 2.9 + 0.8: significant at the 5 % level).
The mean escape latencies for the R H A and R C A strains showed a significant interaction reflecting a longer mean latency for R C A males (10.0 _+ 0.9 sec) than for the other subgroups, all of which have the same means of 9.4 + 0.6 or 0.7. There was no overall effect of dose or interval, though a trend analysis showed suggestions of a significant linear upward trend for the former (Fleming, 1968). The measure of exploratory activity provided by the number of crossings during the habituation period of 2 rain before the first CS was examined for all three strains. A significantly greater mean number of crossings by R H A animals (9.9 + 2.7) was found than with the other two strains (RCA, 7.3 + 2 . 5 ; RLA, 7.1 + 2.6) which did not differ significantly from each other. The main effect for dose is also significant at the 1% level and in a trend analysis the linear component reflects a more marked decrease in crossings for the two highest doses relative to the others including the control group. [ntertrial crossings gave a second measure of activity which showed that, while there was a marked difference, obvious from inspection, between the
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Psychopharmacologia (Berl.), Vol. 42, Fasc. 2 (1975)
strains in the mean number of intertrial crossings (RHA, 10.7 _+ 8.2; RCA, 2.9 + 4.2; RLA, 2.7 + 3.3), there were no other significant effects. The failure to make either an avoidance or an escape response occurred with any frequency only among R L A subjects. Typically the rat, after the first few trials, would freeze, remaining motionless near a wall or corner of the box, at the CS buzzer onset and throughout the duration of the CS with prancing on one spot being often observed during the shock (usages of Miller and Barry, 1960). A significant sex • dose interval interaction was detected in the A N O V A with males showing an increase in freezing from the 0- to 15-rain intervals and then a decrease, and the females freezing less at the 15-rain than at the other two intervals. Finally there were obvious physical effects of the largest dose, the 0.8 mg/kg, including, in some animals, flaccidity, ataxia and tremors within 1 rain of injection which presumably represented the first stages of nicotine-induced convulsions (Orcutt et al., 1963). With the 0.4 dose flaccidity was observed in some animals. These side effects doubtless accounted for some of the decrease in exploratory behaviour noted with this dose. Summarizing, then, the results: the main measure was the number of avoidance responses, in which the expected strain differences were found. Neither dose nor delay interval affected this measure. Of the other measures analysed some effect of dose, dose interval, and their interactions with strain and sex were noted in the avoidance and escape latencies and in intertrial crossings. Dose was an important determinant of exploratory activity but its effect varied between the strains. There was a marked sex • delay interaction in the number of "no responses" made by the RLA strain. Discussion
The results do little to resolve contradictions in the literature regarding the effects of nicotine on learned responses noted above and commented on by reviewers such as McGaugh (1973). On the contrary, they add yet another instance of the failure to find significant effects of nicotine, despite the range of doses and intervals employed. Indeed, the absence of significant effects was so striking as to lead us to suspect artefacts and we investigated two possibilities. The first related to the relatively slight difference in buzzer intensity in the three shuttle boxes we used (see "Apparatus" above). While there was some indication of a differential response in respect of the number of pre-shock avoidances made, it was limited to one strain (the RCA) and was in a direction opposite to that to be
expected if a louder buzzer were occasioning greater response. As regards the number of avoidance responses recorded, the situation was similar, and, moreover, the amount of the variance which could be attributed to the apparatus variable was very small A second possibility was that, despite the apparent facilitation of avoidance by nicotine demonstrated in the RLA strain by Bignami et aL (1965), in our laboratory and many generations later the strains had somehow become unresponsive to the drug's effects, given in the usual doses and tested after typical intervals. We were disabused by the results of a small operant conditioning study using six R H A and two RLA male rats, given either a dose of 0.2 mg/kg nicotine hydrogen tartrate or saline immediately prior to a session of bar pressing in a Skinner box for food reinforcement on an FR10 schedule lasting 1 hr. As may be seen from Figs. 3 and 4, clear effects were found among the R H A subjects, the first three periods and the last showing significant differences from the saline control values, with the single RLA plot exemplifying a similar if somewhat lower overall pattern. The failure in the present study of the RLA strain to show any appreciable acquisition of the avoidance responses at any dose level, including saline, does not necessarily contradict previous findings of greater nicotine-induced facilitation of avoidance acquisition with low performing groups (Bovet, 1965; Bignami et al., 1965). The fact that the RLA strain not only failed to respond to the CS, but frequently failed to respond to the UCS also, invalidates any comparison. Considering also the number of differences between the procedures used by Bovet (1965), Bovet et al. (1961, 1963) and those we used, this lack of confirmation of the specific finding of facilitation of acquisition of the conditioned avoidance response in rats by nicotine with a 15-rain interval is not altogether surprising. A comparison of the findings with respect to acquisition with those of Domino (1965, 1967) where a pole-jumping response was primarily used further illustrates the inconsistency of nicotine effects. Such variable results may be an indication of marked individual differences in dose response relationships which are not uncommon in other, including cholinergic, drugs (Stone, 1964). With respect to the other measures analysed there is little in the literature with which to compare. Avoidance conditioning latency measures are necessarily confounded with frequency of response measures. Thus Broadhurst and Bignami (1965) found a sizeable (0.61) negative correlation between the number of avoidances and their latency in S~ of the R H A and R L A strains. This confounding could account for the shorter mean latency of the R H A strain, and it would
J. C. Fleming and P. L. Broadhurst: Nicotine and Avoidance Conditioning in Selected Strains of Rats
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fect of nicotine on the avoidance conditioning of guinea pigs. Both stimulant (Bovet et al., 1967) and depressant (Morrison and Armitage, 1967; Bhagat and Wheeler, 1973; Stolerman et al., 1974) effects of nicotine on spontaneous and other activity in rats and mice have been reported, which makes it relevant to stress that our subsidiary operant study clearly showed a general stimulant effect of nicotine (0.2 mg/kg s.c.) following the initial depressant effect (Figs. 3 and 4). These findings are in agreement with the results of Morrison (1967) and Morrison and Armitage (1967), and the continuing sensitivity of both strains to the stimulant effect of nicotine and of the R L A ' s to that of amphetamine (Coyle et al., 1973) seems well established.
Acknowledgements. This study was supported by a grant from the Tobacco Research Council of Great Britain, and was facilitated by P.L.B.'s Resident Fellowship for 1973-1974 at NIAS (Netherlands Institute for Advanced Study). Dr. G. Bignami kindly commented on an early draft of the MS. References
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Fig. 4. Effects of nicotine on operant responding. The graph shows the mean number of bar presses (ordinate) by a single RLA rat after one dose of either 0.2 mg/kg of nicotine (solid line) or of saline (dotted line) as a function of time in minutes after injection (abscissa). Compare Fig. 3
seem that, within the R H A strain at least, the avoidance latency measure is more sensitive to drug effects than the frequency measure, such a general sensitivity of latency measures being not uncommon (Kimble, 1961). The two measures of activity (preliminary exploration and intertrial crossings) showed markedly more among the R H A strain, in agreement with the findings of Broadhurst and Bignami (1965) and Holland and Gupta (1966). Indeed, there is evidence that a major difference between the Roman strains is in respect of activity level (Broadhurst and Bignami, 1965; see also Wilcock and Broadhurst, 1967). While there was a decrease in exploratory activity with higher doses of nicotine, there was some increase in activity with the smaller doses though it appeared to be confined to the R C A and possibly R H A strains. Driscoll and B~ttig (1974) have stressed the utility of measures of intertrial crossing and of latencies in detecting the el-
Bhagat, B., Wheeler, N.: Effect of nicotine on the swimming endurance of rats. Neuropharmacology 12, 1161-1165 (1973) Bignami, G.: Selection for high rates and low rates of conditioning in the rat. Anim. Behav. 13, 221-227 (1965) Bignami, G., Bovet, D.: Exp6rience de s61ectionpar rapport h une r6action condition6e d'6vitement chez le rat. C. R. Acad. Sci. (Paris) 260, 1239-1244 (1965) Bignami, G., Robustelli, F., Jankfi, I., Bovet, D.: Action de l'amph6tamine et de quelques agents psychotropes sur l'aquisition d'un conditionnement de fuite et d'6vitement chez des rats selectionn6s en fonction du niveau particuli6rement bas de leurs performances. C. R. Acad. Sei. (Paris) 260, 4273-4278 (1965) Bovet, D.: Action of nicotine on conditioned behaviour in naive and pretrained rats. In: Tobacco alkaloids and related compounds, U. S. v. Euler, ed., pp. 125-136. Oxford: Pergamon 1965 Bovet, D., Bignami, G., Robustelli, F.: Action de la nicotine sur le conditionnement ~t la r6action d'6vitement chez le rat. C. R. Acad. Sci. (Paris) 256, 778-780 (1963) Bovet, D., Bovet-Nitti, F., Oliverio, A.: Action of nicotine on spontaneous and acquired behavior in rats and mice. Ann. N.Y. Acad. Sci. 142, 261-267 (1967) Bovet, D., Gatti, G. L., Pecori-Geraldi, J., Frank, M.: Methode d'enregistrement des r6actions condition6es de fuite dans la cage de Warner. In: Neuropsychopharmacology, E. Rothlin, ed., Vol, 2, pp. 142-146. Amsterdam: Elsevier 1961 Bovet-Nitti, F.: Facilitation of simultaneous visual discrimination by nicotine in rat. Psychopharmacologia (Berl.) 10, 59-66 (1966) Broadhurst, P. L.: Applications of biometrical genetics to the inheritance of behaviour. In: Experiments in personality: Psychogenetics and psychopharmacology. H.
152 J. Eysenck, ed., Vol. 1, pp. 1-102. London: Routledge and Kegan Paul 1960 Broadhurst, P.L.: A note on further progress in a psychogenetic selection experiment. Psychol. Rep. 10, 65-66 (1962) Broadhurst, P.L., Bignami, G.: Correlative effects of psychogenetic selection: A study of the Roman High and Low Avoidance strains of rats. Behav. Res. Ther. 2, 273-280 (1965) Coyle, J. T., Jr., Wender, P., Lipsky, A.: Avoidance conditioningin different strains of rats. Psychopharmacologia (Berl.) 31, 25-34 (1973) Domino, E. F.:Some behavioral actions of nicotine. In: Tobacco alkaloids and related compounds, U. S. v. Euler, ed., pp. 145-166. Oxford: Pergamon 1965 Domino, E.F.: Electroencephalographic and behavioral arousal effects of small doses of nicotine: A neuropsychopharmacological study. Ann. N.Y. Aead. Sci. 142, 216-244 (1967) Driscoll, P., B~ttig, K.: Effects of nicotine on the shuttle box behavior of trained guinea pigs. Psychopharmacologia (Berl.) 38, 47-54 (1974) Fleming, J.: The effects of nicotine in strains of rats selected for escape-avoidance conditioning. Unpublished master's thesis, University of Birmingham (1968) Fleming, J., Broadhurst, P. L.: Nicotine effects on animal behaviour. Paper read to 29th Int. Congr. on Alcoholism and Drug Dependence. Sydney, Australia (1970) Garg, M.: The effect of nicotine on rearing in two strains of rat. Life Sci. 7, 421-429 (1968) Garg, M.: The effect of nicotine on two different types of learning. Psychopharmacologia (Berl.) 15, 408-414 (1969a) Garg, M.: The effects of some nervous system stimulant and depressant drugs on rearing activity in rats. Psychopharmacologia (Berl.) 14, 150-156 (1969b) Garg, M.: Variation in effects of nicotine in four strains of rats. Psychopharmacologia (Berl.) 14, 432-438 (1969 c) Garg, M., Holland, H. C.: Consolidation and maze learning: A comparison of several post-trial treatments. Life Sci. 6, 1987-1997 (1967) Garg, M., Holland, H. C.: Consolidation ~lnd maze learning: A further study of post-trial injections of a stimulant drug (nicotine). Int. J. Neuropharmacol. 7, 55-59 (1968) Garg, M., Holland, H. C.: Consolidation and maze learning: A study of some strain/drug interactions. Psychopharmacologia (Berl.) 14, 426-431 (1969) Holland, H. C., Gupta, B. C.: Some correlated measures of activity and reactivity in two strains of rats selectively Jan C. Fleming, P.O. Box 174, Dickson, ACT 2602, Australia
Psychopharmacologia (Berl.), Vol. 42, Fasc. 2 (1975) bred for differences in the acquisition of a conditioned avoidance response. Anim. Behav. 14, 574-580 (1966) Keenan, A., Johnson, F. N.: Development of behavioural tolerance in the rat. Experientia (Basel) 28, 428-429 (1972) Kimble, G.A.: Hilgard and Marquis' conditioning and learning. New York: Appleton-Century-Crofts 1961 Levine, S., Broadhurst, P. L.: Genetic and ontogenetic determinants of behavior in the rat. J. comp. physiol. Psychol. 56, 423-428 (1963) Linuchev, M. N., Michelson, M. J.: Action of nicotine on the rate of elaboration of food motor conditioned reflexes in rats of different ages. Activ. nerv. sup. (Praha) 7, 25-30 (1965) McGaugh, J. L.: Drug facilitation of learning and memory. Ann. Rev. Pharmacol. 13, 229-241 (1973) Miller, N. E., Barry, H., III.: Motivational effects of drugs: Methods which illustrate some general problems in psychopharmacology. Psychopharmacologia (Berl.) 1, 169-199 (1960) Morrison, C. F.: Effects of nicotine on operant behaviour of rats. Int. J. Neuropbarmacol. 6, 229-240 (1967) Morrison, C. F.:The effects of nicotine on punished behaviour. Psychopharmacologia (Berl.) 14, 221-232 (1969) Morrison, C. F., Armitage, A. K.: Effects of nicotine upon the free operant behavior of rats and spontaneous motor activity of mice. Ann. N.Y. Acad. Sci. 142, 268-276 (1967) Orcutt, J. A., Michaelson, S. M., Prytherch, J. P.: The inhibition of nicotine-induced convulsions in the rat. Arch. int. Pharmacodyn. 146, 238-244 (1963) Robustelli, F.: Azione della nicotina sul condizionamento di salvaguardia di ratti di un mese. Rend. Accad. Naz. Lincei, C1. Sc. fis. mat. nat. 40, 490-497 (1966) Silverman, A. P.: Behaviour of rats given a "smoking dose" of nicotine. Anita. Behav. 19, 67-74 (1971) Stolerman, I. P., Bunker, P., Jarvik, M. E.: Nicotine tolerance in rats; Role of dose and dose interval. Psychopharmacologia (Berl.) 34, 317-324 (1974) Stone, G. C.: Effects of drugs on non-discriminated avoidance behavior. I. Individual differences in dose-response relationships. Psychopharmacologia (Berl.) 6, 245-255 (1964) Wilcock, J., Broadhurst, P. L.: Strain differences in emotionality: open-field and conditioned avoidance behavior in the rat. J. comp. physiol. Psychol. 63,335-338 (1967) Wraight, K. B., Weldon, E., Gupta, B. D., Holland, H. C.: The effects of post-trial injections of nicotine on the learning of an underwater discrimination task by rats. Anita. Behav. 15, 287-290 (1967)
The Effects of Nicotine on Two-Way Avoidance Conditioning in Bi-Directionally Selected Strains of Rats* JAN C. FLEMING and P. L. BROADHURST University of Birmingham, England Received July 29, 1974; Final Version January 13, 1975 Abstract. Rats of both sexes from the genetically selected Roman High Avoidance (RHA), the Roman Low Avoidance (RLA) and the Roman Control (RCA) strains were given one 30-min session of two-way escapeavoidance conditioning (30 trials) in a shuttle box with a buzzer as the conditioned stimulus and shock as the unconditioned stimulus in a factorial design involving three time intervals (0, 15 and 30 rain) following one subcutaneous injection of saline or of nicotine in five doses (0.05, 0.1, 0.2, 0.4, or 0.8 mg/kg of body weight). Six measures relating to performance were analysed in whole or part. While the
number of avoidance responses showed the expected strain differences, no effect of dose or delay interval could be detected. Avoidance and escape latencies and intertrial activity showed some effects of these variables, especially in interaction with sex and strain. Dose determined pre-sessional activity, but its effect was strain dependent. The failure to confirm previous findingsis discussedin the context of other instances in the literature, and the results of an operant experiment confirmingthe continuing sensitivityof the Roman strains to the effects of nicotine are reported.
Key words: Nicotine - Selection - Avoidance Conditioning - Rat - Latency - Freezing.
Reviews suggest that the effects of nicotine on learning in rats has generally been facilitatory (Bovet, 1965; McGaugh, 1973). But there are indications that its extent is strain dependent and limited by temporal factors such as the delay time interval after administration, especially in pre-test treatments. Thus, the Maudsley strains of rats, bidirectionally selected for extremes of emotional elimination (Broadhurst, 1960, 1962), showed differential response to 0.Smg/kg nicotine tartrate in frequency though not duration of rearing, the emotionally non-reactive strain (MNR) increasing (Garg, 1969b, 1969c), but in a study in which small doses were administered prior to operant responding, the number of rats from the two strains was probably too small to show differential effects (Morrison, 1969). Post-trial administration had no effect on scores in swimming an underwater Y-maze (Wraight et al., 1967), though some decrease in errors of the MNR strain in the Hebb-Williams maze are * Thisstudy, ofwhichapreliminaryaccounthasalready been given (Fleming and Broadhurst, 1970), formed the basis of a thesis submitted by the first author for the degree of MScof the University of Birmingham. The second author is responsible for the present report.
variously reported by Garg and Holland (1967, 1968, 1969). The Roman strains, developed by Bignami and Bovet (1965) and Bignami (1965) for differences in speed of acquisition of two-way avoidance conditioning, have also been studied. Garg (1968, 1969b) reported a similar increase in frequency only of rearing in the high avoidance (RHA) strain. Keenan and Johnson (1972) found a reduction in rearing in the Roman control strain (RCA), not used by Garg. Bovet-Nitti (1966) showed females of both the two selected strains responded to 0.2 mg/kg of nicotine sulphate given prior to a visual discrimination task with a significant improvement of performance in one experiment. But in the conditioned avoidance task for which these strains had originally been selected, Bignami et al. (1965) showed that 0.2 mg/kg of nicotine improved the performance of the low avoidance strain. In contrast is Garg's report (1969a) that 0.8 mg/kg given immediately after a series of single trials in the shuttle box improved performance in neither strain, though it appeared to cause a significant decrease in both strains' scores in the Hebb-Witliams maze (Garg, 1969a; Garg and Holland, 1967).
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These discrepant findings suggested that a systematic study of the effects of nicotine at several time delay intervals in animals of all three R o m a n strains, high, low and control a v o i d a n c e - R H A , R L A and R C A respectively-prior to performance in two-way avoidance conditioning would be of interest. The doses were chosen to span the range from the rather large dose of 0.8 mg/kg typically used by Garg to one of the same order of size (0.05) as that said to approximate the "smoking" dose in man (Silverman, 1971). The delay intervals also ranged over the post-treatment periods typically employed in the literature. A 60-rain period of drug action was investigated with double coverage of the important period 15-45 rain post-administration.
Method Experimental Design. Equal numbers drawn from both sexes within the three Roman strains of rats were used in an avoidance conditioning session of 30 trials lasting30min. The delay intervals were 0, 15, and 30 min and for each interval two rats of each sex and strain were assigned to each of five doses of nicotine, the saline-injected control group being treated as the sixth. The doses used were 0, 0.05, 0.10, 0.20, 0.40, and 0.80 mg/kg of nicotine hydrogen tartrate. Thus with 6 dose levels, 3 delay intervals, 2 sexes and 3 strains the design is a 6 • 3 • 2 • 3 factorial with two replications, requiring a total of 216 subjects who were randomly assigned to treatments within strain and sex, except that no litter mates of like sex received the same treatment, to avoid confounding litter similarity with treatment. Subjects. The albino strains of rats used were founded by Bignami and Bovet, and the development in Rome by bidirectional selective breeding for avoidance response acquisition of the first five generations is described elsewhere (Bignami and Bovet, 1965; Bignami, 1965). After the fifth generation of selection ($5) was tested, the two strains were moved to Birmingham (Broadhurst and Bignami, 1965), followed later by some Wistar rats from the original breeder. These animals were randomly bred in parallel with the sixth generation ($6) of RHA and RLA animals to found the control strain (RCA) which has subsequently been maintained by random breeding, to enable a check to be kept on environmental, including apparatus, constancy. Since S5, selection has been on the basis of the number of avoidances in 25 trials of escape-avoidance conditioning, delivered in one session with a buzzer as the conditioned stimulus (CS) in a Miller-Mowrer shuttle-box. Husbandry and testing are standardized. Fig. 1 indicates the development of the three strains. The RHA and RLA rats used were from $13 whereas the RCA's were from $12. While testing and rearing procedures were otherwise the same as those used in the ongoing selection experiment, these animals, especially bred for this experiment, were tested between 72 and 80 days of age, earlier than usual in the selection experiment because of suggestions of a greater facilitatory effect of nicotine on escape-avoidance conditioning (Robustelli, 1966) and maze learning (Linuchev and Michelson, 1965) in younger rats. The average weights were RHA males 253.2g, females
171.7, RCA males 300.3, females 200.7, and RLA males 230.1, females 165.4.
Apparatus. The apparatus consisted of three Miller-Mowrer shuttle boxes, as described by Levine and Broadhurst (1963), operated simultaneously. The unconditioned stimulus (UCS) was an electric shock of 50 Hz a.c. set at 0.25 mA delivered through a scrambler to the grid floor. The conditioned stimulus (CS) was a buzzer with a fundamental frequency of 100 Hz and measurement, using a Dawe Sound Level Meter, Type 1400F, of the CS in each box with the ventilation blower operating gave sound levels of 88, 92 and 95 db (ref. 0.0002 dynes/sq, cm) for the three boxes.
Procedure. The CS buzzer duration was limited to 18.0 sec, with the UCS (shock) duration 10.0 sec, and a CS-UCS interval of 8.0 sec. Both CS and UCS could be terminated by a rat crossing into the other compartment. Thus a conditioned avoidance response was any crossing with latency less than 8.0 sec, and an escape response was any crossing with a latency between 8.0 and 18.0 sec. If a subject failed to cross within 18.0 sec a "no response" was recorded. Intertrial intervals averaged 60 sec, varying pseudo-randomly between 40 and 80 sec: crossings during them were not punished. These are the parameters employed in the genetic selection of these strains, except that the present subjects were given 30 trials lasting 30 rain instead of 25, in order to achieve an hour's coverage with the delay intervals of 0, 15, and 30rain. Test order was partly determined by age. Treatments were allocated randomly within age and sex, and groups of three having the same delay interval were formed, also determining the allocation to the three boxes. The doses used were 0, 0.05, 0.10, 0.20, 0.40, and 0.80 mg/kg of nicotine hydrogen tartrate, with 0 the designation for normal saline. The nicotine, obtained commercially, was dissolved in 0.9 % physiological (normal) saline in concentrations of 0.025, 0.02, 0.10, 0.20, and 0.40 mg/ml. Injection volume was therefore 2 ml/kg of body weight throughout. Injections were made subcutaneously in the right flank using 1 ml tuberculin syringes and size 20 hypodermic needles. Subjects assigned to the 15- or 30-rain intervals were replaced in their home cages and not disturbed again until the time for testing. In the shuttle boxes, the first CS was delivered after a 2-min habituation period during which the number of crossings was recorded. Trial 1 of the conditioning session was the trial on which a rat first received the shock. The measures obtained were (a) number of avoidance responses, (b) avoidance latencies, (c) escape latencies, (d) number of crossings during the habituation period, (e) number of intertrial crossings, and (f) number of trials with no response. All testing was done between 0830 and 1800hr G.M.T., and in all 256 animals were tested, 88 RCA, 89 RHA, and 79 RLA, of which data for two were rejected because of experimenter error and 38 for apparatus failure. While rejections were spread randomly over treatments, they constituted a not negligible percentage (15.6) of those tested. This conservative approach to possible artefacts caused by apparatus failure was, however, deemed prudent. Results The maximum number of avoidance responses possible was 29, since trial 1 by definition could not result
J. C. Fleming and P. L. Broadhurst: Nicotine and Avoidance Conditioning in Selected Strains of Rats 8G
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in such a response. The anticipated strain differences were striking, with means of 19.0 + (SD) 3.9 for the R H A strain, 11.0 _+ 6.9 for the R C A and 1.6 _+ 3.3 for the RLA, or 65.5 %, 37.9 % and 5.5 % respectively which agree well with the percentages for the rest of the animals of the corresponding generations shown in Fig. 1. Animals of variance ( A N O V A ) showed the main effect for strain was significant beyond the 0.1% level, confirming the difference between the means above, but no effect of sex or dose or delay interval was detected. Fig. 2 presents, in part, the data on which this analysis was based, from which it will be clear that few consistent variations are apparent for either dose or delay interval. This impression was confirmed by extensive trend analyses reported by Fleming (1968) together with the details of this and subsequent ANOVA's. The mean avoidance latency of the R H A ' s of 2.8 + 0.8 sec was shorter than that for the R C A rats of 3.8 + 1.2. But the A N O V A revealed no significant main effect for dose or dose interval, though R H A males had shorter latencies than R H A females (2.5 + 0.8 sec vs. 2.9 + 0.8: significant at the 5 % level).
The mean escape latencies for the R H A and R C A strains showed a significant interaction reflecting a longer mean latency for R C A males (10.0 _+ 0.9 sec) than for the other subgroups, all of which have the same means of 9.4 + 0.6 or 0.7. There was no overall effect of dose or interval, though a trend analysis showed suggestions of a significant linear upward trend for the former (Fleming, 1968). The measure of exploratory activity provided by the number of crossings during the habituation period of 2 rain before the first CS was examined for all three strains. A significantly greater mean number of crossings by R H A animals (9.9 + 2.7) was found than with the other two strains (RCA, 7.3 + 2 . 5 ; RLA, 7.1 + 2.6) which did not differ significantly from each other. The main effect for dose is also significant at the 1% level and in a trend analysis the linear component reflects a more marked decrease in crossings for the two highest doses relative to the others including the control group. [ntertrial crossings gave a second measure of activity which showed that, while there was a marked difference, obvious from inspection, between the
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strains in the mean number of intertrial crossings (RHA, 10.7 _+ 8.2; RCA, 2.9 + 4.2; RLA, 2.7 + 3.3), there were no other significant effects. The failure to make either an avoidance or an escape response occurred with any frequency only among R L A subjects. Typically the rat, after the first few trials, would freeze, remaining motionless near a wall or corner of the box, at the CS buzzer onset and throughout the duration of the CS with prancing on one spot being often observed during the shock (usages of Miller and Barry, 1960). A significant sex • dose interval interaction was detected in the A N O V A with males showing an increase in freezing from the 0- to 15-rain intervals and then a decrease, and the females freezing less at the 15-rain than at the other two intervals. Finally there were obvious physical effects of the largest dose, the 0.8 mg/kg, including, in some animals, flaccidity, ataxia and tremors within 1 rain of injection which presumably represented the first stages of nicotine-induced convulsions (Orcutt et al., 1963). With the 0.4 dose flaccidity was observed in some animals. These side effects doubtless accounted for some of the decrease in exploratory behaviour noted with this dose. Summarizing, then, the results: the main measure was the number of avoidance responses, in which the expected strain differences were found. Neither dose nor delay interval affected this measure. Of the other measures analysed some effect of dose, dose interval, and their interactions with strain and sex were noted in the avoidance and escape latencies and in intertrial crossings. Dose was an important determinant of exploratory activity but its effect varied between the strains. There was a marked sex • delay interaction in the number of "no responses" made by the RLA strain. Discussion
The results do little to resolve contradictions in the literature regarding the effects of nicotine on learned responses noted above and commented on by reviewers such as McGaugh (1973). On the contrary, they add yet another instance of the failure to find significant effects of nicotine, despite the range of doses and intervals employed. Indeed, the absence of significant effects was so striking as to lead us to suspect artefacts and we investigated two possibilities. The first related to the relatively slight difference in buzzer intensity in the three shuttle boxes we used (see "Apparatus" above). While there was some indication of a differential response in respect of the number of pre-shock avoidances made, it was limited to one strain (the RCA) and was in a direction opposite to that to be
expected if a louder buzzer were occasioning greater response. As regards the number of avoidance responses recorded, the situation was similar, and, moreover, the amount of the variance which could be attributed to the apparatus variable was very small A second possibility was that, despite the apparent facilitation of avoidance by nicotine demonstrated in the RLA strain by Bignami et aL (1965), in our laboratory and many generations later the strains had somehow become unresponsive to the drug's effects, given in the usual doses and tested after typical intervals. We were disabused by the results of a small operant conditioning study using six R H A and two RLA male rats, given either a dose of 0.2 mg/kg nicotine hydrogen tartrate or saline immediately prior to a session of bar pressing in a Skinner box for food reinforcement on an FR10 schedule lasting 1 hr. As may be seen from Figs. 3 and 4, clear effects were found among the R H A subjects, the first three periods and the last showing significant differences from the saline control values, with the single RLA plot exemplifying a similar if somewhat lower overall pattern. The failure in the present study of the RLA strain to show any appreciable acquisition of the avoidance responses at any dose level, including saline, does not necessarily contradict previous findings of greater nicotine-induced facilitation of avoidance acquisition with low performing groups (Bovet, 1965; Bignami et al., 1965). The fact that the RLA strain not only failed to respond to the CS, but frequently failed to respond to the UCS also, invalidates any comparison. Considering also the number of differences between the procedures used by Bovet (1965), Bovet et al. (1961, 1963) and those we used, this lack of confirmation of the specific finding of facilitation of acquisition of the conditioned avoidance response in rats by nicotine with a 15-rain interval is not altogether surprising. A comparison of the findings with respect to acquisition with those of Domino (1965, 1967) where a pole-jumping response was primarily used further illustrates the inconsistency of nicotine effects. Such variable results may be an indication of marked individual differences in dose response relationships which are not uncommon in other, including cholinergic, drugs (Stone, 1964). With respect to the other measures analysed there is little in the literature with which to compare. Avoidance conditioning latency measures are necessarily confounded with frequency of response measures. Thus Broadhurst and Bignami (1965) found a sizeable (0.61) negative correlation between the number of avoidances and their latency in S~ of the R H A and R L A strains. This confounding could account for the shorter mean latency of the R H A strain, and it would
J. C. Fleming and P. L. Broadhurst: Nicotine and Avoidance Conditioning in Selected Strains of Rats
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fect of nicotine on the avoidance conditioning of guinea pigs. Both stimulant (Bovet et al., 1967) and depressant (Morrison and Armitage, 1967; Bhagat and Wheeler, 1973; Stolerman et al., 1974) effects of nicotine on spontaneous and other activity in rats and mice have been reported, which makes it relevant to stress that our subsidiary operant study clearly showed a general stimulant effect of nicotine (0.2 mg/kg s.c.) following the initial depressant effect (Figs. 3 and 4). These findings are in agreement with the results of Morrison (1967) and Morrison and Armitage (1967), and the continuing sensitivity of both strains to the stimulant effect of nicotine and of the R L A ' s to that of amphetamine (Coyle et al., 1973) seems well established.
Acknowledgements. This study was supported by a grant from the Tobacco Research Council of Great Britain, and was facilitated by P.L.B.'s Resident Fellowship for 1973-1974 at NIAS (Netherlands Institute for Advanced Study). Dr. G. Bignami kindly commented on an early draft of the MS. References
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seem that, within the R H A strain at least, the avoidance latency measure is more sensitive to drug effects than the frequency measure, such a general sensitivity of latency measures being not uncommon (Kimble, 1961). The two measures of activity (preliminary exploration and intertrial crossings) showed markedly more among the R H A strain, in agreement with the findings of Broadhurst and Bignami (1965) and Holland and Gupta (1966). Indeed, there is evidence that a major difference between the Roman strains is in respect of activity level (Broadhurst and Bignami, 1965; see also Wilcock and Broadhurst, 1967). While there was a decrease in exploratory activity with higher doses of nicotine, there was some increase in activity with the smaller doses though it appeared to be confined to the R C A and possibly R H A strains. Driscoll and B~ttig (1974) have stressed the utility of measures of intertrial crossing and of latencies in detecting the el-
Bhagat, B., Wheeler, N.: Effect of nicotine on the swimming endurance of rats. Neuropharmacology 12, 1161-1165 (1973) Bignami, G.: Selection for high rates and low rates of conditioning in the rat. Anim. Behav. 13, 221-227 (1965) Bignami, G., Bovet, D.: Exp6rience de s61ectionpar rapport h une r6action condition6e d'6vitement chez le rat. C. R. Acad. Sci. (Paris) 260, 1239-1244 (1965) Bignami, G., Robustelli, F., Jankfi, I., Bovet, D.: Action de l'amph6tamine et de quelques agents psychotropes sur l'aquisition d'un conditionnement de fuite et d'6vitement chez des rats selectionn6s en fonction du niveau particuli6rement bas de leurs performances. C. R. Acad. Sei. (Paris) 260, 4273-4278 (1965) Bovet, D.: Action of nicotine on conditioned behaviour in naive and pretrained rats. In: Tobacco alkaloids and related compounds, U. S. v. Euler, ed., pp. 125-136. Oxford: Pergamon 1965 Bovet, D., Bignami, G., Robustelli, F.: Action de la nicotine sur le conditionnement ~t la r6action d'6vitement chez le rat. C. R. Acad. Sci. (Paris) 256, 778-780 (1963) Bovet, D., Bovet-Nitti, F., Oliverio, A.: Action of nicotine on spontaneous and acquired behavior in rats and mice. Ann. N.Y. Acad. Sci. 142, 261-267 (1967) Bovet, D., Gatti, G. L., Pecori-Geraldi, J., Frank, M.: Methode d'enregistrement des r6actions condition6es de fuite dans la cage de Warner. In: Neuropsychopharmacology, E. Rothlin, ed., Vol, 2, pp. 142-146. Amsterdam: Elsevier 1961 Bovet-Nitti, F.: Facilitation of simultaneous visual discrimination by nicotine in rat. Psychopharmacologia (Berl.) 10, 59-66 (1966) Broadhurst, P. L.: Applications of biometrical genetics to the inheritance of behaviour. In: Experiments in personality: Psychogenetics and psychopharmacology. H.
152 J. Eysenck, ed., Vol. 1, pp. 1-102. London: Routledge and Kegan Paul 1960 Broadhurst, P.L.: A note on further progress in a psychogenetic selection experiment. Psychol. Rep. 10, 65-66 (1962) Broadhurst, P.L., Bignami, G.: Correlative effects of psychogenetic selection: A study of the Roman High and Low Avoidance strains of rats. Behav. Res. Ther. 2, 273-280 (1965) Coyle, J. T., Jr., Wender, P., Lipsky, A.: Avoidance conditioningin different strains of rats. Psychopharmacologia (Berl.) 31, 25-34 (1973) Domino, E. F.:Some behavioral actions of nicotine. In: Tobacco alkaloids and related compounds, U. S. v. Euler, ed., pp. 145-166. Oxford: Pergamon 1965 Domino, E.F.: Electroencephalographic and behavioral arousal effects of small doses of nicotine: A neuropsychopharmacological study. Ann. N.Y. Aead. Sci. 142, 216-244 (1967) Driscoll, P., B~ttig, K.: Effects of nicotine on the shuttle box behavior of trained guinea pigs. Psychopharmacologia (Berl.) 38, 47-54 (1974) Fleming, J.: The effects of nicotine in strains of rats selected for escape-avoidance conditioning. Unpublished master's thesis, University of Birmingham (1968) Fleming, J., Broadhurst, P. L.: Nicotine effects on animal behaviour. Paper read to 29th Int. Congr. on Alcoholism and Drug Dependence. Sydney, Australia (1970) Garg, M.: The effect of nicotine on rearing in two strains of rat. Life Sci. 7, 421-429 (1968) Garg, M.: The effect of nicotine on two different types of learning. Psychopharmacologia (Berl.) 15, 408-414 (1969a) Garg, M.: The effects of some nervous system stimulant and depressant drugs on rearing activity in rats. Psychopharmacologia (Berl.) 14, 150-156 (1969b) Garg, M.: Variation in effects of nicotine in four strains of rats. Psychopharmacologia (Berl.) 14, 432-438 (1969 c) Garg, M., Holland, H. C.: Consolidation and maze learning: A comparison of several post-trial treatments. Life Sci. 6, 1987-1997 (1967) Garg, M., Holland, H. C.: Consolidation ~lnd maze learning: A further study of post-trial injections of a stimulant drug (nicotine). Int. J. Neuropharmacol. 7, 55-59 (1968) Garg, M., Holland, H. C.: Consolidation and maze learning: A study of some strain/drug interactions. Psychopharmacologia (Berl.) 14, 426-431 (1969) Holland, H. C., Gupta, B. C.: Some correlated measures of activity and reactivity in two strains of rats selectively Jan C. Fleming, P.O. Box 174, Dickson, ACT 2602, Australia
Psychopharmacologia (Berl.), Vol. 42, Fasc. 2 (1975) bred for differences in the acquisition of a conditioned avoidance response. Anim. Behav. 14, 574-580 (1966) Keenan, A., Johnson, F. N.: Development of behavioural tolerance in the rat. Experientia (Basel) 28, 428-429 (1972) Kimble, G.A.: Hilgard and Marquis' conditioning and learning. New York: Appleton-Century-Crofts 1961 Levine, S., Broadhurst, P. L.: Genetic and ontogenetic determinants of behavior in the rat. J. comp. physiol. Psychol. 56, 423-428 (1963) Linuchev, M. N., Michelson, M. J.: Action of nicotine on the rate of elaboration of food motor conditioned reflexes in rats of different ages. Activ. nerv. sup. (Praha) 7, 25-30 (1965) McGaugh, J. L.: Drug facilitation of learning and memory. Ann. Rev. Pharmacol. 13, 229-241 (1973) Miller, N. E., Barry, H., III.: Motivational effects of drugs: Methods which illustrate some general problems in psychopharmacology. Psychopharmacologia (Berl.) 1, 169-199 (1960) Morrison, C. F.: Effects of nicotine on operant behaviour of rats. Int. J. Neuropbarmacol. 6, 229-240 (1967) Morrison, C. F.:The effects of nicotine on punished behaviour. Psychopharmacologia (Berl.) 14, 221-232 (1969) Morrison, C. F., Armitage, A. K.: Effects of nicotine upon the free operant behavior of rats and spontaneous motor activity of mice. Ann. N.Y. Acad. Sci. 142, 268-276 (1967) Orcutt, J. A., Michaelson, S. M., Prytherch, J. P.: The inhibition of nicotine-induced convulsions in the rat. Arch. int. Pharmacodyn. 146, 238-244 (1963) Robustelli, F.: Azione della nicotina sul condizionamento di salvaguardia di ratti di un mese. Rend. Accad. Naz. Lincei, C1. Sc. fis. mat. nat. 40, 490-497 (1966) Silverman, A. P.: Behaviour of rats given a "smoking dose" of nicotine. Anita. Behav. 19, 67-74 (1971) Stolerman, I. P., Bunker, P., Jarvik, M. E.: Nicotine tolerance in rats; Role of dose and dose interval. Psychopharmacologia (Berl.) 34, 317-324 (1974) Stone, G. C.: Effects of drugs on non-discriminated avoidance behavior. I. Individual differences in dose-response relationships. Psychopharmacologia (Berl.) 6, 245-255 (1964) Wilcock, J., Broadhurst, P. L.: Strain differences in emotionality: open-field and conditioned avoidance behavior in the rat. J. comp. physiol. Psychol. 63,335-338 (1967) Wraight, K. B., Weldon, E., Gupta, B. D., Holland, H. C.: The effects of post-trial injections of nicotine on the learning of an underwater discrimination task by rats. Anita. Behav. 15, 287-290 (1967)
