Time of Day as a Conditioned Stimulus for the Effects of Nicotine, in Rats

Abstract

Research has shown that rats are able to discriminate time of day as well as demonstrate environmentally specific tolerance.  It could then, arguably, be assumed that rats could use time of day as a cue for predicting a drug, such as nicotine.  Based on this premise, 7 Sprague-dawley rats were randomly assigned to one of two squads.  The first squad (n=4), was injected with 0.4 mg/kg of nicotine bitartrate at either 09:30 hrs. or 17:30 hrs..  The second squad (n=3) was injected with the same dosage under the same conditions, at 09:30 hrs. and 17:30 hrs., but they were not assigned to always receive it at one time or the other.  On the test day the dosage was increased to 0.6 mg/kg of nicotine.  For squad one, the rat’s time of nicotine injection was switched.  The rats in squad two’s dosage was also increased, and continued to be injected on the same schedule.  The results of this study found that the rats that had been conditioned to a time of day had less of a tolerance to the drug when the time of day was switched, than the rats who had gone through no conditioning.  These results suggest that the rats in squad one, during their conditioning trials associated time of day with receiving nicotine, which is a conditioned compensatory response.  These findings are valuable in a practical sense because they can be applied to human smoking patterns.

 

                                                      Methods                                               

Subjects

Eight adult, male Sprague-Dawley rats, of varying weight, weighing between 370-496g were used in this study. The eight rats were randomly placed in two squads, the first squad was made up of four rats (n=4).  The second squad was made up of three rats (n=3).  This study was conducted under the schools animal care committee guidelines, and was umbrellaed under the animal lab. 

Materials

The apparatus used to hold the rats during sessions were individual black cubes with no top, measuring 18x18x12.  These cubes were videotaped by a small camera mounted on a movable pole.  The coding process of this study involved a questionnaire, which is seen in the appendix, that requires the observer to count the number of center movements and ataxic behavior that the rat displays in fifteen minutes.  They are then asked to rate the intoxication level of each rat, based on these observations.      

Procedure

Conditioning took place over fourteen days.  Each day the rats were injected twice, once at 09:30 hrs. and once at 17:30 hrs.  During the morning conditioning session, each rat was removed from its cage, and weighted on an electronic scale with a small metal basket.  After being weighed the rat was returned to its cage.  Following the weighing of all the rats, each rat was again individually removed from its cage and injected with either 0.4 mg/kg of nicotine or saline.  The first squad, the experimental group, received nicotine and saline at specific times of day, two received nicotine in the morning and saline in the afternoon, and two received saline in the morning and nicotine in the afternoon. A control group, received nicotine and saline at specific times but the drug received was random.   The experimental group was always injected and observed first and the control group was always injected and observed second.  Each rat was injected intraperitoneally (IP), and was placed into his own individual black cube.  The behaviors of the rats were then observed, measured, and video taped for approximately fifteen minutes after each injection.  The behaviors that were measured were ataxia, center-movement, and general intoxication level.  In the testing session the exact same procedure was used but the time of day that the drug was administered was switched and the dose used was increased from 0.4 mg/kg to 0.6 mg/kg.   

 

Results

Figure 1

            Figure 1. Level of intoxication as a function of

            time of day and dosage.  In sessions 1 and 4 rat

            received nicotine in the AM.  On test day, time of day

            was switched, rat received nicotine in the PM, and

            dosage was increased to 0.6 mg/kg.

 

 

 

 

Figure 2

 

             Figure 2.  Level of intoxication as a function of

             time of day and dosage.  In sessions 1 and 4 rat

             received nicotine in the AM.  On test day, time of day

             was switched, rat received nicotine in the PM, and

             dosage was increased to 0.6 mg/kg.

 

 

 

 

Figure 3

            Figure 3.  Level of intoxication as a function of time

            of day and dosage.  In sessions 1 and 4 rat received

            nicotine in the PM.  On test day, time of day was

            switched, rat received nicotine in the AM, and dosage

            was increased to 0.6 mg/kg.

 

 

 

 

Figure 4

 

          Figure 4.  Level of intoxication as a function of time

             of day and dosage.  In sessions 1 and 4 rat received

             nicotine in the PM.  On test day, time of day was

             switched, rat received nicotine in the AM, and dosage

             was increased to 0.6 mg/kg.

 

 

 

 

Figure 5

 

            Figure 5.  Level of intoxication as a function of time

            of day and dosage.  In sessions 1 and 4 rat received

            nicotine in either AM or PM.  This was continued on

            test day, and dosage was increased to 0.6 mg/kg. 

 

 

 

 

Figure 6

 

                   Figure 6.  Level of intoxication as a function of time

                        of day and dosage.  In sessions 1 and 4 rat received

                        nicotine in either AM or PM.  This was continued on

                        test day, and dosage was increased to 0.6 mg/kg.

 

 

 

 

Figure 7

             Figure 7.  Level of intoxication as a function of time

             of day and dosage.  In sessions 1 and 4 rat received

             nicotine in either AM or PM.  This was continued on

             test day, and dosage was increased to 0.6 mg/kg.

 

 

 

                                        Discussion                                

            On a purely descriptive level these results were in support of the hypothesis. It was shown that after tolerance was obtained at a specific time of the day, a switch in the time of day when the drug was presented, with a greater dose, caused the rat to revert back to baseline or greater drug effects.  This would suggest that the rat had become conditioned to receiving the drug only at the time of day that it was conditioned to. 

In analyzing the results it is apparent that nicotine had a much greater effect than saline on the rat’s intoxication levels, at both times of day.  The intoxication levels of the rats dropped in the pretest trials because tolerance, as defined by Colman (2001), to the drug was achieved.  Therefore, the effect of the nicotine decreased over time because the dose remained unchanged throughout pretest sessions.  At baseline the levels of intoxication were moderate to high.  This was because the rats had never before been exposed to the drug.  Intoxication levels in session 4 were lower because the rats had been exposed to the nicotine and become tolerant (i.e. compensatory response) to its effects.  Tolerance to a specific stimulus, time of day, could not be shown until the test day, when the time of the drug was switched.  These results support the literature on the effect of nicotine on rat’s activity levels, by Ksir (1994).  In his study he showed that relatively low dosed of nicotine cause increased activity.  He also showed that doses higher than 0.4 mg/kg would decrease the activity level of the rat.  It is important to acknowledge however, that the level of activity in his study does not have the same implications as the level of intoxication has in this study.

The change in the level of intoxication between sessions 1 and 4 shows the tolerance that was achieved in this time.  On the test day when the time of drug administration was switched the rats showed an increase in drug effect.  This was because during the conditioning sessions, of rats 9 – 12, a conditioned compensatory response was elicited, to the time of day. These findings are supported by Epstein, Caggiula, and Stiller’s (1989) study, which explains that specific environments can be a cue for the presence of a drug, in this study the environmental cue was time of day.  It is unlikely that any other environmental cues caused the reappearance of the drug effect because all other variables were held constant throughout all sessions.  These findings were not only supported by the literature but also by the results of the second squad of rats, 13-15.  These results show that the rats that had not been conditioned to receive nicotine at a specific time of day, did not have as great of a drug effect on the test day, as the rats that were conditioned.  This is because they had not conditioned a compensatory response.  The establishment of this conditioned compensatory response is possible because, as seen in Mellgren, May, and Haddad (1983), rats have the ability to discriminate time.  Mean, Arolfo, Ginn and Pence (2000) also contributed to this by showing that rats use time of day as an occasion setter.  These studies support the theory that rats have an internal clock, which allows them to decipher when they are receiving a drug.              

Although this study does show that time of day can act as a conditioned stimulus for the compensatory response to the nicotine, some of the results do not support the findings of the study.  These findings are included in the study to ensure validity.  These discrepancies are likely to be due to human error.  Although two different observers coded each session of videotape, to ensure inter-observer reliability, differences in some of the observations may have caused these findings.  Another factor that may have contributed to these discrepancies is the change of time for day light savings.  This occurred the night before the test day.  This was however compensated for by injecting the rats and hour earlier, as though the time change had not occurred.  These discrepancies are not strong enough to change the findings of the study but should be accounted for because of their presence.                                       

  The implications of this study have many practical applications.  It suggested that these findings can be applied to human behavior, and by finding that these rats associate time of day with nicotine, it can be assumed that humans have the same associative tendencies.  What this suggests is that humans may associate smoking cigarettes with a specific time of day.  It also addresses the issue of cravings, the feeling of need, that people tend to have when addicted to cigarettes.  For example, an individual may always smoke a cigarette in the morning when they wake up.  Then one morning they do not smoke, they will crave the cigarette. This is because of their body’s conditioned compensatory response to the nicotine in the morning.  Their body is conditioned to receiving nicotine every morning so it prepares itself for the presence of the drug, and when it is not presented the body has over compensated which causes cravings.  Knowledge of information such as this can help in preventing drug and nicotine use in humans.  By knowing when they will be more attracted to the drug, health professionals can better aid them in overcoming their addictions.

                                                                                                   

References

Colman, A. M. (2001). A dictionary of psychology. New York:

Oxford University Press. 

Epstein, L. H., Caggiula, A. R., & Stiller, R. (1989).

Environment-specific tolerance to nicotine. Psychopharmacology, 97, 235-237.

Ksir, C. (1994). Acute and chronic nicotine effects on measures  

of activity in rats: A multivariate analysis. Psychopharmacology, 115, 105-109.

Means, L.W., Arolfo, M.P., Ginn, S.R., Pence, J.D., &

Watson, N.P. (2000). Rats more readily acquire a time-of-day go no-go discrimination than a time-of-day choice discrimination. Behavioural Processes, 52(1), 11-20.

Melligren, R. L., Mays, M. Z., & Haddad, N. F. (1983).

Discrimination and generalization by rats of temporal stimuli lasting for minutes. Learning and Motivation. 14(1), 75-91. 

Siegel, S., Baptista, M. A.S., Kim, J. A., McDonald, R. V., &

Weise-Kelly, L. (2000). Pavlovian psychopharmacology: The associative basis of tolerance. Experimental and Clinical Psychopharmacology, 8(3), 276-293.

Tolerance and Conditioned tolerance.

http://ibs.derby.ac.uk/~keith/b&b/lect5.html.

 

Underlying Processes in Classical Conditioning Theory.

http://www.radford.edu/~pjackson/cctheory.ppt.