Plasma prolactin levels during conditioned avoidance behavior in rats

Physiology & Behavior, Vol. 34, pp. 441--443.Copyright©PergamonPress Ltd., 1985. Printedin the U.S.A.

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Plasma Prolactin Levels During Conditioned Avoidance Behavior in Rats D. B. YELVINGTON, G. K. W E I S S A N D A. R A T N E R 2 Department o f Physiology, University o f N e w Mexico School o f Medicine, Albuquerque, N M 87131 R e c e i v e d 27 F e b r u a r y 1984 YELVINGTON, D. B., G. K. WEISS AND A. RATNER. Plasma prolactin levels during conditioned avoidance behavior in rats. PHYSIOL BEHAV 34(3) 441-443, 1985.--In these experiments, we examined the prolactin (PRL) response during the acquisition of a conditioned avoidance response (CAR). Rats were tested daily in a two-way shuttle box. They were presented with a light stimulation followed by an electric footshock. During each trial period, the rats were given the opportunity to escape the footshock by moving to a safe side of the box. Movement to the appropriate location after the warning signal (light) begins, but before the onset of the footshock, constitutes a CAR. Blood samples were collected from an indwelling cannula and analyzed by radioimmunoassay. PRL levels increased during early acquisition testing, when the rats had not learned to avoid the shock. After one week of testing, acquisition performance increased considerably (70% CARs) while PRL levels remained unchanged. Thus, we were able to show that as rats learned to modify their behavior in response to a stressful situation, they could also modify their PRL response to the stressor. Prolactin

Stress

Conditioned avoidance response

Avoidance behavior

IN addition to physical stressors, stimuli which seem to arouse fear in animals, or what are termed "psychological stressors," have also been shown to induce release of prolactin (PRL) [3, 8, 9, 10]. In a recent study, we examined the PRL response to a conditioned stress paradigm. For two weeks rats received a light stimulation which was always paired with an inescapable electric footshock. After training, the animals showed a PRL stress response when presented with the light alone. The rats had learned to associate the light with a fearful situation. In this study, we examined the PRL response during the acquisition of a conditioned avoidance response (CAR). In our experimental paradigm, rats were presented with a light stimulation followed by an electric footshock, but in each trial, the rats were also given the opportunity to escape the footshock. We were interested to determine if rats, when they learned to modify their behavior in response to a stressful situation, would also modify their PRL response to the stressor. When the light changes from eliciting fear to becoming a safety signal [2], will the PRL response also disappear?

appropriate times and allowed two days to recover from surgery before experiments were performed. During the experiments, blood draws were made by means of a heparinized syringe (15 U/ml saline) which was connected to a polyethylene extension of the indwelling cannula. This allowed the rat free movement and permitted the experimenter to draw blood without disturbing the animal. To ensure that the animals were kept close to an isovolemic state, fluid replacement was made with each blood draw, using Plasmanate TM (Cutter Labs, Berkeley, CA). All experiments were performed between 1300 and 1730 hr. Blood samples were centrifuged at 3500 rpm for 20 minutes and the plasma separated and stored at -20°C until assayed. Plasma PRL levels were determined in duplicate by radioimmunoassay utilizing a double antibody technique. Rat PRL antibody (rabbit) and rat PRL reference preparations were provided by the Hormone Distribution Program of the NIAMDD. Iodine-labeled PRL was obtained from New England Nuclear Corp. (Boston, MA). The limit of sensitivity of the assay was 3 ng/ml and the interassay variation was 9 %. Statistical analysis was performed by Duncan's multiple range test. A p-value of less than or equal to 0.05 was considered statistically significant. Two groups of rats were used in this study. The first group was tested over a 3-week period. These rats were cannulated 2 days prior to testing, and blood draws were made 10 minutes before and 5, 10, 16, and 25 minutes following placement of the rat in the testing box on day 1. Another group of rats was cannulated after 5, 12, or 20 days of testing, and similar blood draws were made after 7, 14, and 22 days of testing.

METHOD Male Sprague-Dawley rats (400-500 g) obtained from Simonsen Labs (Gilroy, CA) were used in this study. Rats were housed in a climate-controlled area (24_+2°C) with a fixed light:dark cycle. Lights were on at 0500 hr and off at 1900 hr. Rats were given food and water ad lib. Rats were acclimated to the presence of the experimenter by being handled daily for at least 2 weeks prior to experimentation. Animals were fitted with a right atn.'al cannula at

1Preliminary reports of this work were presented at the meetings of the Federation of American Society for Experimental Biology, Chicago, 1983. 2Requests for reprints should be addressed to A. Ratner.

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FIG. 1. The acquisition of avoidance behavior during eight days of training. Rats were tested each day (as described in the Method section) over a three week period. Values are given as mean % conditioned avoidance responses (CARs) for the first eight days (N=8).

FIG. 2. Plasma prolactin (PRL) levels during acquisition of avoidance behavior. At time zero, rats were placed in a two-way shuttle box and tested for acquisition performance (as described in the Method section). Blood draws were made 10 minutes before and 5, 10, 16 and 25 minutes following placement of the rat in the testing box on day 1, day 7, day 14, and day 22 of testing. Values indicate mean plasma PRL levels_+SEMs (N=7-12).

On the first day of testing, each rat was removed from its home cage and placed in a rectangular wooden box equipped with a metal grid floor and a light on each side of the box. The animal was allowed 60 seconds to acclimate to the box. At this time, the light (the conditioning stimulus) on the side of the box where the rat was located was turned on. If the rat moved to the opposite side of the box within 5 seconds of the presentation of the light, the light was turned off and the rat was not shocked. Such a response is termed a conditioned avoidance response (CAR). If however, the rat did not move to the opposite side of the box within 5 seconds, the rat was subjected to a 0.3-miUiamp (mA) footshock. The current and light remained on until the rat moved to the opposite side of the box or until 10 seconds of footshock had elapsed. After a predetermined time (the "intertrial interval"), the light was once again turned on and the procedure repeated. Ten such trials were presented to the rat on each day of testing. The time latency between the light and response was recorded for each trial. In a few cases where the rat did not move to avoid the shock, the latency for that trial was assigned a value of 15 seconds. Latencies of less than or equal to 5 seconds were counted as a CAR and the percentage of CARs performed per day was plotted over time. The curve that resulted is indicative of the rat's ability to learn and/or perform the CAR.

testing at various days during the testing period. On the first day of testing, rats showed a significant, 15-fold change in PRL levels in response to testing. Although the mean values of PRL were still increased some 2- to 3-fold after 1, 2, or 3 weeks of testing, this was not shown to be a statistically significant change.

RESULTS Figure 1 shows the average daily percentage of CARs performed by rats during the first 8 days of acquisition testing. During the first week of testing, rats showed improved acquisition with CARs reaching a level of 70% after 8 days. This level of performance did not change considerably for the remaining days of the experiment. Figure 2 shows the PRL response of rats to acquisition

DISCUSSION Since acquisition testing with a CAR paradigm is a highly stressful experience, it is not surprising that, during the early trials, PRL levels would increase during the testing period. We have recently shown that a PRL response occurs when rats are given only a conditioning stimulus of light that had been previously paired with an unavoidable footshock (manuscript in press). During the early trials in the CAR paradigm described in the present report, the light produces a fear response, since the rat associates it with receiving a footshock. The rats have not learned to avoid the shock, as indicated by the low percent of CARs. After one week of training, the rats have learned to avoid the shock 65% of the time. These rats did not show a significant PRL response during the test periods. At this time, the light has taken on a different characteristic; it no longer produces a fear situation but now becomes a safety signal [2]. The light no longer produces a PRL response as it did when fear resulted with its presentation even though 35% of the time they receive the shock in conjunction with the light. Our results are similar to those of investigators looking at the adrenocortical response to avoidance testing [I, 5, 7]. Berger et al. [1] demonstrated that the reduced levels of plasma corticosterone in rats avoiding 60% of the time was not due to having received fewer shocks and therefore habituating to the stress. A reduced PRL response in "avoiders" is also unlikely to be the result of habituation due to the animal receiving fewer shocks.

PROLACTIN DURING AVOIDANCE BEHAVIOR

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Gentsch et al. [6] recently compared the PRL response to shuttle-box exposure in Roman high-avoidance (RHA) and low-avoidance (RLA) rats. However, they did not subject the animals to acquisition testing during this experiment, but merely used the shuttle box as a "novel environment" in one case, and as an inescapable footshock apparatus in another. Interestingly, RHA and RLA rats both showed PRL increases in response to being shocked while in the box, but only the RLA rats showed an increase in PRL levels in response to merely being placed in the box. We feel that the classification by Gentsch et al. of the shuttle box as a "novel environment" in this situation is a misnomer. Both RHA "rod RLA rats had been exposed to the box prior to experimentation and the difference in PRL responsiveness to merely being placed in the box were due to the fact that RHA rats had learned how to avoid the footshock during prior acquisi-

tion testing in the box, whereas the RLA rats did not. Thus, it is possible that the RHA rats did not see the shuttle box as "threatening," while the RLA rats associated the box with footshock. An important question that still remains: what is the significance of the PRL response that occurs during early training? A reasonable hypothesis is that the PRL increase seen in response to acquisition testing plays a role in facilitating the acquisition-performance of a CAR. A recent attempt to test this hypothesis was made by Drago et al. [4]. They reported that hyperprolactinemic rats showed a significant increase in the acquisition of avoidance behaviors in both pole-jumping and shuttle box testing paradigms. Such results strengthened the idea that PRL released during fearmotivated stress can play a role in facilitating the acquisition of an avoidance behavior.

REFERENCES I. Berger, D. F., J. J. Starzec and E. B. Mason. The relationship between plasma corticosterone levels and leverpress avoidance vs. escape behaviors in rats. Physiol Psychol 9: 81-86, 1981. 2. Bolles, R. C. Species-specific defense reaction and avoidance learning. Psychol Rev 77: 32-48, 1970. 3. Brown, G. M. and J. B. Martin. Corticosterone, prolactin, growth hormone response to handling and new environment in the rat. Psychosom Med 36: 241-247, 1974. 4. Drago, F., B. Bohus and J. A. Mattheij. Endogenous hyperprolactinemia and avoidance behaviors of the rat. Physiol Behav 28: 1-4, 1982. 5. Feldman, J. and G. M. Brown. Endocrine responses to electric shock and avoidance conditioning in the rhesus monkey: cortisol and growth hormone. Psychoneuroendocrinology l: 231242, 1976.

6. Gentsch, C., M. Lichtsteiner, P. Driscoll and H. Feeri. Differential hormonal and physiological responses to stress in Roman high- and low-avoidance rats. Physiol Behav 28: 259-263, 1982. 7. Mason, J. W. A review of psychoendocrine research on the pituitary-adrenal cortical system. Psychosom Med 30: 576-607, 1968. 8. Miyabo, S., T. Asato and M. Mizushima. Prolactin and growth hormone responses to psychological stress in normal and neurotic subjects. J Clin Endocrinol Metab 44: 947-951, 1977. 9. Noel, G. L., H. K. Suh, G. Stone and A. G. Frantz. Human prolactin and growth hormone release during surgery and other conditions of stress. J Clin Endocrinol Metab 35:840-85 l, 1972. 10. Seggie, J. A. and G. M. Brown. Stress response patterns of plasma corticosterone, prolactin, and growth hormone in the rat following handling or exposure to novel environment. Can J Physiol Pharmacol 53: 629-637, 1975.