Atropine

As nonprecocial animals mature, their levels of behavioral arousal and locomotor activity change in consistent and predictable ways. Increases in behavioral arousal early in life have been functionally related to the maturation of dopamine (DA) systems (Campbell, Lytle, & Fibiger, 1969), and drugs that stimulate these systems (e.g., cocaine, amphetamine, apomorphine) typically result in increases in locomotor activity and stereotypical behaviors (Barrett, Caza, Spear, & Spear, 1982; Costall, Naylor, & Neumeyer, 1975). As the subject matures, this DA-related arousal wanes, apparently, with the development of forebrain cholinergic (ACh) systems (Blozovski & Bachevalier, 1975; Fibiger, Lytle, & Campbell, 1970). Thus, these two systems seem to interact in affecting an animal's ability to cope with its environment by modulating its arousal levels.

Behaviors associated with DA- and ACh-related arousal vary with maturation and tend to be age appropriate at different stages in the animal's life. Brewster and Leon (1980) described one such behavior in young rats, the "transport response" (TR), which helps a dam carry the pup from one location to another more easily. In response to the mother's grasping the pup by its dorsal surface, the young rat actively flexes and adducts its hindquarters, extends and adducts its forelimbs, and adducts its tail, producing a rather compact package for transport (Brewster & Leon, 1980).

The TR typically can first be elicited at about 8 days of age; it increases in intensity until approximately 22 days and then declines until about 30 days of age. Thereafter, it is difficult to elicit (Brewster & Leon, 1980). Because the TR occurs when the pup is fairly active and relatively unwieldy (Brewster & Leon, 1980; Wilson, 1985; Wilson, Cullen, & Sendell, 1984), its obvious ecological import is that it enables the mother to quickly and efficiently transport her litter when this would otherwise be very difficult to do.

The TR may be a form of directed behavioral arousal (Wilson, 1985; Wilson, Cromey, & Kramer, 1989). The TR can be suppressed with the DA antagonist haloperidol (Wilson et al., 1984) and, under some circumstances, with the beta-adrenergic antagonist propranolol (Wilson, Koontz, & Seymour, 1994); and it can be induced with the DA agonist apomorphine (Wilson, 1985). It can also be potentiated with external tactile stimuli (Wilson, 1988) like those that induce other goal-directed behaviors - for example, feeding, gnawing, and licking (Antelman & Szechtman, 1975; Antelman, Szechtman, Chin, & Fisher, 1975). In addition, ACh agonists result in decreases in TR intensity, whereas antagonists increase response intensity (Wilson & Cromey, 1989a, 1989b).

Although Wilson (1985, 1988) likened the TR to behavioral arousal, Meyer, Smith, and Van Hartesveldt (1984) suggested that the response may be a form of behavioral inhibition, perhaps related to the dorsal immobility response (DIR) in rats. In fact, given the complexity of the TR, the TR and the DIR may share some common characteristics. The DIR requires that the animal remain immobile and quiescent while being gently grasped and suspended by the nape of the neck, whereas the TR requires that the pup remain quiescent while being firmly grasped by the dorsal surface. Furthermore, Brewster and Leon (1980) reported that they could reliably induce the TR in 40-day-old rats, animals typically too old to show the response, if the animals had been handled for a mere 20 s daily between the ages of 20 and 40 days. Brewster and Leon (1980) asserted that the reliable elicitation of the TR in these animals was attributable to a reduction in the rats' typical defense reactions, such as struggling and biting. Thus, it may be that a reduction in aggressive behaviors and a concomitant increase in quiescence are common variables in both TR intensity and DIR duration.

From a pharmacological viewpoint, ACh appears to be involved in both DIR and TR. The administration of the ACh agonist pilocarpine causes reductions in DIR durations in adult ovariectomized rats, but pretreatment with the antagonist scopolamine blocks this effect (Potter, Cottrell, & Van Hartesveldt, 1990). Similarly, administration of pilocarpine reduces TR intensity in 23-day-old rats (Wilson & Cromey, 1989a). These results, the decrease in limb adduction reported by Wilson and Cromey (1989a), and the decrease in DIR with pilocarpine, led Potter et al. (1990) to suggest that the two responses are closely related.

However, from a pharmacological standpoint, there appears to be at least one major difference in the TR and DIR. Several authors have reported increases in DIR durations with DA antagonists (Byrnes, Ughrin, & Bruno, 1996; Meyer, 1990; Meyer et al, 1984; Van Hartesveldt & Meyer, 1993). Wilson et al. (1984), on the other hand, reported a suppression of the TR with the DA antagonist haloperidol. Thus, it would appear from these reports that the two responses are in conflict, the DIR perhaps being a manifestation of quiescence, and the TR being a manifestation of arousal.

Interestingly, Wilson and Cromey (1989b) showed that the ACh antagonist atropine sulfate produced a reinstatement of the TR in 30-day-old rats. These results led Wilson and Cromey (1989a, 1989b) to propose that the gradual reduction and eventual disappearance of the TR in juvenile rats may be mediated by the development of telencephalic ACh systems and the consequent suppression of DA system(s) responsible for the TR. An alternative explanation for this effect is that blocking the ACh systems may have less to do with the active flexion-extension and adduction of the animals' limbs than with inducing quiescence, a component of the TR necessary for its elicitation. If this is the case, then the TR and DIR may be related through behavioral suppression - that is, quiescence, perhaps subserved by central ACh systems.

The present study was designed to gain a better understanding of the nature of the physiological precursors of arousal-related behaviors that nonprecocial animals go through during adolescence. Specifically, we investigated the effects of varying doses of the central ACh antagonist attopine sulfate on both DIR duration and TR intensity in 40-day-old rats. If there is an increase in quiescence with atropine, then both DIR and TR should increase with administration of the drug. In addition to DIR and TR, we elected to use a third measure of activity, vertical cling catalepsy (VCC), "a failure to correct an externally imposed posture" (Meyer, 1990, p. 533). If atropine increases quiescence, one would expect that administration of the drug would necessarily lengthen the duration of VCC. Therefore, we thought this an appropriate measure for our study. Additionally,we used 40-day-old rats because we wished to reinstate the TR with ACh antagonism, and the response would have already waned considerably by this age.

Because atropine sulfate works in both the central and peripheral nervous systems and we wished to be able to tease apart the central and peripheral effects of this drug on these behaviors, we chose also to test the effects of atropine methylnitrate, an ACh antagonist that does not cross the blood brain barrier. We hypothesized that atropine sulfate would increase both TR intensities and DIR and VCC durations and that there would be a positive correlation among these measures.

Method

Subjects

The subjects in this experiment were 55 Sprague-Dawley albino rats, 40 days of age at the time of testing. Litters were derived from established breeding colonies in the Department of Psychology at Sam Houston State University. All litters were housed in clear Plexiglas breeding cages in a room kept on a 12:12 light:dark schedule with lights on at 0700 h. All testing was done between 0900 h and 1100 h, with each pup being tested at only one drug dosage. During the experimental procedure, the subjects were randomly assigned to one of five drug - dosage groups, each group containing 11 rats.

Materials

Atropine sulfate and attopine methylnitrate were purchased from Sigma Chemical Co., St. Louis, MO. All drugs were dissolved in isotonic saline.

Procedure

Before parturition, pregnant female rats were placed in Plexiglas breeding cages containing wood shavings as nesting material. The cages were checked daily at 0900 h and 1600 h for the presence of newborn litters. The first day a litter was present was recorded as Postpartum Day 0, with litters being culled to 8 to 10 pups on Postpartum Day 1. Pups were weaned from their mothers on Postpartum Day 21 but remained in groups in the breeding cages until the day of testing.

On Postpartum Day 40, the rats were removed from their home cages and placed in breeding cages containing fresh litter. The animals were then randomly assigned to drug groups, with each rat being given an intraperitoneal administration of either isotonic saline, atropine sulfate (7.5 or 15 mg/kg/5 ml), or atropine methylnitrate (7.5 or 15 mg/kg/5 ml). Immediately after each drug administration, the rat was taken to a separate room and marked with a felt-tipped marker for later identification.

Fifteen min after drug administration, the animals were tested for TR intensity, then for vertical cling catalepsy, and finally, for DIR duration. (Specific procedures for each task are presented in the sections that follow.) During testing, the investigator applying the stimulation and the experimenter scoring the responses were unaware of the group to which any animal had been assigned.

Behavioral Testing

Transport response. Testing for TR intensity followed procedures outlined by Wilson et al. (1984). Briefly, the rat was grasped by the nape of the neck between the thumb and first two fingers of an experimenter, suspended above a table top, and firmly squeezed. The subject's response was graded on a scale of 0 to 5; 1 point was awarded for each hind limb, forelimb, and tail that came into contact with the pup's ventrum. Each subject was given three trials with intertrial intervals of approximately 2 min.

Vertical cling catalepsy. Testing for vertical cling catalepsy followed the procedure described by Van Hartesveldt and Meyer (1993). Each rat was placed on a wire mesh screen positioned at an 85 [degrees] angle, and the time it took the animal to move one limb was recorded.

Dorsal immobility. The DIR was measured according to the procedure outlined by Meyer et al. (1984). The rat was gently grasped by the nape of the neck between an experimenter's thumb and index finger and suspended above a tabletop. Timing was begun immediately upon the subject's being suspended and ended when the animal made a directed escape movement, defined as an abrupt turning of its body around toward the experimenter's hand. The animals were given three trials with intertrial intervals of approximately 3 min. The maximum duration for each trial was set at 300 s.

Data Analysis

Data were analyzed with analyses of variance (ANOVAs) and Pearson product-moment correlation coefficients. With respect to the effect of a specific drug regimen on TR and DIR, repeated-measures ANOVAs were used to detect both the effects of the drug and any trends that were apparent over the repeated trials. A one-way ANOVA was used to determine effects of the drags on vertical cling catalepsy. Post hoc tests were carried out using Newman-Keuls a posteriori procedures.

In correlating specific relations among the various behavioral measures, we analyzed data for each drug-dosage group separately. Additionally, when DIR duration or TR intensity was examined, the mean of each measure was used in the analysis.

Results

Data for TR intensity are reported in Table 1. A two-factor ANOVA on these data revealed a significant drug effect, F(4, 50) = 6.29, p [less than] .05, a reliable trials effect, F(2, 100) = 8.55, p [less than] .05, and a nonreliable Drug x Trials interaction effect, F(8, 100) = 1.43, p [greater than] .05. When the data were collapsed across trials, subjects in the 7.5 and 15 mg/kg/5 ml atropine sulfate groups showed significantly more intense TRs than subjects in the saline group. Subjects in the 7.5 mg/kg atropine sulfate group showed more intense TRs than subjects in both atropine methylnitrate groups, and subjects given 15 mg/kg of atropine sulfate had reliably stronger TRs than subjects that received 15 mg/kg of the methylnitrate solution. Finally, rats that received 7.5 mg/kg atropine methylnitrate showed more intense TRs than rats in the saline-control condition. No other differences were statistically reliable. With respect to the repeated trials effect, post roc analyses on these data collapsed across groups revealed that TR intensity in the first trial was significantly weaker than in the second and third trials, which were not different.

Data for vertical cling catalepsy are reported in Table 2. An ANOVA on these data revealed that differences among the groups of this study were not statistically reliable, F(4, 50) = 0.47, p [greater than] .05.

Data for the DIR are presented in Table 3. A two-factor ANOVA revealed that the repeated measures (trials) effect was statistically reliable, F(2, 100) = 14.17, p [less than] .05, but that the drug effect was not significant, F(4, 50) = 1.20, p [greater than] .05. Additionally, the Drug x Trials interaction effect also was not statistically reliable, F(8, 100) = 0.48, p [greater than] .05. When the data were collapsed across drug conditions, post hoc analyses revealed that the DIR duration of the initial trial was longer than the durations of Trials 2 and 3, which did not differ.

Intraclass correlations among the three behavioral measures studied here are reported in Table 4. The only reliable correlation was in the saline-control condition between mean TR intensity and DIR duration, [r.sub.xy](9) = 0.67, p [less than] .05. None of the other relationships among the behavioral variables was statistically reliable.

Discussion

The primary loci of this study were to investigate physiological precursors of arousal-related behaviors; to determine if relationships exist among TR, DIR, and VCC; and if so, to determine if those relationships are based on quiescence induced by cholinergic antagonists.

Results from this study replicated the report by Wilson and Cromey (1989b) that administration of atropine sulfate can produce a reinstatement of the TR in rats typically too old to show the response. Generally, in this study, the rats that received atropine sulfate showed TRs significantly more intense than the rats that received the vehicle solution and those that received the peripherally acting atropine methylnitrate. Administration of the methylnitrate solution also resulted in increases in TR intensity, compared with the saline-treated subjects. That administration of centrally acting atropine sulfate produced reliable increases over and above the increments seen with methylnitrate indicates that there is a central mechanism involved in the reinstatement of the response.

With repeated stimulation, the TR became stronger, regardless of the drug condition. Conceivably, the handling involved in eliciting the TR inadvertently calmed the animals. If this had been the case, though, one might assume that the DIR durations also would have increased with repeated stimulation, but those durations actually decreased over trials. Another possibility is that the tactile stimulation of pinching the nape of the neck induced the release of dopamine (or norepinephrine) and that this resulted in the trial-dependent increases in TR strength. We are in the process of determining how to assess these possibilities.

Because Potter et al. (1990) reported reductions in DIR durations following administration of the ACh agonist pilocarpine, we predicted increases in DIR with the ACh antagonist attopine. No such result was obtained in this study. The discrepancy in our results and those of Potter et al. (1990) may have been attributable to the large intragroup variability. This variability could be a function of inconsistent handling by the experimenter, and we are now reassessing our procedures and remeasuring the effects of blocking (and stimulating) ACh systems on DIR durations in developing animals.

There was no effect of any of the drug conditions on VCC duration reported in this study. Therefore, we propose that ACh systems are not involved in this behavior.

Thus, it appears that administration of atropine affects TR and that increases in TR intensity are not a function of quiescence. If the effect of the drug had been to increase quiescence, thus increasing the probability of induction of the TR, then one would have expected similar increases in DIR and VCC durations. This was not the case. In fact, in the correlations among the various behaviors, the only reliable correlation coefficient recorded was between TR and DIR in the saline-control condition. This relationship disappeared with administration of either atropine methylnitrate or atropine sulfate, and that disappearance occurred in what appears to be a dose-dependent manner.

We are somewhat reticent to put a great deal of stock in the lack of correlation, given that each intraclass correlation contained only 11 observations, but we would like to offer some thoughts. It appears that the loss of relationship between DIR and TR can be accounted for by consistent increases in TR intensities with little change in DIR following administration of atropine. Because the reductions occurred with both the atropine sulfate and the peripherally acting methylnitrate, the decreases in correlation between the two measures may be attributable to peripheral system(s). In this light, the increases in TR intensity with the methylnitrate solution may be the result of having inadvertently induced some amount of sympathetic arousal by blocking the parasympathetic system. Given that Wilson et al. (1989) asserted that the TR is a manifestation of behavioral arousal, it is not surprising that even a modicum of sympathetic arousal could induce the response. In fact, Wilson et al. (1989, 1994) reported that in some instances - for example, when the animal is stressed or is receiving supplemental stimulation - the TR is under control of noradrenergic systems. We currently are investigating the possibility that adrenergic arousal is responsible for the results reported in the present study.

In conclusion, we report here that blocking cholinergic systems causes a reinstatement of the TR with little or no effect on DIR and VCC durations. The effect on TR appears to be attributable to both central and peripheral mechanisms and may be attributable, in part, to indirect adrenergic stimulation. There is little evidence of the TR and DIR being related through quiescence, but any relation between the two may have been hidden by peripheral sympathetic arousal.

The authors wish to thank Rowland S. Miller and A. Jerry Bruce for their assistance in the preparation of this article.

REFERENCES

Antelman, S. M., & Szechtman, H. (1975). Tail pinch induces eating in sated rats which appears to depend on nigrostriatal dopamine. Science, 189, 731-733.

Antelman, S. M., Szechtman, H., Chin, P., & Fisher, A. E. (1975). Tail pinch-induced eating, gnawing and licking behavior in rats: Dependence on the nigrostriatal dopamine system. Brain Research, 99, 319-337.

Barrett, B. A., Caza, P., Spear, N. E., & Spear, L. P. (1982). Wall climbing, odors for the home nest and catecholaminergic activity in rat pups. Physiology & Behavior, 29, 501-507.

Blozovski, D., & Bachevalier, J. (1975). Effects of atropine on behavioral arousal in the developing rat. Developmental Psychobiology, 8, 97-102.

Brewster, J., & Leon, M. (1980). Facilitation of maternal transport by Norway rat pups. Journal of Comparative & Physiological Psychology, 94, 80-88.

Byrnes, E. M., Ughrin, Y., & Bruno, J. P. (1996). Developmental plasticity in the D1- and D2-mediation of motor behavior in rats depleted of dopamine as neonates. Developmental Psychobiology, 29, 653-666.

Campbell, B. A., Lytle, L. D., & Fibiger, H. C. (1969). Ontogeny of adrenergic arousal and cholinergic inhibitory mechanisms in the rat. Science, 166, 637-638.

Costall, B., Naylor, R. J., & Neumeyer, J. L. (1975). Differences in the nature of stereotyped behaviour induced by apomorphine derivatives in the rat and their actions in extrapyramidal and mesolimbic brain areas. European Journal of Pharmacology, 31, 1-16.

Fibiger, H. C., Lytle, L. D., & Campbell, B. A. (1970). Cholinergic modulation of adrenergic arousal in the developing rat. Journal of Comparative & Physiological Psychology, 72, 384-389.

Meyer, M. E. (1990), Dorsal pressure potentiates the duration of tonic immobility and catalepsy in rats. Physiology & Behavior, 47, 531-533.

Meyer, M. E., Smith, R. L., & Van Hartesveldt, C. (1984). Haloperidol differentially potentiates tonic immobility, the dorsal immobility response, and catalepsy in the developing rat. Developmental Psychobiology, 17, 383-389.

Potter, T. J., Cottrell, G. A., & Van Hartesveldt, C. (1990). Effects of cholinergic agonists on the dorsal immobility response. Pharmacology, Biochemistry, & Behavior, 36, 77-80.

Van Hartesveldt, C., & Meyer, M. E. (1993). Differential effects of SCH 23390 on immobility behaviors in developing rats. Developmental Psychobiology, 26, 335-343.

Wilson, C. (1985). The effects of apomorphine and isoproterenol on the "transport response" in the white rat. International Journal of Developmental Neuroscience, 3, 279-284.

Wilson, C. (1988). The effects of sensory stimulation in inducing or intensifying the transport response in white rats. Animal Learning & Behavior, 16, 83-88.

Wilson, C., & Cromey, A. (1989a). The effects of varying doses of pilocarpine on the transport response in white rats. Bulletin of the Psychonomic Society, 27, 138-140.

Wilson, C., & Cromey, A. (1989b). Evidence of a cholinergic input into the suppression of the transport response in white rats. Psychobiology, 17, 43-48.

Wilson, C., Cromey, A., & Kramer, E. (1989). Tactile, maternal, and pharmacologic factors involved in the transport response in rat pups. Animal Learning & Behavior, 17, 373-380.

Wilson, C., Cullen, E., & Sendell, K. (1984). A pharmacologic investigation of the "transport response" in the white rat. International Journal of Developmental Neuroscience, 2, 323-329.

Wilson, C., Koontz, D., & Seymour, T. (1994). Effects of haloperidol and propranolol on rats' transport response to maternal stimuli. The Journal of General Psychology, 121, 147-156.

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