Atropine

ABSTRACT. Two experiments investigated the mechanism for changes in measures of behavioral arousal inhibition in rats following administration of atropine. In Experiment 1, 40-day-old rats were given administrations of atropine sulfate, the [alpha]-, [beta]-adrenergic blocker labetalol, or both. The drugs, either alone or in combination, increased transport response intensity, whereas both together increased dorsal immobility durations. In Experiment 2, rats were given atropine, the [beta]-adrenergic antagonist propranolol, the [alpha]-adrenergic antagonist phentolamine, or a combination of two of the drugs. Propranolol blocked atropine-induced increases in transport response, and phentolamine was without effect. Phentolamine, when combined with atropine, increased dorsal immobility durations. Results are discussed with respect to aspects common to both transport response and dorsal immobility.

LEVELS OF AROUSAL in non-precocial animals lead to consistent and predictable changes in behavior. Many of the behaviors associated with increased arousal early in life appear to be under the influence of catecholaminergic systems, specifically dopamine (DA) and norepinephrine (Barrett, Caza, Spear, & Spear, 1982; Campbell, Lytle, & Fibiger, 1969; Costall, Naylor, & Neumeyer, 1975), whereas modulation of these behaviors and the related systems occurs later on and appears, at least in part, to be a function of developing cholinergic systems (Blozovski & Bachevalier, 1975; Fibiger, Lytle, & Campbell, 1970). Understanding how these various systems interact may enable the investigator to better understand the effects of particular psychoactive (e.g., cocaine, amphetamine, apomorphine) drugs at different times in an individual's life.

Brewster and Leon (1980) described one arousal-related behavior in young rats, the transport response (TR), which enables the mother to carry her pup more easily. The TR is elicited by the mother's grasping a pup by the nape of the neck with her mouth, suspending it, and firmly biting down. In response, the pup extends and adducts its forelimbs, flexes and adducts its hindlimbs, and adducts its tail, producing a compact package for transport. The TR first occurs at about 8 days of age, increases in intensity until approximately 20 days, then progressively weakens until about 30 days of age, after which it is difficult to elicit (Brewster & Leon). Because the response occurs at a time when the pup ambulates easily and is behaviorally quite active, the mother's ability to quickly subdue the young rat and efficiently transport it has obvious ecological import.

Wilson and his colleagues (Wilson, 1985, 1988; Wilson, Cromey, & Kramer, 1989) proposed that the TR may be a manifestation of behavioral arousal. It can be suppressed with the DA antagonist haloperidol (Wilson, Cullen, & Sendell, 1984), reinstated with the DA agonist apomorphine (Wilson, 1985), and potentiated with presentation of external stimuli (Wilson, 1988), such as those that induce other goal-directed responses, namely, licking, gnawing, and feeding (Antelman & Szechtman, 1975; Antelman, Szechtman, Chin, & Fisher, 1975).

In contrast, Van Hartesveldt and her colleagues (Meyer, Smith, & Van Hartesveldt, 1984; Potter, Cottrell, & Van Hartesveldt, 1990) suggested that the TR might be a form of behavioral inhibition related to the dorsal immobility response (DIR) in rats. Administration of the cholinergic (ACh) agonist pilocarpine produces reductions in DIR durations (Potter et al., 1990) as well as TR intensity (Wilson & Cromey, 1989a). Additionally, Brewster and Leon (1980) reported that the TR could be elicited in 40-day-old rats, animals typically too old to show the response, if the pups had been handled for a mere 20 s daily between the ages of 20 and 40 days. They proposed that this was attributable to a reduction in the animal's typical defense reactions, such as struggling and biting, making elicitation of the TR much more probable.

It may be that the TR comprises two distinct components, an initial quiescence followed by an activation of distinct muscle groups resulting in the characteristic limb adduction. The animal's initial quiescence, a requisite for the TR, may depend on suppression of escape, defensive behaviors, or both, linking it directly to the DIR. If this is the case, these behaviors may be linked through developing ACh systems.

Wilson, Cswaykus, Brocher, and Koenning (1998) investigated the possibility of a cholinergic relationship between TR and DIR by administering atropine to 40-day-old rats and testing for both TR and DIR. They reported that the drug produced increases in TR intensities with little or no effect on DIR, results that were disconcerting given the report of decreases in DIR with pilocarpine administration (Potter et al., 1990), Wilson et al. (1998) proposed that this lack of effect on DIR may have been attributable to indirect adrenergic stimulation resulting from parasympathetic antagonism, and that if the two behaviors are related through quiescence, then this relationship may have been masked by peripheral sympathetic arousal.

To test this possibility, we performed two experiments. In Experiment 1, we administered atropine sulfate ([ATRSO.sub.4]) in combination with the [alpha]-, [beta]-adrenergic receptor blocker labetalol (LAB) to 40-day-old rats and tested for TR and DIR. In Experiment 2, we administered ([ATRSO.sub.4]) in combination with the [alpha]-adrenergic blocker phentolamine (PHENT) or the [beta]-blocker propranolol (PROP) to 40-day-old rats and again measured TR intensity and DIR duration. We hypothesized that if the effects obtained by Wilson et al. (1998) are the result of indirect sympathetic arousal, then we should be able to negate those effects with adrenergic blockers.

Doses of the drugs used in these experiments were based on previous research (Wilson et al., 1984; Wilson et al., 1998; Wilson & Cromey, 1989b). In addition, the dose of ([ATRSO.sub.4]) employed throughout these experiments was used to ensure that we replicated the effects reported by Wilson et al. (1998) and to ensure that we maximized our primary variance.

Method

Subjects

Subjects in these experiments were 120 Sprague-Dawley albino rats, 40 days of age at the time of testing. Animals were derived from litters raised from established breeding colonies at the first author's home institution. Litters were housed on an ad libitum food and water schedule in clear Plexiglas cages in a room kept on a 12 hr light:12 hr dark schedule, with lights on at 7:00 a.m. All testing occurred between 9:00 a.m. and 12:00 p.m., with each pup being tested under only one drug condition.

Materials

([ATRSO.sub.4]), LAB, and PROP were purchased from Sigma Chemical Co., St. Louis, MO. PHENT (Regitine) was kindly supplied by CIBA-GEIGY. All drugs were dissolved in isotonic saline (SAL) prior to administration.

Procedure

Pregnant female rats were placed in Plexiglas breeding cages prior to parturition, and cages were checked daily at 9:00 a.m. and 4:00 p.m. for the presence of newborn litters. The first day a litter was present was recorded as post-partum day 0, and on post-partum day 1, litters were culled to 8 to 10 rats. Pups were weaned from their mothers at 21 days of age but continued to be housed in groups in breeding cages until the day of testing.

At 40 days of age, pups were removed from their home cages, placed in breeding cages containing fresh litter, and randomly assigned to various drug/dosage groups, based on a split-litter design.

Forty-five min prior to testing, each pup was given a subcutaneous administration of either SAL or LAB (Experiment 1) or SAL, PHENT, or PROP (Experiment 2). Thirty min prior to testing, each pup was given an intraperitoneal administration of either SAL or [ATRSO.sub.4]. Following the second drug administration, the pup was removed to a second room and marked with a felt-tipped marker for later identification. At the time of testing, neither the individuals handling the subjects nor the experimenters scoring the responses knew to which group any particular animal had been assigned.

Behavioral Testing

Transport response. Testing for TR intensity followed procedures outlined by Wilson et al. (1984). Briefly, an experimenter grasped the pup by the nape of the neck between the experimenter's thumb and first two fingers and firmly squeezed. Response intensity was recorded on a scale of 0 to 5 with one point awarded for each forelimb, hindlimb, and/or tail that was adducted to the subject's ventrum. The rat was given three trials with approximately 2-min intertrial intervals.

Dorsal immobility. DIR duration was measured using the procedure outlined by Meyer et al. (1984). The pup was gently suspended by the skin of the nape of the neck between the experimenter's thumb and index finger and held firmly without squeezing. Duration was measured from the time of suspension until the pup made an escape response, defined as an abrupt jerking of its body directed at the experimenter's hand. Subjects were given three trials with intertrial intervals of approximately 5 min and a maximum of 300 s for each trial.

Data Analysis

Data were analyzed using standard parametric procedures. Analyses of variance (ANOVAs) were used to determine main and interactive effects, and Newman-Keuls a posteriori procedures were used to determine differences between specific groups. With both TR intensity and DIR duration, repeated measures ANOVAs were used to detect any trends that occurred over the repeated trials.

Experiment 1

LAB is a combined [alpha]-, [beta]-adrenergic receptor blocker (Gilman, Goodman, & Gilman, 1980). If the reinstatement of the TR with atropine in 40-day-old rats is a function of sympathetic arousal, then administration of LAB should negate this effect.

Subjects

Subjects in this experiment were 48 forty-day-old rats. We used a split-litter design, and each animal was randomly assigned to one of four drug/dosage groups (n = 12).

Procedure

The procedure followed that outlined previously. Subjects were given an initial administration of either SAL or LAB (15.0 mg/kg/5ml) with a second administration of either SAL or ATRSO [4] (15.0 mg/kg/5ml). Therefore, this experiment contained the following groups: SAL-SAL, SAL-[ATRSO.sub.4], LAB-SAL, and LAB-[ATRSO.sub.4].

Results and Discussion

Data for TR intensities are presented in Figure 1. A repeated-measures ANOVA revealed a reliable drug effect, F(3, 44) = 5.02, p [less than] .05, and a reliable trials effect, F(2, 88) = 10.72, p [less than] .05. The Drug x Trials interaction effect was not significant, F(6, 88) = 0.68. Post hoc tests on these data collapsed over trials revealed that the animals in the SAL-[ATRSO.sub.4], LAB-[ATRSO.sub.4], and LAB-SAL groups had reliably greater TR intensities than those in the SAL-SAL group. When the data were collapsed across groups, response intensities in Trial 3 were stronger than those in Trial 1. No other differences between groups were statistically reliable.

Mean DIR durations for this experiment are presented in Figure 2. An ANOVA revealed a reliable drug effect, F(3, 44) = 5.36, p [less than] .05, a significant trials effect, F(2, 88) = 13.67, p [less than] .05, and a reliable Drug x Trials interaction, F(6, 88) = 2.42, p [less than] .05. Post hoc analyses revealed that the animals in LAB-[ATRSO.sub.4] had reliably longer DIR durations than all the other groups in Trial 1. In Trial 2, the rats in the LAB-[ATRSO4 group had longer DIRs than those in the SAL-SAL and LAB-SAL groups, and the rats in the SAL-[ATRSO.sub.4] group had longer durations than those in the LAB-SAL group. In Trial 3, the animals in the SAL-[ATRSO.sub.4] and LAB-[ATRSO.sub.4] groups had longer DIRs than those in the SAL-SAL and LAB-SAL groups. No other differences were statistically reliable.

These data show that administration of [ATRSO.sub.4] resulted in a reinstatement of the TR and increases in DIR and that administration of LAB did not suppress these effects. Interestingly, LAB given alone resulted in a reinstatement of the TR, and when LAB was combined with [ATRSO.sub.4], it resulted in an increase in DIR in Trial 1.

With respect to the TR, behavioral inhibition may have occurred, making elicitation of the response much more likely, or the TR may have somehow been stimulated (see Experiment 2), an effect perhaps relating to an intrinsic function of the drug itself. Concerning DIR, it appears that LAB failed to suppress the effect of atropine on the response. However, there may have been a synergism between LAB and [ATRSO.sub.4], an effect resulting in an increase in DIR duration in Trial 1. At this time the mechanism is not known. Regardless, these data lead us to suspect that the TR and DIR are related, perhaps through quiescence, and that the mechanism may involve ACh systems.

Experiment 2

As mentioned, an alternative explanation for the changes reported in Experiment 1 may relate to the intrinsic properties of the drug itself. Even though LAB is classified as a combined [alpha]-, [beta]-adrenergic antagonist, it does not bind to receptors as strongly as other adrenergic antagonists (Gilman et al., 1980). Additionally, [alpha]-adrenergic antagonists have been shown to enhance "the stimulation-induced overflow of NE at sympathetic junctions" (Feldman, Meyer, & Quenzer, 1997, p. 328). More specifically, LAB has been shown to block norepinephrine reuptake (Gilman et al., 1980) and induce [beta]2 agonist activity in low doses (Physicians' Desk Reference, 1996). Therefore, the increase in TR may have been attributable to an increase in [beta]-adrenergic activity.

PROP is a general, non-selective, [beta]-adrenergic antagonist approximately 3 times as potent as LAB with no agonistic properties; PHENT is an [alpha]-adrenergic blocker 10 times as potent as LAB (Gilman et al., 1980). If the effects of LAB reported in Experiment 1 are attributable to the aforementioned properties of that drug, and the increase in TR with [ATRSO.sub.4] is attributable to adrenergic arousal, then antagonists with stronger binding affinities than LAB, that is PROP and PHENT, should block any effects of [ATRSO.sub.4] on the response.

Subjects

Subjects in this experiment were 72 forty-day-old rats, each randomly assigned to one of six drug/dosage groups (n = 12).

Procedure

The procedure in this experiment was identical to that described in Experiment 1 except that the initial drug administration was either SAL, PHENT (15.0 mg/kg/5ml), or PROP (15.0 mg/kg/5m1), followed by a second administration of either SAL or [ATRSO.sub.4] (15 mg/kg/5m1). Thus, this experiment contained subjects in the following groups: SAL-SAL, SAL-[ATRSO.sub.4], PROP-SAL, PROP-[ATRSO.sub.4], PHENT-SAL, and PHENT-[ATRSO.sub.4]. All animals were tested as described previously.

Results and Discussion

Data for TR intensities are presented in Figure 3. A repeated-measures ANOVA revealed a reliable drug effect, F(5, 66) = 9.38, p [less than] .05, and a reliable trials effect, F(2, 132) = 27.04, p [less than] .05. The Drug x Trials interaction was not statistically significant, F(10, 132) = 1.63. Post hoc tests on these data collapsed over trials revealed that subjects in the SAL-[ATRSO.sub.4] and the PHENT-SAL groups had reliably greater TR intensities than subjects in the SAL-SAL, PROP-SAL, and PROP-[ATRSO.sub.4] groups. TRs were stronger for subjects in the PHENT-[ATRSO.sub.4] group when compared with rats in the SAL-SAL, PROP-SAL, PROP-[ATRSO.sub.4], and PHENT-SAL groups. No other differences between groups were statistically reliable. When the data were collapsed across trials, TR intensities in Trial 3 were stronger than in Trial 2, which were stronger than in Trial 1.

Data for DIR durations are presented in Figure 4. A repeated-measures ANOVA revealed a reliable drug effect, F(5, 66) = 7.19, p [less than] .05, and a reliable trials effect, F(2, 132) = 7.44, p [less than].05. The Drug x Trials interaction was not significant, F(10, 132) = 0.83. Collapsing across trials, post hoc tests revealed that subjects in the PHENT-[ATRSO.sub.4] group had longer DIR durations than rats in all other groups. In addition, DIR durations were significantly longer in Trial 1 than in Trials 2 and 3, which did not differ. No other differences were statistically reliable.

In this experiment, [ATRSO.sub.4] again resulted in a reinstatement of the TR. [ATRSO.sub.4] when combined with PHENT resulted in large increases in DIR durations, whereas [ATRSO.sub.4] administered alone caused intermediate, albeit nonreliable, increases in DIR. Any effects of [ATRSO.sub.4] were effectively negated with the [beta]-adrenergic blocker PROP.

General Discussion

The primary focus of this research was to determine if changes in TR intensities and DIR durations following administration of atropine were attributable to indirect induction of sympathetic systems. With respect to TR, labetalol was ineffective in suppressing atropine-induced increases in response intensities (Experiment 1), whereas propranolol abolished those increases (Experiment 2). With respect to DIR, atropine increased response durations. Labetalol failed to negate these increments and even seemed to work synergistically with atropine in Trial 1 to produce large increases in DIR durations. In the second experiment reported here, propranolol appeared to suppress DIR durations, but those effects were not statistically reliable.

Administration of the [alpha]-blocker phentolamine, whether given alone or in combination with atropine, resulted in increases in TR intensities. As with labetalol, phentolamine appeared to work synergistically with atropine, resulting in increases in DIR durations.

The differences between labetalol and propranolol, at least with respect to their effects on TR intensity, may have to do with the natures of the drugs themselves. Labetalol's blocking capabilities are approximately one third of propranolol's (Gilman et al., 1980), and labetalol's lack of effect may have been attributable to its incomplete antagonism. An alternative explanation is that there may have been an interaction between the two effects of labetalol, [alpha]- and [beta]-antagonism. This is an interesting possibility given the increases and decreases in TR intensities recorded with phentolamine and propranolol, respectively, in Experiment 2.

Given the increments in DIR durations reported here, one might argue that increases in TR intensities with phentolamine may have been attributable to the induction of quiescence, a condition that would necessarily make elicitation of the TR much more probable (see Brewster & Leon, 1980). If this is the case, then the two behaviors may be related through both adrenergic and cholinergic systems.

The second component of the TR, the active adduction of the animal's limbs, may require [beta]-noradrenergic activation. This final proposal is not without precedent. Propranolol has been shown to block increments in TR intensity following presentation of supplemental tactile stimulation (Wilson et al., 1989) and following presentation of maternal stimuli (Wilson, Koontz, & Seymour, 1994). Therefore, increments in TR intensity with atropine in this study may be attributable to induction, or release, of [beta]-noradrenergic systems, thereby innervating the behavior.

Potter et al. (1990) stated that "cholinergic agonists and dopamine antagonists have opposite effects on the DIR" and that it is difficult "to characterize dopaminergic-cholinergic interactions with respect to behavior" (p. 80). On the other hand, Wilson and Cromey (1989a, 1989b) stated that cholinergic agonists and dopamine antagonists have the same effects on TR. They argued that the development of cholinergic systems may act to suppress the already-extant DA systems, resulting in reductions in the TR. Data presented here indicate that administration of cholinergic blockers such as atropine induces changes in behaviors and that these changes may be a function of [beta]-adrenergic systems. Therefore, it appears that cholinergic, [beta]-adrenergic (and perhaps [alpha]-adrenergic), and dopaminergic systems all may play pivotal roles in the elicitation of the TR and induction of the DIR. The nature of the interactions of these systems and the sites of their actions are now under investigation.

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