Haloperidol

VERY YOUNG NONPRECOCIAL ANIMALS must coordinate their behaviors with their mothers' behaviors to ensure their survival. Brewster and Leon (1980) reported that infant rats, in response to being grasped on their dorsal surfaces by their mothers, flex and adduct their hindlimbs and extend and adduct their forelimbs. By assuming this compact package, the infant rats enable their mothers to transport them with relative ease. This transport response, which occurs in rat pups between 8 and 28 days of age, can also be elicited with a dorsal pinch by an experimenter.

Wilson, Cromey, and Kramer (1989) proposed that the transport response is a form of directed, or discrete, behavioral arousal. They proposed that a general behavioral arousal, evident in rats within the first week, may become channeled into a very specific, directed transport response when the rat pups reach 8 to 10 days old. As evidence for this assertion, Wilson (1988) reported that tactile stimulation in the form of an air puff, water puff, or tail pinch to the ventrum was able to elicit a transport response in rat pups when a pinch to the nape of the neck was inadequate. These types of stimuli have been shown to induce specific behavioral arousal in developing rats (Szechtman & Hall, 1980).

Wilson, Cullen, and Sendell (1984) reported that administration of haloperidol to 19-day-old rats suppressed the transport response. In addition, administration of apomorphine to 40-day-old rats (rats normally too old to show a response) reinstated the transport response (Wilson, 1985). Wilson asserted that the primary neurochemical underlying the transport response was dopamine, a neurotransmitter system purported to be excitatory to behavior (Sobrian, Weltman & Pappas, 1975). Interestingly, Wilson (1988) reported that under certain circumstances, such as maternal or littermate deprivation and presentation of additional tactile stimulation, haloperidol was ineffective in suppressing the pup's transport response potentiation. Wilson proposed that this result indicated that a second neurotransmitter system may be involved in potentiation of the transport response under certain circumstances.

Various forms of biologically significant maternal stimuli, including vigorous stroking, presentation of maternal saliva, and delivery of milk increase behavioral activity in infant rats (Hall, 1979; Pederson & Blass, 1982; Sullivan, Hofer & Brake, 1986). On the other hand, passive maternal stimuli have been shown to produce emotional quieting in infant rats. This phenomenon, referred to as a "comfort response," occurs in a variety of forms. Research has shown reductions in both glucocorticoid levels (Kuhn, Pauk, & Schanberg, 1990) and cardiac responses (Richardson, Siegel, & Campbell, 1989) in rat pups placed in stressful situations, including maternal separation, or shock, when presented with an anesthetized dam. Additionally, presence of the dam produces reductions in ultrasonic vocalizations (Hofer, Shair, & Murowchick, 1989) and locomotor responses (Richardson et al., 1989) in rat pups placed in stressful situations. Thus, presence of the mother can attenuate arousal, as measured both physiologically and behaviorally.

Presentation of maternal stimuli to infant rats potentiates the transport response (Wilson & Gibson, 1991). Young rat pups, given an inadequate stimulus to induce the response, would assume the response when presented with the mother or with passive stimuli from the mother.

Our purpose in the present study was to investigate neurotransmitter mechanisms underlying potentiation of the transport response following maternal separation and presentation of maternal stimuli in rat pups. Experiment 1 was designed to investigate the effect of blocking dopaminergic systems on transport response potentiation in 16-day-old rats with haloperidol, a dopaminergic antagonist. Experiment 2 was designed to investigate the effect of blocking beta-noradrenergic systems on transport response potentiation in 16-day-old rat pups with propranolol, a noradrenergic antagonist. We used 16-day-old rats in these experiments because pilot work has shown that they are significantly affected by the maternal stimuli we used in this study.

Method

Subjects

In both experiments, we used 16-day-old Sprague-Dawley albino rat pups (N = 240). Litters were derived from breeding colonies maintained in the Division of Psychology and Philosophy at Sam Houston State University. Procedures for the preparation of litters used in these experiments have been described in detail previously (Wilson et al., 1989). All litters were housed with their mothers in clear Plexiglas breeding cages in a room kept at 22 [degrees] C on a 12:12 hr light:dark cycle, with lights on at 0700 hr. All testing occurred between 1000 and 1300 hr.

Apparatus

In these experiments, we used incubators constructed from 10-gal aquaria. The incubators, capable of maintaining an internal temperature of 34 [degrees] C [+ or -] 1 [degree] C with between 40% and 50% humidity, have also been described previously (Wilson et al., 1989).

Procedure

We tested 16-day-old rat pups for presence and intensity of transport response under various drug and stimulus conditions. In both Experiment 1 (n = 120) and Experiment 2 (n = 120), litters were split into two groups 12 hr before testing. One group of pups was placed in the incubators in separate glass compartments. Pups not placed in the incubators at this time remained with the dam in the litter box. Then, 6 hr before testing, the pups that remained with the dam were separated into two groups; one group was placed into separate compartments in the incubators, the other group remained with the dam.

All pups were subjected to drug regimens 30 min before testing. Following these administrations, the pups that had been separated from the dam were returned to their separate containers and placed back into the incubators. Those pups that had remained with the dam were placed together in a clean Plexiglas cage without the dam. Each pup was subjected to only one deprivation condition and one drug condition with only one dose of that drug.

We gave the dam an intraperitoneal administration of ketamine HCI (1.1cc/kg) 15 min before testing. The dam's ventral surface then was shaved, and the hair was put into a covered container.

Pups were removed singly from their containers and tested for transport response potentiation. During the testing, an experimenter held the pup loosely by the nape of the neck to induce dorsal immobility. The pup was then presented with a series of stimuli to determine their effect on induction of the transport response. After all pups had been given their first stimulus presentation, the procedure was repeated until each pup had received its entire sequence of stimulus presentations. During the testing phase, stimuli for each pup were presented in random order. There were four stimulus conditions. In the first condition (M-PRES), the mother's snout was lightly stroked against the pup's snout area. In the second condition (HAIR), an artist's brush that had been covered with the mother's hair was lightly stroked against the pup's snout area. In the third condition (CL-BR), a clean brush was lightly stroked against the pup's snout area. The fourth condition (NO-STIM) was used as a control; therefore, no stimulus was presented. Drug and deprivation conditions remained blind to the experimenters. Scoring of the response intensities was performed by an independent observer.

Transport response intensities were scored according to a schema developed by Brewster and Leon (1980). Each pup's response was graded on a scale of 0-5, with 1 point being awarded for each forelimb, hindlimb, or tail that the pup brought into contact with its ventral surface.

Data Analysis

We used standard parametric procedures to analyze the data (Kirk, 1968). Differences between conditions were assessed using Newman-Keuls post hoc tests. Differences with a chance probability of less than .05 were considered statistically reliable.

Experiment 1

Wilson et al. (1984) reported that administration of haloperidol in rat pups disrupts the transport response. Wilson (1988) reported that under some circumstances (i.e., presentation of specific types of additional tactile stimuli to the pup's ventral area) haloperidol was ineffective in blocking the response. Experiment 1 of the present study was designed to test the effects of haloperidol on a rat pup's ability to show a transport response to maternal stimuli, stimuli that most probably are biologically important.

Procedure

In this experiment, 120 16-day-old rats were deprived of their mothers and littermates for 0, 6, or 12 hr as previously described. Pups were treated with subcutaneous administrations of varying doses (0.0, 1.0, 2.0, and 4.0 mg/kg) of haloperidol 30 min before testing. The pups' transport response intensities then were tested using the four stimulus conditions (M-PRES, HAIR, CL-BR, and NO-STIM).

Results

Data for Experiment 1 are presented in Figure 1. A three-way analysis of variance (ANOVA) revealed reliable drug dose effect, F(3, 108) = 3.44, p [is less than] .05; a reliable deprivation effect, F(2, 108) = 81.57, p [is less than] .05; and a reliable stimulus condition effect, F(3, 324) = 171.95, p [is less than] .05. In addition, the Deprivation X Stimulus Condition interaction was statistically significant, F(6, 324) = 14.90, p [is less than] .05. Neither the Deprivation X Drug Dose nor the Drug Dose X Stimulus Condition interactions were reliable, F(6, 108) = .51, p [is greater than] .05, and F(9, 324) = 1.55, p [is greater than] .05, respectively. Finally, the Drug Dose X Deprivation X Stimulus Condition interaction was not statistically significant, F(18, 324) = .67, p [is greater than] .05.

Post hoc tests revealed larger transport response potentiation for pups that received 2 mg/kg and 4 mg/kg of haloperidol than for pups that received either 1 mg/kg of haloperidol or no haloperidol. Post hoc tests also revealed that increasing deprivation produced increases in response potentiation with each of the stimulus conditions. Presentation of the mother produced greater transport response intensities than did the clean brush and hair brush conditions at any level of deprivation. Presentation of the brush covered with the mother's hair produced a more intense response than presentation of the clean brush at 6 hr of deprivation.

Experiment 2

Wilson et al. (1989) found that the transport response potentiation seen with additional stimulation in rat pups could he partially blocked by treating the pups with propranolol prior to testing. However, Wilson et al. used stimuli--a water squirt and a water puff from an atomizer--that are probably not biologically significant. We designed Experiment 2 of the present study to test the effects of blocking beta-noradrenergic systems with propranolol on transport response potentiation with biologically significant stimuli.

Procedure

As in Experiment 1, 120 16-day-old rats were deprived of their mothers and littermates for varying amounts of time (0, 6, or 12 hr). Pups were treated with intraperitoneal administrations of varying doses (0.0, 10.0, 15.0, and 20.0 mg/kg) of propranolol 30 min before testing. The pups' transport response intensities then were tested using the same additional stimuli as in Experiment 1.

Results

Data from this experiment are presented in Figure 2. A three-way ANOVA revealed a reliable drug dose effect, F(3, 108) = 10.15, p [is less than] .05; a reliable separation effect, F(2, 108) = 34.53, p [is less than] .05; and a reliable stimulus condition effect, F(3, 324) = 99.46, p [is less than] .05. The Drug Dose x Deprivation interaction was significant, F(6, 108) = 3.43, p [is less than] .05, as were the Drug Dose x Stimulus Condition, F(9, 324) = 2.90, p [is less than] .05, and the Deprivation x Stimulus Condition, F(6, 324) = 9.04, p [is less than] .05, interactions. The Drug Dose x Deprivation x Stimulus Condition interaction was not statistically significant, F(18, 324) = 1.62, p [is greater than] .05.

Post hoc tests revealed that all additional stimuli at both 6 hr and 12 hr increased transport response intensities. At 6 hr of deprivation, these increases in response intensity did not arise with any dose of propranolol. At 12 hr of deprivation, transport response potentiation was negated in pups that received 20 mg/kg of propranolol. In all cases, these decreases in response intensity appeared to be dose dependent.

Presentation of the mother to the pup produced stronger transport responses than did the no-stimulus situation and the clean brush condition at all levels of deprivation. At 6 hr of deprivation, presentation of the brush covered with the mother's hair produced greater increases than did the no-stimulus and clean brush conditions. Also, 6 hr and 12 hr of deprivation produced increases in transport response intensity in the mother presentation, hair brush, and clean brush conditions, compared with the 0-hr deprivation condition.

With respect to the Drug Dose x Stimulus Condition interaction, at 0 mg/kg, presentation of the mother produced a reliably greater response potentiation than presentation of the clean brush. Fifteen and 20 mg/kg of propranolol were able to suppress response increments seen with presentations of the mother, the hair brush, and the clean brush.

Discussion

Presentation of maternal stimuli, in the form of the mother or a brush covered with the mother's hair, was able to potentiate the transport response beyond that of the clean brush. This points to the conclusion that, in potentiation of the response, there is more involved than just the tactile stimulation afforded by the brush. Wilson et al. (1989) reported that tactile stimulation alone, in the form of an air puff or tail pinch, could potentiate the transport response. The effect on potentiation with the "clean" stimuli used in the present study clearly was not of the magnitude as that produced by maternal stimuli combined with light tactile stimulation.

Data from the present study also indicate that maternal and littermate deprivation prior to stimulus presentation is an important variable in potentiating the response. Apparently, as time away from the nest passes, the pup becomes more sensitive to stimuli associated with the mother, an effect with obvious survival value. Increasing the probability of the transport response occurring with passive maternal stimuli would aid the mother in being able to efficiently transport the pup back to the nest.

With respect to possible neurochemical systems underlying the response potentiation reported in the present study, haloperidol was ineffective in attenuating the effect of deprivation. In fact, in the present study, pups given 2 and 4 mg/kg of haloperidol showed slight increases in transport response intensity. These results are similar to those reported by Wilson et al. (1989), who found that response potentiation with an air puff to the ventrum in 20-hr deprived rats was not affected by haloperidol. Thus it appears that dopaminergic systems are not involved or are, at best, only partly involved in the potentiation of the transport response with presentation of maternal stimuli.

The data are much more interesting for propranolol than for haloperidol. Propranolol was able to attenuate transport response potentiation with deprivation. One might assume from this finding that the beta-noradrenergic system subserves the effect reported here. Reliable attenuations occurred with all doses of propranolol at 6 hr of deprivation. At 12 hr of deprivation, there was an attenuation of the response potentiation at 15 and 20 mg/kg; however, 10 mg/kg of the drug showed basically no effect at this level of deprivation. Perhaps, with 10 mg/kg of propranolol, either some beta-noradrenergic systems are still intact or many of the systems are partially intact. With respect to the effect of propranolol on individual stimulus conditions, higher doses (15 and 20 mg/kg) were able to attenuate the response potentiation seen with additional stimulation. Again, these reductions were dose dependent for each of the conditions presented.

The results of this study are interesting in a number of ways. First, presentation of maternal stimuli in addition to very light tactile stimuli to rat pups can potentiate the transport response over and above the potentiation seen with tactile stimulation alone. Second, this effect appears to be subserved by beta-noradrenergic systems. Wilson et al. (1989) proposed that potentiation of the transport response with additional tactile stimulation and little or no maternal deprivation was subserved by dopamine systems and that beta-norepinephrine systems served, perhaps, a secondary role in transport response potentiation. Data from the present experiments suggest that, with maternal stimuli, dopamine is not primarily involved in potentiation of the transport response but that beta-norepinephrine is. Disruption of dopamine systems was ineffective in attenuating the potentiation, whereas disruption of beta-norepinephrine systems was effective in disrupting the response potentiation seen with presentation of maternal stimuli.

REFERENCES

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