Effects of serotonergic and cholinergic antagonists on suckling behavior of neonatal, infant, and weanling rat pups

BEHAVIORAL AND NEURAL BIOLOGY

41, 99-126 (1984)

Effects of Serotonergic and Cholinergic Antagonists on Suckling Behavior of Neonatal, Infant, and Weanling Rat Pups LINDA A . RISTINE AND LINDA PATIA SPEAR 1

Department of Psychology and Center for Neurobehavtoral Sciences, State University of New York at Binghamton, Binghamton, New York 13901 In Experiment 1, Sprague-Dawley rat pups at postnatal days 3-4, 7-8, 1011, 15-16, and 23-24 were tested for suckling behavior on their anesthesized multiparous dams following administration of metergoline or scopolamine. The serotonergic antagonist, metergoline, inhibited suckling in 3- to 4- and 7- to 8day-old rat pups, but was not found to influence suckling in older pups. Scopolamine, a cholinergic antagonist, reduced suckling primarily in 3- to 4-day-old pups. In Experiment 2, parity of the maternal female was mampulated to assess whether this variable would influence suckling behavior and the effects of metergoline on suckling of weanlings. Baseline levels of suckling were reduced in 23- to 24day-old pups of primiparous dams when compared with multiparous-derived offspring. Administration of metergoline increased the amount of time that these primiparous-derived pups spent attached to nipples, but did not influence suckling of offspring of multiparous dams. perhaps as a result of a ceiling effect on suckling behavior in these animals.

Suckling is one of the most characteristic and complex of the behavior patterns of neonatal mammals. It is a robust and pervasive sequence of behaviors that is absolutely essential for survival in neonates of most mammalian species. Yet, this behavioral sequence completely disappears later in life as suckling becomes gradually superseded by adult-typical forms of ingestion of liquids and solid foods. Suckling may differ from ingestive behaviors in adults not only in terms of the actual motor movements involved, but also in terms of the brain mechanisms subserving these behaviors. For instance, lesions of the ventromedial hypothalamus in adulthood induce hyperphagia, but : This research was supported in part by National Institute of Mental Health Grant R01MH3576101 and a BRSG Grant S07RR07149-09 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institutes of Health. We thank Farmitalia Pharmaceutical Company (Milan, Italy) for their generous supply of the metergoline used in these experiments. Requests for reprints should be sent to Linda Patia Spear, Department of Psychology, SUNY-Binghamton. Binghamton. NY 13901. 99 0163-1047/84 $3.00 Copyrxght © 1984 by Academic Press, lnc, All rights of reproductton lrt any form reserved.

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when the lesions are made within 24 h of birth, they induce retarded growth for the first I0 days of life, which is only then followed by accelerated growth (Hill, Almli, & Williams, 1978). Also, while lateral hypothalamic lesions induce profound aphagia in rats lesioned in adulthood, extensive damage to this area in neonatal rat pups has little influence on food intake at any subsequent age, although such lesions may produce transient growth retardation and an increase in latency to attach to nipples in suckling latency tests. If the damage occurs after the first week of life, however, these same lesions produce an immediate cessation of suckling (Almli, 1978). While brain lesions that influence ingestive behavior in adults are generally ineffective in altering suckling behavior of neonates, there are a few incidental reports that the strong suckling propensity of neonates can be reduced by other means. For example, although Adrien (1978) primarily examined the influence of electrolytic lesions of raphe nuclei in neonates on the ontogeny of sleep, he also mentioned that these lesions disrupted suckling in neonatal rat pups. Similarly, intracisternal administration of the serotonergic neurotoxin 5,6-dihydroxytryptamine on postnatal day 5 has been observed to disrupt suckling behavior of rat pups to the extent that it was often necessary to hand-feed the pups for several days to maintain their viability (Isaacson, Fish, Lanier, & Dunn, 1977). These results suggest that intact serotonergic systems may be critical for the maintenance of suckling during the early postnatal period. Psychopharmacological work supports this suggestion. We have observed that the serotonergic antagonists metergoline, methiothepin, and methysergide all block suckling behavior of 3- to 4-day-old rat pups (Spear & Ristine, 1982). The actual process of suckling consists of a number of different behaviors (e.g., MacFarlane, Pedersem Cornell, & Blass, 1983). Pups first search for a nipple, using wide sweeping movements of the snout. This is followed by probing around the nipple, then mouthing and licking of the nipple which directly precedes attachment itself. Following attachment, pups periodically exhibit sucking (chewing-like movements) of the nipple (e.g., Brake, Sager, Sullivan, & Hofer, 1982) and, during a milk letdown, emit a series of behaviors that presumably facilitates ingestion, including gaping the mouth and a body stretch (Drewett, Statham, & Wakerley, 1974). Serotonergic antagonists disrupt the behavioral sequence of suckling very early in the sequence--prior to attachment. Pups given serotonergic antagonists search and probe for the nipple, but they do not mouth and lick the nipple, nor do they attach (see Spear & Ristine, 1982). Serotonergic antagonists not only attenuate mouthing that precedes nipple attachment, but also mouthing that is elicited in neonates by other stimuli as well. Mouthing induced in neonatal rat pups by intraoral infusions of milk (Caza & Spear, 1982) or by treatment with the serotonergic

SEROTONERGIC AND CHOLINERGIC ANTAGONISTS& SUCKLING 101 agonist quipazine (Ristine & Spear, 1984) is similarly markedly attenuated by serotonergic antagonists. Thus, serotonergic antagonists appear to attenuate mouthing elicited by a variety of situations in the neonatal rat pup, and may block the process of suckling by interfering with the mouthing and licking components of the sequence of behaviors leading to nipple attachment in neonates. The attenuation of suckling by serotonergic antagonists appears to be centrally mediated given that intracisternal administration of small doses of these compounds (much less than would be required to attenuate suckling following systemic administration) blocks suckling in the neonates (Spear & Ristine, 1982). The reduction in the amount of suckling behavior induced by these antagonists does not appear to be a result of a debilitating effect of the drugs on behavior, alterations in body temperature (Spear & Ristine, 1982), nor any drug-induced anosmia (Ristine, Spear, & Spear, 1984). These effects appear to be somewhat specific to the serotonergic system, given that opiate, dopaminergic, a-adrenergic, and/3-adrenergic antagonists do not consistently produce alterations in suckling behavior (Spear & Ristine, 1982). In this work, however, central or peripheral injection of the cholinergic antagonist, scopolamine, was also observed to reduce the amount of time that neonates spent attached to their dams' teats, an effect that did not seem to be related to a drug-induced decrease in salivation, alterations in body temperature, or the elicitation of behaviors incompatible with suckling. While this work suggests that serotonergic antagonists attenuate suckling attachment in neonates, other investigators have reported that serotonergic antagonists increase the propensity of preweanling and weanling rat pups to suckle. Williams, Rosenblatt, and Hall (1979) observed that serotonergic antagonists such as methysergide stimulated suckling in rat pups 15 days of age and older, while serotonergic agonists such as quipazine inhibited suckling in deprived 10- and 21-day-old pups, an effect blocked by methysergide. Taken together, these results suggest the intriguing possibility that an early-maturing serotonergic system may be critical for the initiation of suckling in the neonate, while one or more serotonergic systems maturing later in life may be involved in the normal inhibition of suckling behavior at weaning. To begin to assess the validity of this suggestion, it is essential to examine, within the same laboratory, the transitions in the response to serotonergic antagonists upon suckling behavior throughout the postnatal period. Given that suckling behavior in neonates has also been shown to be influenced by administration of cholinergic antagonists, it also is of interest to examine the ontogeny of this response. The present experiments examine the psychopharmacology of suckling behavior during the crucial developmental interval between the early postnatal period and weaning.

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GENERAL METHODS

Subjects All rat pups used in these experiments were offspring of SpragueDawley-derived breeding pairs bred in our laboratory. The day of birth for a litter was designated as postnatal day 0 (P0) and all litters were culled to 10 pups within 24 h after birth. Litters containing less than 8 pups were not used in these experiments. Subjects were housed with their parents in plastic breeding cages until the time of testing. The colony room was maintained on a 12/12-h light/dark cycle with lights on at 0700 h. All testing occurred between 1100 and 1600 h.

Testing Procedure For the suckling tests, the pups' own dam was used as a suckling stimulus. To remove the active maternal contribution to suckling, the dams were anesthetized prior to the suckling test. This procedure was developed by Hall, Cramer, and Blass (1975, 1977) and has been frequently used in suckling experiments (see Blass, Hall, & Teicher, 1979, for a review). The dams were subcutaneously injected 1 h prior to the initiation of testing with Chloropent (Fort Dodge, 3 cc/kg), an anesthetic that blocks milk ejection (e.g., Williams et al., 1979). During this hour, the litter was removed from the home cage and pups that were 16 days and younger were communally housed in a temperature- and humidity-controlled incubator maintained at an ambient temperature of 31-33°C. Older pups, being nearly homeothermic, were not placed in an incubator and were maintained with littermates at room temperature (24-26°C). Prior to testing, the pups were weighed and numbered on their backs or tails with a marking pen. Half of each litter was tested at one time and each pup was only tested once. In these experiments all drug and vehicle solutions were coded such that the experimenter testing the animals was unaware of the contents of any given injection. The anesthetized dam was placed on her side and tilted at approximately a 45 ° angle toward her back in a test chamber (37 x 19.2 x 10 cm) with a layer of paper towels on the floor. For pups of all ages, the test chamber was maintained at ambient room temperature (approximately 25°C). The suckling test began 5 min after the pups were placed in close proximity to the nipples on the ventrum of their anesthetized dam. Pups were checked for suckling behavior (defined as being attached to a nipple) and other behaviors at the beginning of the test and every 1 min thereafter for a duration of 60 min (a total of 61 checks). Internal body temperatures were taken with a rectal probe (YSI 511) immediately prior to drug injection and after the suckling test. EXPERIMENT 1 In this experiment, the effects of the serotonergic antagonist, metergoline, and the cholinergic antagonist, scopolamine, on suckling behavior were

SEROTONERGIC AND CHOLINERGIC ANTAGONISTS & SUCKLING

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assessed in neonatal, infant, preweanling, and weanling rat pups. Because pharmacological manipulations could interfere with the suckling process by eliciting competing behaviors or through debilitating the animals, the effects of the drugs on other behaviors as well as internal body temperatures were also assessed in this study. Methods

In this experiment 300 offspring of multiparous rat dams were given suckling tests on either postnatal days 3-4, 7-8, 10-11, 15-16. or 2324. Prior to the suckling test, animals were subcutaneously injected with either 0 (0.2% ascorbate vehicle), 1, 2.5, 5, or 10 mg/kg/5 cc of the serotonergic receptor antagonist, metergoline (Farmitalia), or 0 (0.9% saline vehicle), 0.2, 0.8, 1.4, or 2.0 mg/kg/5 cc of the muscarinic cholinergic antagonist, scopolamine (Sigma). Metergoline was injected 25 rain and scopolamine injected 5 min before the beginning of the test session. Suckling test procedures described under General Methods were used. At each check, in addition to assessing whether pups were attached, the behaviors of both attached and unattached pups were recorded. The behavioral categories included lying still (when nonattached), probing (with the snout against an object, predominantly the dam), locomotion (movements translocating the body in space), nipple shifting (disattaching and reattaching to the same or a different nipple within a l-rain time period), twitching, paddling with forelimbs, and treading with hindlimbs. The latter three behavioral categories included behaviors that were recorded when the pups were both attached and nonattached to the nipples. An " o t h e r " category was used to describe other noncategorized behaviors emitted by the pups during the sampling periods. Unattached pups were prompted to suckle at 5-rain intervals by returning pups that were found to be away from the nipples back in close proximity to a nipple, as well as gently rotating the snouts of hand-held pups around the teat at periodic intervals. One animal in each litter was assigned to each of the 10 treatment conditions (5 drug doses for each of the two drugs) at a given testing age. Six animals were tested under each dose and drug condition at each age. Results

Separate 5(age) x 5(drug dose) analyses of variance (ANOVAs) were conducted for each of the two sets of drug conditions for each target behavior. Post hoc tests were then employed to clarify further the results. Where specification of main or interactive effects of Dose were indicated, Dunnett post hoc tests were used. Where specification of Age effects were indicated, Tukey A tests were applied. Given that there may well be ontogenetic differences in the potency of these drugs across age, no direct post hoc comparisons were made between the effects of a particular dose across age, except in the case of zero-dose control animals. ANOVAs

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for main effects of Age were conducted on data from zero-dose controls under both drug conditions in order to evaluate normal developmental changes in each behavior. For each drug condition, a 2 (pretest vs posttest) x 5(age) x 5(drug dose) ANOVA was calculated on the body temperature data. All analyses were specificed as significant at o~ = .05. To the extent that the drug treatments decrease the amount of time pups spend attached to the dam, it might be expected that a variety of behaviors that are only displayed when not attached would be correspondingly increased in these animals when they are compared with control animals that suckle virtually continuously during the 1-h test. Consequently, drug-induced alterations in the elicitation of behaviors displayed while not attached should not be construed to suggest that the drugs would necessarily increase the incidence of these same behaviors in a testing situation where suckling was not an available behavioral option. Yet, it is important to examine the effects of the drugs on behaviors other than suckling in order to assess whether the drugs might attenuate suckling through the elicitation of behaviors incompatible with suckling. If a drug elicits a behavior that competes with suckling, then drug-induced decreases in suckling behavior should be systematically associated with a concomitant drug-induced increase in another behavior. Thus, in the ANOVA results described below, the patterns of drug-induced alterations in suckling were compared with the pattern of drug effects on other behaviors. Metergoline. Figure 1 presents the effect of metergoline on suckling behavior. There were main effects of both Dose (F(4, 125) = 10.73, p ~< .001) and Age (F(4, 125) = 6.57, p ~< .001) on suckling behavior. Three-to four-day-old pups given doses of 2.5, 5, and l0 mg/kg metergoline spent significantly less time attached to the dam than control animals of the same age. Metergoline also decreased suckling in 7- to 8-day-old pups at doses of 5 and 10 mg/kg when compared with controls. Metergoline did not significantly influence suckling at any other age. There were significant Age F(4, 125) = 12.845, p ~< .001), Dose (F(4, 125) = 10.762, p ~< .001), and Dose x Age interaction (F(16, 125) = 2.384, p ~< .01) effects of metergoline on probing behavior. As can be seen in Table 1, doses of 2.5, 5, and 10 mg/kg metergoline increased probing behavior in 3- to 4-day-old pups and the 10 mg/kg dose of metergoline increased probing at 7-8 days of age. Hence, while 3- to 4- and 7- to 8-day-old rat pups rarely attached to their dam's nipples, they did exhibit a great deal of probing, a behavior that is one of the normal sequence of behaviors that leads to attachment in normal animals (see MacFarlane et al., 1983~. There were significant Dose effects (F(4, 125) = 6.513, p ~< .001) of metergoline on locomotor behavior; at doses of 5 and 10 mg/kg, metergoline increased locomotor behavior in all ages of animals tested (see Table 2).

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Fro. 1. Effects of 0, 1, 2.5, 5, and 10 mg/kg metergoline on suckhng behavior of multiparous-derived rat pups at 3-4, 7-8, 10-11, 15-16, and 23-24 days postnatally. ~Slgnificantly different at p ~< .05 from vehicle control pups using Dunnett tests.

Because suckling was not reduced in pups older than P7-8, whereas locomotion was increased in pups of all ages, it does not appear that the reduction in suckling induced by metergoline in 3- to 4- and 7- to 8day-old rat pups is a result of potential drug-induced increases in activity. Additionally, examination of the mean amount of time pups were engaged in locomotor behavior suggested that this behavior occurred relatively infrequently. For instance, at the highest dose of metergoline, P3-4 pups were found to exhibit locomotion only during an average of 4.9% of the sampling periods. Hindlimb treading behavior was also influenced by metergoline (see Table 3). With this behavior, there were significant effects of Age (F(4, 125) = 22.710, p ~< .001) and Dose (F(4, 125) = 7.991, p ~< .001), as well as a significant Age x Dose interaction (F(16, 125) = 2.505, p ~< .025). In 7- to 8-day-old pups, treading was increased by 1 mg/kg metergoline and was decreased by the 10 mg/kg dose of the drug. In addition, P1011 pups decreased treading at doses of 5 and 10 mg/kg metergoline when compared with controls. The effect of metergoline on treading behavior

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SEROTONERGIC AND CHOLINERGICANTAGONISTS & SUCKLING 109 does not appear to account for reductions in suckling behavior given that metergoline markedly reduced suckling, but had no effect on treading, in 3- to 4-day-old pups, and conversely reduced treading, but had no effect on suckling, in 10- and 11-day-olds. Although there was relatively little nipple shifting observed in this testing situation, there were significant Age F(4, 125) = 12.28, p <~ .001) and Dose x Age interaction (F(16, 125) = 1.783, p ~< .05) effects of metergoline on this behavior. As can be seen in Table 4, P23-24 pups given doses of 2.5 and 10 mg/kg metergoline exhibited more nipple shifting than control animals. There were no significant effects of metergoline on body temperature, although there were significant effects of Pretest vs Post-test (F(I, 125) = 102.021, p ~ .001), Age (F(1, 125) = 30.271, p ~< .001), and a Pretest vs Post-test interaction (F(4, 125) = 11.897, p ~< .001) in this analysis. Post-test body temperatures were less than pretest body temperatures in 3- to 4-, 7- to 8-, 10- to 11-, and 15- to 16-day-old animals. Scopolamine. In scopolamine-treated animals, there were significant Age (F(4, 125) = 12.121, p ~< .001), Dose (F(4, 125) = 5.052, p ~< .001), and Age × Dose (F(16, 125) = 1.834, p ~< .05) effects on suckling behavior. At P3-4, doses of 0.8, 1.4, and 2.0 mg/kg scopolamine significantly decreased the number of time periods pups spent attached to the dam (see Fig. 2). Suckling was also decreased by the highest dose of scopolamine in 15- to 16-day-old pups. Scopolamine did not have a significant influence on suckling behavior at the other testing ages. There were also significant Age (F(4, 125) = 5.386, p ~< .001) and Dose (F(4, 125) = 4.458, p <~ .005) effects on probing behavior. Pups at 3-4 days of age probed more frequently than those at 10-11 and 2324 days postnatally, and 15- to 16-day-old pups probed more frequently than 10- to 11-day-old pups (see Table 1). Also, probing was increased at doses of 0.2, 1.4, and 2.0 mg/kg scopolamine when compared with saline control pups. In 3- to 4-day-old pups, the effects of scopolamine appear to be largely restricted to the suckling process because no behaviors other than suckling and probing were significantly affected by the drug. In 15- to 16-day-old pups, the only other behavior affected by scopolamine was locomotion. Analysis of locomotor behavior of scopolamine-treated animals revealed main effects of Dose (F(4, 125) = 2.624, p ~ .05) and Age (F(4, 125) = 15.03, p ~< .01), as well as a Dose x Age interaction (F(16, 125) = 1.815, p ~< .05). At the lowest dose of scopolamine, pups 15-16 days of age increased locomotor behavior and, at doses of 0.8 and 1.4 mg/kg~ pups at 23-24 days of age increased locomotor behavior relative to controls (see Table 2). Scopolamine increases locomotor behavior in adults; hence these effects of scopolamine may represent the emergence of the adult-typical response of scopolamine when measured in terms of

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locomotor activity. One should be cautious, however, with this interpretation, given that this testing situation was not designed to examine the emergence of scopolamine-induced hyperactivity due to the presence of suckling as a behavioral option during testing. The reduction in suckling by scopolamine at 3-4 and 15-16 days of age does not seem to be a result of a drug-induced elicitation of behaviors incompatible with suckling. Scopolamine influences the incidence of only two behaviors other than suckling. Probing was increased by scopolamine at all ages. Scopolamine increased locomotor behavior of 15- to 16-dayold animals only at the lowest dose, whereas suckling was disrupted only at the highest dose of scopolamine at this age. Moreover, scopolamine increased locomotor activity, but did not alter the amount of time that pups spent attached to the dam in 23- to 24-day-old animals. Scopolamine had no significant effect on body temperature. As with metergoline-treated animals, analysis of the body temperature data revealed only significant effects of Pretest vs Post-test (F(1, 125) = 72.781, p ~< .001), Age (F(1, 125) = 31.566, p ~< .001), and a Pretest vs Post-test ×

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Age interaction (F(4, 125) = 7.810, p ~< .001). Post-test body temperatures were less than pretest body temperatures for all age groups except for the 23- to 24-day-old animals. Behaviors e x p r e s s e d as percentage o f time while not attached. Another approach was used to examine potential competing behaviors at ages where the drug manipulations were found to significantly influence suckling. These analyses examined whether drug-treated and control animals spent an equivalent percentage of time while not suckling exhibiting behaviors mutually exclusive with attachment. Because these analyses obviously required that animals be unattached for at least one sampling period, some animals could not be included. Due to the small number of animals representing some of the treatment conditions, Mann-Whitney U tests were used for these comparisons between each drug-treatment group and the control group (pooled across the two vehicle conditions). These analyses revealed only two significant differences: 7- to 8-day-old pups given 10 mg/kg metergoline spent a greater percentage of time locomoting while not attached than control animals (U -- 1, p ~< .016) and 15- to 16-day-old pups given 2 mg/kg scopolamine spent a great percentage of nonattachment time probing than control animals (U = 0, p ~ .036). Given that there were so few significant differences in these analyses, it appears that drug-treated and control animals do not differ substantially in their proportioning of time to specific behaviors when not attached. Hence, these results reinforce the conclusions reached above that the antagonists do not appear to interfere with suckling through eliciting competing behaviors. However, this interpretation should be tempered by the consideration that there was only a limited amount of data available on the behavior of nonattached control animals; as seen below, most control animals were attached throughout the entire test. For instance, in 3- to 4-day-old control animals, 9 out of 12 pups were attached at all sampling periods, with the remaining 3 animals being unattached for only I, 6, and 33 sampling periods, respectively. Control behavior across age. In addition to assessing psychopharmacological responsiveness during ontogeny, this experiment provided the opportunity to observe the behavioral profile of suckling rat pups during ontogeny. For these age comparisons across control animals, data from the two vehicle control groups (saline and ascorbate vehicle) were collapsed as preliminary analyses revealed no significant differences among these two control groups at any age. As is evident from Table 5, suckling was the prominent behavior of all control pups in this testing situation. Control pups of all ages spent between 77 and 100% of the 1-h test time attached to their dam's nipples, despite the fact that they were receiving no milk. In this study, age did not appear to have a significant effect on the absolute amount of suckling exhibited by these animals. The incidence of nipple shifting was low at

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all ages in this testing situation. For 15- to 16- and 23- to 24-day-old pups, 4 pups out of 12 and 3 pups out of 12, respectively, nipple shifted, whereas only 1 pup nipple shifted at each of the three younger ages (P34, P7-8, P10-11). This low incidence of nipple shifting is not surprising, given that the pups were separated from the dam only 1-2 h prior to testing. Cramer, Blass, and Hall (1980) have reported that the incidence of nipple shifting is very low throughout the preweaning period in nondeprived and 4-h-deprived rat pups tested in a situation without milk letdowns, whereas a marked ontogenetic increase in nipple shifting is seen beginning at approximately 15 days of age in pups deprived of the dam and food for 8 or 24 h prior to testing. There was an increased incidence of twitching in 7- to 8-day-old control pups when compared with pups of other ages. Twitching is presumably indicative of active sleep early in life (Jouvet-Mounier, Astic, & LaCote, 1970). The difference in the magnitude of twitching behavior between P7-8 and older pups may be a result of the emergence of slow-wave sleep around 10 days postnatally (see Gramsbergen, 1974). In this test situation, control pups at 7-8 and 10-11 days of age were found to exhibit more hindlimb treading and forelimb paddling movements when compared with younger and older animals. These behaviors were observed most frequently in pups that were suckling. Treading and paddling may aid in maintaining contact with the nipple and proximity to the dam's ventrum. The increase in treading and paddling during the first week of life may reflect the development of greater motoric competence or the ability to sustain movements for a longer period of time, while the diminution of these behaviors in 15- to 16- and 23- to 24-day-old pups may be related to increased body size and more refined motor movements that may serve to facilitate maintenance of suckling postures. Under this test condition, weanling control pups (P23-24) exhibited more locomotor activity than younger pups. However, the absolute amount of locomotion exhibited by control pups was low (less than 3% of the total test time even in 23- to 24-day-old control animals). This low incidence of locomotion is not surprising, given that control pups spent the majority of the test time attached to their dam's nipples. Interpretation of the developmental changes in behaviors other than suckling must be tempered by the observation that in this testing situation suckling is the predominant response in pups of all ages. While we observed that several behaviors showed predictable alterations with age, the overall incidence of these behaviors may well be less than would be predicted in a testing situation where suckling was not an available behavioral option. Discussion

In this experiment, the serotonergic antagonist, metergoline, and the cholinergic antagonist, scopolamine, were observed to decrease attachment

SEROTONERGIC AND CHOLINERGICANTAGONISTS & SUCKLING 115 time primarily in neonatal rat pups. Metergoline was shown to reduce the amount of time that 3- to 4- and 7- to 8-day-old rat pups spent attached to the dams' nipples, while scopolamine produced a reduction in suckling principally in 3- to 4-day-old pups, although the highest dose of scopolamine significantly reduced attachment time in 15- to 16-day-old animals as well. The inhibitory effects of these drugs on attachment did not appear to be a result of the drugs debilitating the animals or inducing behaviors incompatible with the process of attaching to a teat. Indeed, drug-treated animals spent a great deal of time when not attached probing for the nipple, a behavior that is part of the normal sequence of behavior leading to nipple attachment (MacFarlane et al., 1983). Collectively, these data can be interpreted to suggest that both serotonin and acetylcholine may play an important role in facilitating the suckling process in neonatal rat pups. EXPERIMENT 2

We have previously suggested that serotonin may be important in facilitating suckling behavior of the neonatal rat pup, given our observations that serotonergic antagonists potently attenuate suckling attachment in 3- to 4-day-old animals (Spear & Ristine, 1982). The results of Experiment 1 confirm and extend this finding to neonates through the first postnatal week. However, we observed that the serotonergic antagonist, metergoline, had no systematic influence on maintenance of suckling behavior of pups from 10- to 24 days of age. These results in older pups are inconsistent with other reports in which serotonergic antagonists were found to stimulate suckling in animals beginning at 15-20 days postnatally (Williams et al., 1979). One possible reason for the failure to observe a metergolineinduced increase in suckling in weanlings in Experiment 1 may be the high baseline levels of suckling in these animals that could have obscured a potential drug-induced facilitation of suckling behavior. Indeed, in the Williams et al. (1979) study, baseline levels of suckling in weanling control animals were substantially lower than the baseline levels observed in weanlings in Experiment 1. It is possible that some aspect of the test conditions or home environment was influential in producing the high amounts of suckling in the relatively nondeprived weanlings of Experiment I. Thus, in a series of preliminary experiments we attempted to vary individually several aspects of the testing or home environment to observe the effects of these manipulations on suckling behavior in 23- to 24-day-old, non-drug-treated animals. Pups were observed under the following conditions: (1) body temperature was or was not taken prior to testing; (2) testing environment was either quiet or noisy (white noise, human voices, or music); (3) testing apparatus contained clean shavings, shavings from the home cage, or no shavings; (4) pups that wandered away from the nipple were replaced in proximity to a nipple at 5-min intervals or were not handled during the test; (5)

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the father of the pups was present or absent in the home cage during gestation and lactation; (6) pups were taken from our breeding colony or were taken from a different Sprague-Dawley-derived breeding colony; (7) pups were offspring of either dams that had never given birth before (primiparous) or dams that had previously given birth to, and reared, one or more other litters (multiparous). The only condition that was observed to systematically influence the amount of attachment behavior of 23- to 24-day-old pups in these pilot studies was the parity of the maternal female. Offspring of primiparous dams were observed to suckle less than offspring of multiparous dams. Indeed, in Experiment 1, multiparous-derived offspring were used, whereas in the Williams et al. (1979) study, primiparous-derived offspring were tested. Thus, differing parity conditions of the dams may be related to differences in the baseline levels of suckling observed in these two experiments. Consequently, to assess whether parity of the dam, and hence baseline levels of suckling, would influence the effects of serotonergic antagonists on suckling behavior of older rat pups, in this experiment we assessed the effects of metergoline on suckling behavior of preweanling and weanling rat pups from primiparous and multiparous litters.

Method In this experiment, 190 offspring of either multiparous or primiparous dams were tested for suckling behavior at either 15-16 or 23-24 days of age. Pups received subcutaneous injections of 0 (ascorbate vehicle), 1, 2.5, 5, or 10 mg/kg/5 cc metergoline 25 rain prior to the test session. Procedures for the suckling test are described under General Methods. In addition to assessing whether or not pups were attached, instances of locomotion and nipple shifting were also recorded at each behavioral check. The pups were not handled by the experimenter once the test had begun. Each litter was tested at only one of the two testing ages and no more than 2 animals within a given litter were assigned to any of the 5 drug treatment conditions at each age. Nine or ten animals were tested under each of the 20 treatment conditions (2 Age × 5 Drug Dose x 2 Parity).

Results Comparisons of the baseline levels of suckling for vehicle control pups were examined using a 2 (Parity) x 2 (Age) ANOVA and Tukey A post hoc tests. There were significant effects of both Parity (F(1, 34) = 9.74, p ~< .01) and Age (F(1, 34) = 27.88, p ~< .001), as well as a Parity x Age interaction (F(1, 34) = 6.39, p ~< .025). As can be seen in Fig. 3a, multiparous pups did not significantly alter the amount of test time spent attached to the dam across age, whereas primiparous pups at P23-24 were observed to spend significantly less time attached than primiparous

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pups at P15-16. Indeed, parity of the maternal female appears to be a significant factor in influencing the amount of suckling exhibited by P 2 3 24 pups, with offspring of primiparous dams spending less time suckling than offspring of multiparous dams at this age. In contrast, P15-16 vehicletreated offspring f r o m multiparous and primiparous litters did not differ significantly in the a m o u n t of time they spent attached to their m o t h e r s ' nipples. Because of these baseline differences in the amount of suckling behavior in vehicle-treated animals, individual suckling scores for all animals were e x p r e s s e d as a p e r c e n t a g e of the mean amount of suckling for vehicletreated pups under the appropriate parity and age condition. A 5 (Dose) × 2(Parity) × 2(Age) A N O V A on these transformed scores indicated that there were significant main effects of Parity (F(1, 170) = 31.6, p .001) and Age (F(1, 170) = 62.05, p ~< .001), along with several interactions. A significant Dose x Age interaction (F(4, 170) = 4.70, p ~< .01) suggested that at higher doses of metergoline, P23-24 pups increased the a m o u n t of time they were attached to the dam when c o m p a r e d with controls. It is a p p a r e n t f r o m inspection of Fig. 3b that this effect at P23-24 was largely contributed to by a drug-related increase in attachment behavior of offspring of primiparous dams. Metergoline-treated 15- to 16-day-old pups were not found to significantly alter their attachment behavior when c o m p a r e d with controls. There was also a significant interaction of Dose × Parity (F(4, 170) = 2.76, p ~< .05). In general, drug-treated offspring of p r i m i p a r o u s darns spent m o r e time attached to the teats than did drug-

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RISTINE AND SPEAR

treated offspring of multiparous dams. Moreover, a significant Parity x Age interaction (F(1, 170) = 5.99, p ~< .025) suggested that, while drugtreated pups of primiparous dams tended to suckle more than drug-treated pups of multiparous dams, this effect was most pronounced in the older, 23- to 24-day-old pups. In addition to suckling, the occurrence of locomotion and nipple shifting was recorded for all animals during the test. A generally low incidence of these behaviors was observed, similar to that seen in the same-age pups in Experiment 1. There were no apparent differences among offspring of multiparous and primiparous dams on these behavioral measures. Body temperature data were analyzed using a 2 (Pretest vs Post-test) x 5 (Drug Dose) x 2 (Parity) x 2 (Age) ANOVA. As in Experiment 1, there were no significant effects of metergoline on body temperature. There were, however, significant effects of Age (F(1, 150) = 81.753, p <~ .001) and Pretest vs Post-test (F(1, 150) = 45.116, p ~< .001), as well as an Age x Pretest vs Post-test interaction (F(1, 150) = 56.178, p < .001). Post-test body temperatures were less than pretest body temperatures for P15-16 pups but not for animals tested at P23-24 (see Fig. 4). There was also a significant main effect of Parity (F(1, 150) = 23.215, p ~< .001) as well as a Parity x Pretest vs Post-test interaction (F(1, 150) = 4.304, p ~< .05). The body temperatures of primiparous pups were substantially lower than those of multiparous pups. As can be seen in Fig. 4, these effects were more pronounced in body temperature measurements taken pretest than in those obtained post-test. Thus, the effect of parity on body temperature does not appear to be a function of differences in

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SEROTONERGIC AND CHOLINERGIC ANTAGONISTS & SUCKLING

119

the amount of suckling exhibited during testing by pups from the two parity conditions. Discussion

In P23-24 pups of primiparous dams~ baseline levels of suckling are reduced when compared with offspring of multiparous dams. Administration of metergoline appears to result in an elevation in the amount of time that these primiparous-derived weanlings spent attached to the dams' teats. Yet, no differences were observed in this experiment nor in Experiment 1 in the amount of time spent attached by metergoline-treated weanlings of multiparous dams when they were compared with controls. Because weanlings derived from multiparous dams are characterized by high baseline levels of suckling even in control animals, it is possible that these high baselines may have obscured potential facilitatory effects of metergoline on suckling behavior in these animals. In the work showing that serotonergic antagonists attenuate suckling behavior in neonates (Spear & Ristine, 1982; Experiment 1 of the present series), multiparous-derived offspring were used, whereas in this experiment the facilitatory effect of the serotonergic antagonist metergoline on suckling behavior was seen in primiparous-derived, but not multiparous-derived, weanlings. Thus, there is a confound of parity between the experiments showing an attenuating effect of metergoline on suckling in neonates and those showing a facilitatory effect of the drug on attachment in weanlings. To assess the influence of parity on these ontogenetic differences in drug responsiveness, we examined the effects of metergoline on suckling behavior of 3- to 4-day-old offspring of primiparous dams and observed an equivalent reduction in suckling as when the drug was given to 3- to 4day-old pups derived from multiparous dams. Consequently. it appears that the opposite effect of metergoline on suckling of weanlings, when compared to that of neonates, is not a function of the parity condition of the dam. In primiparous-derived pups, metergoline attenuates suckling in neonates while facilitating the elicitation of this behavior pattern in weanlings. Although parity was manipulated in this experiment only to alter baseline levels of suckling in weanling rat pups, the parity results were rather intriguing. Not only did the parity state of the maternal dam influence the incidence of suckling in weanlings, but it also affected internal body temperatures of both preweanling and weanling rat pups. The observation that vehicle control offspring of primiparous dams spent less time attached to the teats at P23-24 than did offspring from multiparous dams could be interpreted to suggest that primiparous-derived pups may be maturationally more advanced than multiparous pups. The opposite suggestion, however, could be derived from consideration of the body temperature data. Offspring of primiparous dams had substantially lower internal body

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temperatures than pups from multiparous dams, suggesting that they may be slower to develop homeothermia. From these data, there does not appear to be a clear difference in rate of maturation in pups from dams of these two parity conditions. Indeed, we examined daily body weights from P1 to P27 of 36 offspring of multiparous and 35 offspring of primiparous dams and found only a significant effect of Age (F(26, 314) = 385.24, p ~< .001) (cell means were derived from litters collapsed within sex for this 2(Sex) x 2(Parity) z 27(Days) ANOVA). However, while these data suggest that there were no overt maturational differences with respect to body weight in offspring of dams from the two parity conditions, this does not eliminate the possibility of more subtle differences in nutritional status between offspring of the two parity conditions that might effect behavior but not body weight during this period of brain development. Indeed, Resnick, Morgane, Hasson, and Miller (1982) have identified a subtle form of prenatal protein malnourishment of the dam involving depletion of protein nutrient reserves prior to pregnancy that does not manifest itself in weight loss of the offspring until about a week postnatally. It is possible that in our multiparous dams, some depletion of protein reserves may have occurred during repeated pregnancies. In these experiments, the procedures used for breeding the animals involved leaving the male in the breeding cage throughout pregnancy and lactation. Under these conditions, many of the multiparous dams are impregnated during postpartum estrus, and thus may have to partition their food-derived nutrients between themselves, developing fetuses, and suckling offspring. Consequently, a depletion of maternal nutrient stores and subtle malnutrition of the offspring might occur when the multiparous dams give birth to litters in rapid succession. In some respects, the behavioral results of the multiparous-derived pups in this experiment are similar to those observed for protein malnourished pups. For example, Massaro, Levitsky, and Barnes (1974) observed that pups malnourished postnatally from P1 to P28 exhibited a developmental delay in the onset of independent feeding and drinking, and in the onset of exploration away from the nest and littermates. In the present experiment, P23-24 control pups from multiparous dams spent more time attached to the nipples of their anesthetized dam than did pups of primiparous dams, perhaps suggesting that their weaning to solid food had been delayed. Clearly, it is not possible from these results to determine whether the parity effects observed in this experiment were a result of parity per se or of some other factor such as differences in maternal behavior or slight malnutrition in multiparous offspring. There is surprisingly little discussion in the literature concerning the effects of parity on offspring behavior in rodents. It has been reported that primiparous dams, when compared

SEROTONERGIC AND CHOLINERGIC ANTAGONISTS & SUCKLING 121 with multiparous dams, exhibit less "lactation behavior" toward preweanling offspring that had been previously stressed (Wright, Bell, Schreiber, Villescas, & Conely, 1977). If primiparous dams begin the weaning process earlier than multiparous dams, this could result in an earlier diminution of the tendency to suckle by primiparous pups in the suckling test in this experiment. Indeed, Henning (1982) suggests that lactational status of the dam is an important variable in the completion of offspring weaning. The effects observed regarding parity and body temperature are rather surprising and difficult to interpret. The pretest body temperature readings, in which the greatest difference between parity conditions was observed, were taken immediately prior to testing, and thus after the litter had been separated from the dam for at least an hour. Thus, it is not known whether these body temperature differences would be seen in the nest, or whether they are a function of alterations in the behavior of the pups after maternal separation. It is possible that body temperature differences between the two parity conditions might have led to differences in the amount of suckling exhibited by the pups. For instance, perhaps primiparous pups suckle less because lower body temperatures depress activity levels, and hence the tendency of these pups to engage in the motor movements necessary for attachment. Indeed, it has been reported that the latency to attach to the dam was longer in pups tested at room temperature than in those tested at nest temperature throughout the age range examined (3-15 days of age) (Cramer & Blass, 1982). Similarly, there is some suggestion that pretest and test exposure to room temperature decreases the amount of time that 17- but not 21- or 24-day-old hypothyroid rat pups spend suckling when they are compared with warmed hypothyroid age-mates (Blake & Henning, 1983). These studies suggest that body temperature may play a role in influencing suckling propensity in preweanling animals. However, in the present study, parity was observed to influence body temperature at both 15-16 and 23-24 days postnatally, but to influence the amount of time that pups spent attached to the dam only at 23-24 days of age. Although it is an intriguing possibility that the lower body temperatures of primiparous-derived pups may depress their propensity to suckle, it is difficult to determine why this effect would be seen only in weanling, and not preweanling, animals. These interpretations of the parity results are most speculative and more work is certainly needed to examine systematically the effects of parity on offspring behavior and physiology during ontogeny. This area of inquiry is particularly important given the scattered reports that parity of the maternal female can influence offspring behavior in adulthood (e.g., Furchtgott, Lazar, & Deitchman, 1969; Wright & Bell, 1978) and even during aging (Elias, Nau, Villescas, & Bell, 1982).

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GENERAL DISCUSSION

In Experiment 1, the serotonergic antagonist, metergoline, and the cholinergic antagonist, scopolamine, were observed to decrease the amount of time that pups spent attached to their dams predominantly when these drugs were administered during the early postnatal period. Specifically, metergoline was shown to reduce suckling in 3- to 4- and 7- to 8-dayold rat pups, while scopolamine produced a reduction in suckling principally in 3- to 4-day-old pups, although the highest dose of scopolamine also significantly reduced attachment in 15- to 16-day-old animals. The inhibitory effects of these drugs on attachment time did not appear to be a result of the drugs debilitating the animals nor inducing behaviors incompatible with suckling. Thus, it appears that both serotonin and acetylcholine may play an important role in facilitating the suckling process in the neonatal rat pup. Although studies which have examined the emergence of adult-typical behaviors in response to serotonergic manipulation have suggested that serotonin does not becomes functional in influencing behavior until the second to third postnatal week (e.g., Mabry & Campbell, 1974), there is neurochemical evidence that serotonin substrates are present a good deal earlier. For example, it has been reported that the amount of serotonin binding as well as serotonin-sensitive adenylate cyclase activity is greater in the early neonatal period than in adulthood in a variety of brain areas (e.g., Uzbekov, Murphy, & Rose, 1979; VonHungen, Roberts, & Hill, 1974), which may indicate the presence of hypersensitive serotonergic receptor-enzyme systems that subserve some special function in immature brain (see VonHungen et al., 1974). Moreover, Gallager (1982) has examined extracellular recordings of the spontaneous activity of neurons in the dorsal raphe of neonates and has reported that the firing pattern and rate of these neurons are similar to those of the adult. Using immunohistochemistry, Lidov and Molliver (1982) have observed that while serotonergic innervation of forebrain is still quite immature in neonates, the pattern of distribution and density of serotonergic nerve terminals in brain stem of newborn rats approaches that of the adult. Thus, by several indices, portions of the serotonergic system, particularly those in caudal brain regions, seem to exhibit impressive maturity during the early postnatal period. Classically, the cholinergic system, like the serotonergic system, has been suggested to become functionally mature with respect to adulttypical behavior patterns only during the third week of life (e.g., Baez, Eskridge, & Schein, 1976; Campbell, Lytle, & Fibiger, 1969). Yet, using other behavioral as well as electrophysiological measures, cholinergic manipulations have been reported to have an impact as early as the first week or two of life (e.g., Blozovski & Blozovski, 1973; LeBlanc & Bland, 1979; Murphy & Nagy, 1976; Smith, Spear, & Spear, 1982). Ample

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postsynaptic substrates are available in the neonate for acetylcholine; brain muscarinic cholinergic receptors have been reported to be at near adult levels at birth in the rat, particularly in caudal brain regions (Rotter, Field, & Raisman, 1979). Moreover, whole brain acetylcholine levels in newborns appear to be approximately 30-50% of adult levels (Coyle & Yamamura, 1976; Mann, Pleul, & Kewitz, 1971), with the concentration of acetylcholine in medulla-pons at birth not significantly different from that seen in adulthood (Coyle & Yamamura, 1976). Thus, it would appear that there are ample neural substrates for both acetylcholine and serotonin in certain brain regions, particularly the brain stem, of neonates. Early maturing portions of these neurotransmitter systems may play a role in facilitating behaviors of adaptive relevance to the neonate such as those involved in suckling. While the serotonergic antagonist metergoline was observed to decrease the time neonates spent attached to the dam in Experiment 1 (see also Spear & Ristine, 1982), metergoline conversely was observed to increase the time weanling offspring of primiparous dams spent attached in Experiment 2. This latter finding is consistent with that of Williams et al. (1979) where serotonergic antagonists were likewise observed to increase suckling behavior in rat pups beginning at 15-20 days postnatally. These results suggest the intriguing possibility that an early-maturing serotonergic system may be important for facilitating portions of the suckling process in neonates and that a later-maturing portion of this anatomically and neurochemically heterogeneous system may be involved in the normal inhibition of suckling during the weaning process. The time course of variations in the responsiveness of rat pups to serotonergic antagonists is paralleled by alterations in the neurochemical responsiveness of the serotonergic system to food deprivation. Scalzo and Spear (1983) observed that 24 h of food deprivation markedly increased levels of serotonin and its metabolite 5-hydroxyindole acetic acid in various brain regions, particularly brain stem, of rat pups during the first week to 10 days of life, but did not increase levels of these indoles in any brain regions examined in older preweanling or weanling animals. The existence of a serotonergic system activated by food deprivation in neonates would be consonant with our suggestion that serotonin is critical for stimulating portions of the suckling process in newborns. The dissipation of this deprivation-enhanced serotonergic system as the animal approaches weaning may be related to the possible emerging role of serotonin in the inhibition of suckling during weaning. If early-maturing serotonergic and cholinergic systems are important for stimulating certain components of the suckling process in neonates, there is still the question of the neural substrates governing suckling in the interim period prior to the (possibly serotonergically facilitated) dissipation of suckling at weaning. Neural processes modulating suckling

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behavior may change with age in concert with the gradual maturation of other physiological controls of the suckling process. The process of attachment and maintenance of attachment to a nipple in the neonatal animal is a prepotent response, one that is difficult to alter by varying the nutritional state of the animal or the nutritive consequences of suckling. During this early postnatal period, serotonin and acetylcholine appear to be critical for stimulating certain components of the suckling process. However, manipulations of these neurotransmitter systems are no longer effective soon after the first postnatal week, at the time that suckling becomes more susceptible to modulation by internal and external stimuli. For instance, control over milk intake volume, with respect to latency to attach and duration of suckling bouts, is only apparent after 10-15 days of age. Before this time, pups will suckle until they become so bloated that they can no longer maintain nipple contact (Hall & Rosenblatt, 1977). One might hypothesize that newly emergent neural systems may be involved in the continuation of suckling, as well as the addition of other physiological controls to the suckling process, after the first postnatal week. Later in life, as the pups approach weaning age, there appears to be a gradual inhibition of suckling behavior as other ingestive processes begin to become predominant. At present, little is known regarding the roles that early-maturing portions of the neurotransmitter systems may play in mediating behaviors of the young organism. Yet, it is evident that the neonatal animal possesses a behavioral repertoire that differs substantially from that of the adult. These critical age-specific behaviors of infancy are presumably neurally mediated, and may be modulated by early-maturing portions of various neurotransmitter systems. The process of suckling appears to be an excellent model system for investigation of nascent brain processes involved in the mediation of age-specific behaviors of infancy and their ontogenetic decline. REFERENCES Adnen, J. (1978). Ontogenesis of some sleep regulations: Early postnatal impairment of the monoaminerglc systems. Progress in Brain Research, 48, 393-403. Almli, C. R. (1978). The ontogeny of feeding and drinking behaviors: Effects of early brain damage. Neuroscience and Biobehavioral Reviews, 2, 281-300. Baez, L. A., Eskridge, N. K., & Schein, R. (1976). Postnatal development of dopaminergic and cholinergic catalepsy in the rat. European Journal of Pharmacology, 36, 155-162. Blake, H. H., & Henning, S. J. (1983). Weaning in the rat: A study of hormonal influences. American Journal of Physiology, 244, R537-R543. Blass. E. M., Hall, W. H., & Teicher, M. H. (1979). The ontogeny of suckling and ingestive behaviors. In J. M. Sprague & A. N. Epstein (Eds.), Progress in psychobiology and physiological psychology (Vol. 8, pp. 243-299t. New York: Academic Press. Blozovski, D., & Blozovski, M. (1973). Effects de l'atropine sur l'exploration, l'apprentissage et l'activite electrocorticale chez le rat au cours du developpement. Psychopharmacologia, 33, 39-52. Brake, S. C., Sager, D. J.. Sullivan, R.. & Hofer, M. (1982). The role of intraoral and

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