Preweanling Rats Research Articles

Repeated maternal separation of preweanling rats attenuates behavioral responses to primary and conditioned incentives in adulthood

Early social experience has profound effects on a wide spectrum of behaviors and neurochemical correlates in the rat. Repeated separation of rat pups from their dam during the early neonatal period causes acute perturbation of neuroendocrine and physiological status. The chronic sequelae of repeated separations have not been studied as extensively as the acute responses. Altered social experience at a later developmental stage, postweaning isolation rearing, is known to induce enduring changes in the behavioral responses to reward and reward-related stimuli in maturity. We have evaluated the influence of repeated early maternal separation on the responses to both primary and conditioned incentives in mature rats. Separated animals showed enhanced weight gain, a blunted locomotor response to a novel environment and a blunting of the response to both negative and positive contrast effects. Female separated animals, but not males, exhibited a profound attenuation of the acquisition of a conditioned anticipatory locomotor response to the presentation of food. These data are discussed with respect to the putative involvement of ventral striatal dopamine systems in reward mechanisms and the potential utility of early maternal separation as an animal model of depression.

Precataractous changes affect lens transparency in the selenite cataract.

Selenite treatment of the preweanling rat stabilized the transparency of the lens nucleus to decreasing temperature. Hence, we compared properties of the cortex and nucleus from lenses of selenite-treated and age-matched control rats. A subcutaneous dose of 30 nmol Na2SeO3/g body weight was administered to 10- to 13-day-old Sprague-Dawley rats. Uninjected, age-matched littermates served as controls. As required, lenses were frozen in liquid N2 and separated into nuclear and cortical-epithelial fractions. Transparency of solutions of lens proteins (90-100 mg per ml) was monitored from 30 to 2 degrees C as percent transmittance (%T) at 490 nm. The critical phase separation temperature, Tc, was the temperature at 80%T. Protein associations were monitored with gel filtration chromatography. The nuclear 'cold cataract', in intact lenses, formed at similar temperatures at 14 and 15 days of age, but at a significantly lower temperature when the lenses were from a selenite-treated rat. The Tc, however, was greater by 1.5-2 degrees C for solutions of proteins isolated from whole lenses or lens nuclei from rats 24 and 48 h after treatment with selenite. Further, less gamma-crystallin was associated with the alpha-crystallin fraction in extracts from the nucleus of lenses from treated rats. Altered phase separation properties occurred as an early event in the etiology of selenite cataract. The different in vivo and in vitro responses to temperature indicated that properties of lens crystallins do not solely establish transparency in the intact lens.

Glucocorticoids, the hippocampus, and behavioral inhibition in the preweanling rat

Endogenous corticosteroids influence brain development and behavioral expression. In rat pups, a corticosteroid-dependent developmental response is behavioral inhibition, which occurs in situations involving threat. Behavioral inhibition consists of freezing and a reduction in ongoing behavior. It is presently unknown which brain region(s) that bind corticosterone (CORT) is involved in the development of freezing. The hippocampus (HC), however, is the principal target site of CORT that regulates the postnatal development of HC dentate granule cells. Therefore, this study examined whether the HC, and in particular, the dentate granule cells, plays a major role in the early appearance of behavioral inhibition. On postnatal day 9, rat pups received bilateral HC electrolytic lesions, or bilateral HC infusions of colchicine, a neurotoxin selective for dentate granule cells, or bilateral HC infusions of kainic acid, a neurotoxin selective for pyramidal cells in the CA3 field. Control rats received sham operations. After the operations, all rats were adrenalectomized (ADX) and injected daily with 3.0 mg/kg CORT, except on the day of the behavioral test. On day 14, all pups were tested for behavioral inhibition, which consisted of removing the pup from the nest box and placing it in a temperature-controlled enclosure subdivided into two compartments by a wire-mesh partition. The pup was placed in one compartment and an unfamiliar anesthetized adult male rat was placed in the adjacent compartment. Results indicated that preweanling rats with electrolytic lesions ranging from the dorsal to the ventral HC exhibited significant deficits in freezing. Importantly, similar deficits in freezing were present in pups treated with colchicine but not KA. Hence, administration of exogenous CORT is not effective in facilitating the occurrence of freezing in preweanling pups lacking dentate granule cells. To determine whether the dorsal HC dentate gyrus is an essential target site of CORT in facilitating freezing, 9-d-old rats were implanted bilaterally with 30 gauge cannula filled with either CORT or cholesterol. After the operation, all rats were ADX and tested for behavioral inhibition on day 14. During testing, ADX pups with CORT-filled cannulae showed significantly higher levels of freezing than ADX control pups. Taken together, results suggest that during the early postnatal period, the action of endogenous CORT in the HC influences the development of dentate granule cells that play an essential role in mediating the appearance of behavioral inhibition.

Effects of dopamine D 1 and D 2 receptor antagonists on cocaine-induced place preference conditioning in preweanling rats

The effects of dopamine D 1 and D 2 receptor antagonists on the reward processes of 10- and 17-day-old rats were assessed using the conditioned place preference paradigm. Conditioning and testing were conducted in a three-compartment chamber, with each end compartment having its own distinct tactile and odor cues (almond and lemon). During six experiments, 10- and 17-day-old rats (age at initial conditioning) were injected intraperitoneally with either saline, the dopamine D 1 receptor antagonist R(±)-SCH 23390 hydrochloride (0.01–1.0 mg/kg), or the dopamine D 2 receptor antagonists (±)-sulpiride (1–100 mg/kg) or S(-)-eticlopride hydrochloride (0.1–0.5 mg/kg) 30 min prior to being injected with cocaine hydrochloride (20 mg/kg) or saline. After the latter injections, rats were immediately confined in the lemon-scented (nonpreferred) compartment for 30 min. On the alternate conditioning day, rats were injected with saline and confined in the almond-scented compartment. On the third day (i.e., the test day), rats were given saline and allowed free access to the entire chamber for 15 min. The results showed that the dopamine D 1 receptor antagonist SCH 23390 blocked the cocaine-induced place preference conditioning of both 10- and 17-day-old rats. Surprisingly, the dopamine D 2 receptor antagonists sulpiride and eticlopride blocked the place preference conditioning of 10-day-old rats, while leaving the 17-day-old rats unaffected. These results indicate that dopamine D 1 receptors are critically involved in the reward processes of preweanling rats, but that the importance of dopamine D 2 receptors changes across ontogeny.

Developmental analysis of habituation of the acoustic startle response in the preweanling and adult rats

Our experiments assessed the build-up of short-term and long-term habituation of the acoustic startle response in preweanling and adult rats. Results showed that the adult rats demonstrated both short-term and long-term habituation of the acoustic startle response (ASR). In contrast, the decreased responsiveness to the auditory stimulations was revealed in the preweanling rats only 2 hours after the training session, whilst 24 hours and 48 hours after learning no significant change of the startle amplitude in the rat pups was detected. The data are discussed with respect to maturational interpretations of age-related differences in learning and retrieval processes.

Ontogenetic development and pentylenetetrazol seizure thresholds in rats

Ontogenetic differences in seizure thresholds to pentylenetetrazol (PTZ) were examined in young, developing rats (ages: 10, 18, 28, and 60 days). A subconvulsive dose of PTZ (15 mg/kg IP) was administered every 10 min and the occurrence of partial and full (clonic tonic) seizures was noted. Ten day old pups displayed complete seizures more rapidly than any of the other groups ( p < .01) without any behavioral indication of going through the partial stages. Among 18 day old pups the development of early seizure stages was followed rapidly by the full seizure, while in the weaned groups the early seizure stages tended to persist much longer before the appearance of the full generalized convulsion. The preweanling rat's immature brain seems less able to suppress PTZ induced generalized convulsions compared to the brain of the older rats.

Behavioral effects of the reversible dopamine antagonist flupenthixol are not potentiated by N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline in the preweanling rat

In the preweanling rat, the irreversible dopamine (DA) receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) does not diminish behaviors induced by the nonselective DA agonist R(−)propylnorapomorphine (NPA). To determine whether EEDQ was simply inactivating an insufficient percentage of DA receptors, the NPA-induced behaviors of 17-day-old rats were assessed after treatment with flupenthixol (a reversible DA receptor antagonist) and/or EEDQ. When given alone, flupenthixol (0.04, 0.1, and 0.4 mg/kg, intraperitoneally [IP]) produced a dose-dependent decrease in the behavioral effects induced by 1.0 mg/kg NPA. Unexpectedly, EEDQ (7.5 mg/kg, IP) did not potentiate flupenthixol's actions. This suggests that EEDQ's inability to block the NPA-induced behaviors of preweanling rats was not the result of an insufficient percentage of DA receptors being inactivated.

Depletion of brain glutathione is accompanied by impaired mitochondrial function and decreased N-acetyl aspartate concentration.

The effect of depletion of reduced glutathione (GSH) on brain mitochondrial function and N-acetyl aspartate concentration has been investigated. Using pre-weanling rats, GSH was depleted by L-buthionine sulfoximine administration for up to 10 days. In both whole brain homogenates and purified mitochondrial preparations complex IV (cytochrome c oxidase) activity was decreased, by up to 27%, as a result of this treatment. In addition, after 10 days of GSH depletion, citrate synthase activity was significantly reduced, by 18%, in the purified mitochondrial preparations, but not in whole brain homogenates, suggesting increased leakiness of the mitochondrial membrane. The whole brain N-acetyl aspartate concentration was also significantly depleted at this time point, by 11%. It is concluded that brain GSH is important for the maintenance of optimum mitochondrial function and that prolonged depletion leads also to loss of neuronal integrity. The relevance of these findings to Parkinson's disease and the inborn errors of glutathione metabolism are also discussed.

Ontogeny of behavioral sensitization in the rat: effects of direct and indirect dopamine agonists.

In the present study, the abilities of NPA (a direct DA receptor agonist) and amphetamine (an indirect DA receptor agonist) to induce short- and long-term behavioral sensitization were assessed in 11- and 17-day-old rats (age at initial injection). Rats were injected on 4 consecutive days with amphetamine (1.0, 2.5, or 5.0 mg/kg), NPA (1.0 mg/kg), or saline. A final test day occurred either 2 days (experiment 1) or 8 days (experiment 2) later. On the test day, rats given successive agonist injections received a single injection of the same agonist again; whereas rats given successive saline injections received either amphetamine or NPA for the first time. Five minutes after injection, locomotor activity (line-crosses), stereotyped sniffing, and vertical activity were measured during a 30-min testing session. The results showed that 11- and 17-day-old rats exhibited behavioral sensitization when tested with NPA or amphetamine after a 2-day interval. In contrast, neither NPA nor amphetamine was able to sensitize the behaviors of preweanling rats when measured 8 days after initial drug treatments. Therefore, these results show that both direct and indirect DA agonists are able to induce short-term behavioral sensitization in preweanling rats, but that the mechanisms responsible for mediating long-term behavioral sensitization have not yet matured.

Context and tone conditioning are selectively impaired by ethanol in the preweanling rat: Effects of dose and time of administration

Depending on dose and task requirements, ethanol can have either a facilitative or an impairing influence on learning. Some explanations for this dichotomy have considered ethanol's suppression of behavioral variability and processing of incidental stimuli (e.g., context). The present study examined the effect of ethanol on context and conditioned stimulus (CS) learning in the preweanling rat. To assess state-dependent effects, a drug dissociation design was used. Learning to both context and CS were analyzed within each dose of ethanol (0, 1.2, 1.6, or 2.0 g/kg) and a trend analysis was conducted to determine dose-response relationships as a function of train-test state. The 1.2 g/kg dose of ethanol did not affect conditioning to either the context or the CS. A 1.6 g/kg dose tended to disrupt context, but not CS, conditioning. The influence of 2.0 g/kg ethanol depended on train-test conditions. Ethanol administration prior to training resulted in the stronger impairment of CS learning while context conditioning was most disrupted if ethanol was given only prior to testing. The results suggest that ethanol selectively attenuates processing of stimuli, possibly dependent on relative saliency at the time of testing.

Organizing action of corticosterone on the development of behavioral inhibition in the preweanling rat

Altricial rat pups develop the ability to freeze and to terminate their emission of ultrasonic vocalizations when exposed to an unfamiliar adult male rat. This developmental competence in expressing behavioral inhibition is impaired when rat pups are adrenalectomized (ADX) on postnatal day 10, a period prior to the emergence of behavioral inhibition. Adrenalectomy, however, fails to induce similar behavioral deficits when performed after behavioral inhibition has developed. Results suggest that adrenal steroids are involved in promoting the development but not the activation of behavioral inhibition. To critically test this hypothesis, four groups of rats were adrenalectomized on day 10 and tested for behavioral inhibition on day 18. Prior to testing, one group of rats received daily s.c. injections of vehicle whereas another group was treated with daily injections of 3.0 mg/kg of corticosterone (B). The other two groups of rats received daily B injections on only days 10–13 or days 14–17. Results indicated that ADX rats treated with B only on days 10–13 as well as throughout exhibited significantly higher levels of freezing than the other two treatment groups. In order to evaluate whether the behavioral inhibitory deficits produced by ADX at 10 days of age are due to a delayed insensitivity to the 3.0 mg/kg dose of B, day 10 pups were ADX and injected on days 14–17 with doses of B as high as 12 mg/kg. When tested for behavioral inhibition on day 18, these high doses of B were found to be ineffective in potentiating freezing above the level of vehicle-treated rats. Together, results suggest that prior to the end of the second postnatal week endogenous adrenal steroids and in particular B, are critically involved in developmental changes necessary for the eventual species typical expression of behavioral inhibition.

Intracranial action of corticosterone facilitates the development of behavioral inhibition in the adrenalectomized preweanling rat.

We tested the hypothesis that in preweanling rats central administration of exogenous corticosterone (CORT) is sufficient to facilitate the development of behavioral inhibition. 28-gauge cannulae containing varying concentrations of CORT (0, 25, 50 and 100%) were implanted unilaterally into the lateral ventricles of 9-day-old rat pups. After a 24-h postoperative recovery period, pups were adrenalectomized. At 14 days of age, pups were tested for behavioral inhibition which consisted of removing the pup from the nest and exposing it to an unfamiliar adult male rat. Pups implanted with cannulae containing 0, 25 and 50% concentrations of CORT spent significantly less time in freezing postures than pups implanted with cannulae containing 100% CORT. These freezing pups also tended to emit fewer ultrasonic vocalizations than pups in the other three implant conditions, albeit the level obtained was not statistically significant. RIAs indicated that, in general, hormone-filled cannulae produced no detectable concentrations of plasma CORT on the day of the test or on days preceding testing. Results suggest that in the early postnatal period endogenous CORT acts centrally to facilitate the development of neural pathways involved in the ontogenetic expression of behavioral inhibition.

Stimulus control of behavioral inhibition in the preweanling rat

Previous studies demonstrate that 14-day-old rats reduce their emission of ultrasonic vocalizations and freeze when exposed to an unfamiliar adult male rat. This study sought to identify the stimulus characteristics of conspecific males that potentiate the display of behavioral inhibition. In Experiment 1, day 14 rats were isolated from the nest and exposed to either an unfamiliar prepubescent male rat or an unfamiliar adult male rat. Pups exposed to the unfamiliar adult male rat exhibited significantly elevated levels of freezing and reduced their emission of ultrasounds. In Experiment 2, pups were exposed to either a familiar or an unfamiliar adult male rat. Although several pups exposed to the familiar adult male rat exhibited freezing, pups tested with the unfamiliar adult rat showed a reliably higher duration of freezing and made fewer vocalizations. Results suggest that neither the unfamiliar factor nor cues associated with male adulthood are sufficient to account for the occurrence of behavioral inhibition when presented separately. However, the combination of unfamiliarity and adult male stimuli are highly potent stimulus features that elicit behavioral inhibition in preweanling rats.

Effect of chronic maternal diazepam treatment on the development of stress-induced antinociception in young rats

The use and abuse of benzodiazepines is widespread and we have begun to address whether maternal exposure to these drugs influences the development of opioid systems. We have studied the effect of maternal diazepam treatment on stress-induced antinociception in the neonatal offspring. Diazepam (1 or 10 mg/kg) was administered twice daily to mothers from conception. Pre- and postweanling rat pups were assessed for opioid-mediated stress-induced antinociception by 3-min swimming and measuring nociception using the tail immersion test. In preweanling rats there was stress-induced antinociception in both vehicle- and diazepam-treated animals but in diazepam-treated groups (1 and 10 mg/kg) this was insensitive to reversal by the opioid antagonist naloxone, suggesting that nonopioid systems are operating this response. In postweanling rats a similar insensitivity to naloxone was observed in 1 mg/kg diazepam-treated groups; with 10 mg/kg diazepam there was no significant antinociception. The results suggest that maternal diazepam treatment interferes with the development of stress-mediated responses and that part of this toxicity is due to actions on opioid systems in the CNS.

Role of the spinal cord in intracisternal β-endorphin-evoked suppression of liver DNA synthesis in 10-day-old rats

Previously, we have shown that intracisternal (i.c.) administration of β-endorphin (an opioid peptide naturally occurring in the brain) to preweanling rats markedly decreases DNA synthesis (an index of cell proliferation) in both brain and liver. This observation is consistent with our hypothesis that endogenous CNS β-endorphin plays an important role in controlling postnatal growth. The current research specifically undertook to investigate, in 10-day-old rats, whether or not i.c. β-endorphin-evoked suppression of liver DNA synthesis is actually mediated by spinal opioid receptors and/or by descending endorphinergic pathways. In contrast to the i.c. route of administration, β-endorphin given directly into the spinal subarachnoid space via intrathecal (i.t.) injection did not alter liver DNA synthesis, yet was able to evoke profound antinociceptive responses. This demonstrates that intracisternally applied β-endorphin exerts its effect on liver DNA by acting at supraspinal sites, and not by directly stimulating spinal opioid receptors after diffusion from its intracerebral site of injection. As it is possible that β-endorphin's supraspinal actions may activate a descending inhibitory endorphinergic pathway to reduce DNA synthesis, we conducted studies in rat pups administered naloxone intrathecally. Naloxone i.t. was completely ineffective in preventing β-endorphin i.c. from inhibiting liver DNA synthesis. On the other hand, i.t. coinjection of naloxone plus β-endorphin was able to block the analgesic response, while their i.c. coinjection reversed the DNA effect. The results from these studies indicate that opioid systems within the spinal cord do not play a major role in mediating CNS β-endorphin's regulation of DNA synthesis in peripheral tissues. However, the data obtained do not necessarily exclude other spinal neurochemical systems (e.g. adrenergic or serotonergic) from participating in this process.

Paradoxical autonomic responses to aversive stimuli in the developing rat.

The goals of this research were to determine (a) the change in heart rate elicited by aversive auditory stimuli in the laboratory rat at different ages and (b) the autonomic origins of those changes at each age. The results of the first 2 experiments showed that aversive white noise stimuli elicited cardiac deceleration in preweanling (16-day-old) rats and cardiac acceleration in weanling (23-day-old), periadolescent (30-day-old), and adult (60-day-old) rats. Subsequent experiments showed that (a) the decrease in heart rate elicited by the noise stimulus in preweanling rats was mediated by parasympathetic activation of the heart, (b) the stimulus-elicited increase in heart rate elicited by the noise in periadolescent rats was mediated by parasympathetic withdrawal of the heart, and (c) the noise-induced increase in heart rate in adult rats was primarily mediated by sympathetic activation of the heart.

Postnatal high-peak blood ethanol concentration and external cue-based discrimination learning and reversal in the preweanling rat: comparison with memory-based discrimination learning.

Postnatal exposure to ethanol that produces high-peak blood ethanol concentrations (HP-BEC) in artificially reared infant rats affects hippocampal neuroanatomy and discrimination learning based on memorial cues from a patterned (single) alternation (PA) schedule in preweanling rats (P. L. Greene, J. L. Diaz-Granados, & A. Amsel, 1992). In the present experiments, discrimination by preweanling rats exposed to ethanol in the same way was tested with nonmemorial, external cues. In this external cue-based discrimination and in its reversal, ethanol-exposed rats were not different from normal or artificially reared controls whether the cues were presented in a PA or random manner, although there was some evidence that the memorial cues from the PA schedule contributed to learning a discrimination based on external cues, suggesting that the deficit reported earlier in ethanol-exposed rats is a memorial deficit and not a general discrimination deficit.

Postnatal high-peak blood ethanol concentration and external cue-based discrimination learning and reversal in the preweanling rat: Comparison with memory-based discrimination learning.

Postnatal high-peak blood ethanol concentration and external cue-based discrimination learning and reversal in the preweanling rat: Comparison with memory-based discrimination learning.

Ontogenetic differences in the effects of EEDQ on dopamine-mediated behaviors

Previous results suggest that 17-day-old rat pups may have substantial reserves of both D 1 and D 2 receptors. To assess this possibility, the behavioral effects of a nonselective dopamine (DA) agonist, R-propylnorapomorphine (NPA), were measured in 11- and 17-day-old rat pups previously treated with the irreversible DA receptor antagonist N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ). Rat pups were treated with EEDQ (7.5 mg/kg) either alone or in combination with the D 1 and D 2 antagonists, SCH 23390 (1.0 mg/kg) and sulpiride (100 mg/kg), respectively. (The SCH 23390 and sulpiride were used to protect. dopamine receptors from EEDQ-induced inactivation.) NPA's effects on stereotype sniffing and locomotor activity were then assessed 1, 2, and 4 days after EEDQ pretreatment. Results showed that NPA (0.01, 0.1, 1.0, or 5.0 mg/kg) produced a dose-dependent increase in the stereotyped sniffing of both aged rats. Unexpectedly, however, EEDQ did not disrupt the NPA-induced stereotyped sniffing or either the 11- or 17-day-old rat pups. Thus a behavior (i.e., stereotyped sniffing) that requires the activation of a large complement of DA receptors was not sensitive to the receptor-depleting actions of EEDQ. Moreover, the behaviors of 11-day-old-rats, which have fewer DA receptors than older pups or adults, were also not susceptible to the effects of EEDQ. When taken together, these results suggest that EEDQ's inability to block the agonist-induced behaviors of preweanling rat pups cannot be explained by ontogenetic changes in DA receptor reserves.

Naltrexone does not increase brain and body development in rats

Two strains of preweanling rats (88 Long-Evans; 120 Sprague-Dawley) were given daily SC injections of either 50 mg/kg naltrexone or the equivalent volume of saline, from birth to postnatal day 21. Brain and body weights were assessed at the end of the injections, and in Long-Evans animals, significant differences were noted on both measures with the outcome favoring the control animals. No significant differences were observed between the naltrexone and control Sprague-Dawley rats. These results disconfirm the hypothesis that increased postnatal growth is accomplished by prolonged early postnatal opioid receptor blockade.