The effect of periaqueductal gray lesions on responses to age-specific threats in infant rats.

During early ontogeny infant rats show specific responses to a variety of age-dependent threatening situations. When isolated from nest and dam, they emit ultrasonic vocalizations and show decreased reactivity to noxious stimulation, or analgesia. When exposed to an unfamiliar adult male, they become immobile and analgesic. The midbrain periaqueductal gray (PAG) is an important area within the circuitry that controls responses to threatening stimuli in the adult. Little is known about the functions of the PAG in early life. It was hypothesized that the PAG mediates the responses to the age-specific threats social isolation and male exposure in the infant rat. Rat pups were lesioned electrolytically either in the lateral or the ventrolateral PAG on postnatal day 7, tested in social isolation on day 10, and exposed to a male on day 14. On day 10 during isolation, ultrasonic vocalizations and isolation-induced analgesia were decreased in both lesion groups. On day 14, male-induced immobility and analgesia were decreased in ventrally lesioned animals. In conclusion, the PAG seems to play a developmentally continuous role in age-specific responses to threat such as ultrasonic vocalization, analgesia, and immobility.

Behavioral and heart rate effects of infusing kainic acid into the dorsal midbrain during early development in the rat.

The dorsal midbrain including the periaqueductal gray (PAG) is involved in the control of threat-induced vocalizations and other behavioral and autonomic defensive responses in adult animals. Little is known of its function early in life. The present study examined the ability of kainate receptor stimulation in these midbrain areas to trigger behavioral and physiological responses during the first three postnatal weeks in the rat. Kainate (0.03-0.3 nmol) was infused into the dorsal midbrain of postnatal day 7 (P7), P14 and P21 rat pups. At P7, subjects exhibited only a brief period of locomotor activation immediately following infusion of kainate. There were no changes in the heart rate or in any other behavioral measures, including their production of ultrasonic vocalizations. At P14, kainate induced adult-typical escape behaviors consisting of running and jumping, increases in the duration of time spent immobile, and increases in heart rate. At P21, subjects given kainate exhibited escape behavior coupled with elevated heart rate and immobility coupled with decreased heart rate. P14 and P21 subjects produced only small, non-significant increases in their production of ultrasonic vocalizations. These results indicate that kainate receptor stimulation in the dorsal midbrain does not mediate most adult-typical, threat-induced responses until sometime during the second postnatal week in the rat.

Evidence for opioid and nonopioid processes mediating adaptive responses of infant rats that are repeatedly isolated.

Previous research examining the ability of neonatal rats to adapt to repeated isolation demonstrated that an opioid-dependent decline in ultrasonic vocalizations occurred across a series of isolations (Goodwin, Molina, & Spear, 1994). These findings were expanded in the present study. In the first experiment, the decline in vocalization rates was found to result from the release of endogenous opioids throughout the series of isolations. Although naltrexone attenuated the decline in calling rates relative to vehicle-treated subjects, there was still a significant decline in calling rates following opioid receptor blockade. In the second experiment, two injections of naltrexone did not attenuate the decline in calling rates any more than a single injection did, suggesting that there must also be some nonopioid process that modulates this decline. In both experiments, activity levels and, in the first experiment, the amount of body heat lost in the repeatedly isolated subjects declined in a nonnaltrexone reversible manner. In a final study, after calling rates had been suppressed by a series of isolations, a brief exposure to the mother was found to restore baseline calling rates, suggesting the decline is not the consequence of fatigue. The attenuation of vocalization rates, activity, and loss of body heat are adaptive responses of infant rats to isolation; however, of these three, only the attenuation of vocalization rates is consistently modulated by the release of endogenous opioids.

Precipitated morphine withdrawal induces a conditioned aversion in the preweaning rat.

Opiate abstinence in the adult of many species, including humans, alters autonomic function and motor behavior, and induces a negative affective state. The neurobehavioral bases of each consequence of opiate withdrawal differs. Little attention has been paid to the issue of drug withdrawal in infants, although it is a common consequence of the maternal use of illegal and legal drugs. Infant rats as young as 7 days of age that experience opiate withdrawal show an abstinence syndrome consisting of developmentally appropriate behaviors that differ from those of the adult rat, including fewer autonomic signs. Unlike the adult, there are no data in the infant on whether or not opiate withdrawal induces a negative affective state. We treated infant rats twice daily for seven days with either morphine or saline. Pups were injected with naltrexone or saline and exposed to a novel odor. After conditioning, pups were given the option of spending time with the conditioned odor or in a neutral environment. Fourteen day old pups, but not 7 day old animals, chronically treated for 7 days with morphine and conditioned with naltrexone, showed a significant avoidance of the conditioned odor. This suggests that a conditioned aversion had formed, a result similar to that shown for adult animals.

Immediate early gene expression to examine neuronal activity following acute and chronic stressors in rat pups: examination of neurophysiological alterations underlying behavioral consequences of prenatal cocaine exposure.

Altered behavioral responses to stressors have been observed in animals exposed to cocaine prenatally. In the present study, both behavioral and physiological responses to repeated and single stressor exposure were measured in animals prenatally exposed to cocaine. Offspring were derived from 3 prenatal treatment groups: dams that were administered 40 mg/kg cocaine from gestational day 8-20 (C40); dams that were pair-fed and -watered to weight-matched C40 dams (PF); and untreated dams (LCC). Starting on postnatal day 16-17 (P16-17), offspring from the 3 prenatal treatment groups were exposed to either footshock or isolation daily for 5 days. Two days after the last day of stressor exposure (P21-22), subjects were given 1 final exposure to the stressor to which they were previously exposed. In addition, at P21-22, littermates of animals given repeated exposure to stressors were exposed to either footshock or isolation for the first and only time. During all footshock sessions, the duration of freezing behavior was recorded. Plasma adrenocorticotrophin (ACTH) and corticosterone levels were determined from blood samples taken immediately following the final stressor session and brains were processed for C-FOS immunoreactivity (FOS-IR). Plasma corticosterone was increased following either single or repeated exposure to either stressor compared to homecage control animals. Plasma ACTH was increased by exposure to both repeated and single footshock exposure, but the increase was not as great following repeated footshock exposure, suggesting adaptation to repeated exposure to this stressor. Following both single and repeated footshock exposure, FOS-IR was increased relative to baseline levels in the paraventricular nucleus of the hypothalamus (PVN) and in the supraoptic nucleus (SON), but not the locus coeruleus (LC). Repeatedly footshocked animals exhibited more time freezing than animals given a single footshock session. Prenatal exposure to cocaine resulted in more time spent freezing in C40 than LCC animals during the chronic footshock exposure period; however, no differences were seen in any of the physiological measures taken from these 2 groups on the final test day. The implications of these findings are discussed in the context of other research examining the effects of prenatal cocaine exposure on stress responses.

Effects of prenatal cocaine exposure on haloperidol-induced increases in prolactin release and dopamine turnover in weanling, periadolescent, and adult offspring.

Offspring of dams given 40 mg/kg cocaine SC on gestational days (GD) 8-20 (E8-20) (C40), dams given 0.9% saline SC on E8-20 that were pair fed and watered to C40 dams (PF), and untreated control dams given ad lib access to food and water (LC) were challenged with haloperidol (0.0, 0.05, 0.10, or 0.50 mg/kg) at either 21, 35, or 60 days postnatally (P21, 35, 60). One hour postinjection, animals were sacrificed, trunk blood collected for assay of prolactin, and the striatum (ST) and nucleus accumbens (NAc) removed. The ratio of the dopamine metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid to dopamine (DA) as well as the ratio of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) to serotonin (5-HT) were determined in these brain regions as an index of DA and 5-HT turnover, respectively. Assessment of 5-HIAA/5-HT ratios did not indicate any reliable dose or prenatal treatment effects. Reminiscent of previous findings obtained in C40 offspring at P11 (35), P21 C40 offspring exhibited a slightly reduced sensitivity to haloperidol relative to LC controls both in terms of DA ratios in the NAc as well as plasma prolactin levels. These findings were also evident in PF controls suggesting that they may be the result of prenatal undernutrition. Furthermore, this reduced sensitivity was not evident at the older test ages. At P60, planned comparisons revealed haloperidol-induced increases in prolactin levels in C40 males but not PF or LC males; these findings could potentially reflect feminization in males following prenatal cocaine exposure.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopaminergic and peptidergic mRNA levels in juvenile rat brain after prenatal cocaine treatment.

The effects of prenatal cocaine treatment on gene expression in dopaminergic pathways of juvenile rats were investigated by in situ hybridization histochemistry. Pregnant rats from gestational day 8 to 20 were administered one of the following treatments: (A) 40 mg/kg cocaine hydrochloride/3 ml/day s.c.; (B) 0.9% saline/3 ml/day s.c. and pair fed to cocaine-exposed dams; (C) 0.9% saline/3 ml/day s.c. and placement on cellulose-diluted diet to match the caloric intake of the cocaine-treated group without explicit food restriction; (D) no injection and lab chow diet. Levels of mRNA for the dopamine transporter, tyrosine hydroxylase, cholecystokinin, D1 and D2 dopamine receptors and enkephalin were quantitated in relevant dopaminergic regions of forebrain and midbrain of offspring that were sacrificed on postnatal day 21. Quantitative analysis revealed no significant changes in mRNA levels in any of the brain regions examined. In the present animal model, cocaine exposure in utero had no significant effect on mRNA levels of the dopamine transporter, D1 or D2 dopamine receptors, enkephalin, tyrosine hydroxylase, or cholecystokinin in juvenile rats.

The 5-HT1A agonist 8-OH-DPAT increases attachment maintenance but decreases suckling-related intake in 17-18-day-old rat pups.

Deprived and nondeprived preweanling (17-18 days of age) Sprague-Dawley rat pups were injected with 0, 0.03, 0.06, 0.1, or 0.5 mg/kg of the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and observed in a suckling test using a milk replete anesthetized dam, with milk let-downs being intermittently precipitated via IV infusions of oxytocin. In experiment 1, the 0.5 mg/kg dose of 8-OH-DPAT was observed to increase the proportion of nondeprived animals which attached to a nipple; no dose effect was seen in deprived animals, who generally all attached. Deprived pups given the 0.5-mg/kg dose exhibited a lower frequency of nipple disattachment/reattachment following milk let-downs and had significantly lower percent body weight gains when compared with saline controls. In experiment 2a, the 0.5-mg/kg dose of 8-OH-DPAT was observed to decrease the overall incidence of nipple disattachment/reattachment as well as to suppress nipple shifting per se in both deprived and nondeprived 17-18-day-old rat pups; this dose also suppressed body weight gains in both the deprived and nondeprived pups. The suppression in weight gain by 8-OH-DPAT does not appear to be primarily related to a drug-induced reduction in nipple shifting. In experiment 2b, where pups were given access to only one nipple, an 8-OH-DPAT-related reduction in body weight gain was still evident. These experiments, which demonstrate that attachment maintenance and suckling ingestion are altered in opposite ways by 8-OH-DPAT, provide strong evidence that these two suckling-related phenomena are subject to different physiological controls.

Repeated exposure of rat pups to isolation attenuates isolation-induced ultrasonic vocalization rates: reversal with naltrexone.

Young rat pups are dependent on the dam for their survival, thus isolation of the neonatal rat pup from the dam presents the young organism with a variety of stressors. The question examined in this study concerns the ability of the young rat pup to modify its response to isolation following repeated exposure to that isolation as well as the role played by endogenous opiates in this process. Following repeated isolations, pups were seen to decrease vocalization rates. Altering the context in an attempt to dishabituate animals failed to reverse the decreased vocalization rate. However, opiate receptor blockade attenuated this decrease when administered subsequent to the first isolation period but not prior to the last isolation period. These results suggest that the development of this attenuated response to isolation stress is opiate-mediated but that once established, its expression is not dependent on endogenous opiate release.

Prenatal cocaine exposure increases the behavioral sensitivity of neonatal rat pups to ligands active at opiate receptors.

Offspring of dams given 40 mg/kg cocaine HCl (C40) from gestational day 8-20 (E8-E20), pair-fed dams injected daily with saline (PF), nutritional control dams placed on a 40% cellulose based diet and injected with saline daily (NC), and untreated dams (LC) were examined. Offspring were given morphine (0.0, 0.1, or 0.5 mg/kg SC) on postnatal day 10-11 (P10-11) in Experiment 1, and isolation-induced ultrasonic vocalizations were measured. Planned comparisons indicated that both C40 and NC offspring exhibited a greater sensitivity to the morphine-related decrease in isolation-induced ultrasounds than LC controls. However, the presence of an anesthetized littermate suppressed isolation-induced ultrasounds equally across all groups, with all groups of offspring spending equal amounts of time in physical contact with the littermate. A tail-flick measure of analgesia indicated that PF animals were hyperalgesic relative to the other prenatal treatment groups; however, no differences in sensitivity to morphine were seen across the prenatal groups. In Experiment 2, animals were given the selective delta, [D-Pen2,D-Pen5]-enkephalin (DPDPE), and mu, [D-Ala2-NMe-Phe4Gly ol]-enkephalin (DAMGO) agonists ICV and ultrasonic vocalizations were recorded. Results indicated that both C40 and NC offspring were more sensitive to the low dose of DAMGO; however, because of the profound suppression of vocalizations seen at both doses of DPDPE, potential differences among the prenatal treatment groups in responsiveness to the delta agonist were difficult to detect.(ABSTRACT TRUNCATED AT 250 WORDS)

A fostering study of the effects of prenatal cocaine exposure: II. Offspring behavioral measures.

The impact of rearing condition was assessed in Sprague-Dawley dams given 40 mg/kg cocaine (C40) or saline (LC control) subcutaneously (SC) from gestational days 8-20 and their offspring. Treated pups reared by their biological dams (LC/LC; C40/C40), treated pups reared by surrogate dams (FOS/LC; FOS/C40), and foster pups raised by treated dams (LC/FOS; C40/FOS) were examined. On postnatal day 7 (P7), pups received either 0 (unpaired) 2, 3, or 4 pairings of an odor and footshock and were tested for their aversion to this odor. Foster and LC pups, regardless of rearing condition, exhibited significant odor aversions following either 2, 3, or 4 training trials. In contrast, C40 pups reared by surrogate dams required 4 trials to acquire the aversion, and C40 pups reared by their own dams did not exhibit conditioning even after 4 trials. At P17, no differences were seen among the groups in the aversion formed to an auditory or an olfactory stimulus that was paired with footshock. At P60, shock-elicited aggression among pairs of siblings was examined. Regardless of prenatal exposure condition, offspring reared by dams given cocaine showed a decreased latency to the first aggressive contact, an effect that was evident without any alteration in shock sensitivity. Together these data suggest that being reared by a dam previously exposed to cocaine has an impact on offspring behavioral function apart from the effects of prenatal cocaine exposure per se. The implications of the data regarding the cognitive performance of pups exposed prenatally to cocaine are also discussed.

Prenatal cocaine exposure attenuates cocaine-induced odor preference in infant rats.

In order to further examine whether prenatal cocaine exposure alters the later reward efficacy of cocaine, exposed offspring were tested for cocaine-induced odor preference early in life. Test offspring were derived from Sprague-Dawley dams that received daily SC injections of 40 mg/kg/3 cc cocaine hydrochloride (C40) from gestational day 8-20, nutritional control dams receiving daily SC saline injections (NC), and nontreated control dams (LC). At testing on postnatal day 8 (P8), both LC and NC offspring were observed to exhibit a preference for the odor that had been paired on P7 with 2.0, 5.0, or 10.0 mg/kg cocaine. In contrast, C40 offspring exhibited a significant odor preference only when the odor had been previously paired with 5.0 or 10.0 mg/kg cocaine. These results, combined with previous work from our laboratory showing that adult offspring exposed gestationally to cocaine did not exhibit a cocaine-induced conditioned place preference, provide evidence that offspring exposed prenatally to cocaine are less likely to develop a preference for stimuli associated with cocaine. Further studies are needed to determine whether these alterations in cocaine preference reflect a learning deficit, pharmacokinetics factors, or an attenuation in the rewarding properties of cocaine.

Psychopharmacological effects of MK-801 in infant and preweanling rat pups.

-Neonatal (3-4-day-old) and preweanling (17-18-day-old) Sprague-Dawley rat pups were tested following SC administration of saline, 0.01, 0.05, 0.1, 0.5, or 1.0 mg/kg MK-801. In neonatal rat pups, reductions in a number of behaviors (forward locomotion, mouthing) were seen at the higher (0.5 and 1 mg/kg) doses. In contrast, evidence of behavioral stimulation in forward locomotion at 30 min postinjection was seen at a lower dose (0.1 mg/kg). In preweanling rat pups, marked sedative effects of MK-801 were seen at higher doses (decreases in forward locomotion, headlift and sniff), with signs of behavioral stimulation (increases in forward locomotion and mouthing) evident at low doses. Thus, as in adults, low doses of MK-801 may be behaviorally stimulatory and higher doses inhibitory to both neonatal and preweanling pups, although the stimulatory effects appear to be somewhat less pronounced in these young animals than has been previously reported in adulthood.