Long-term effects of acute perinatal asphyxia on rat maternal behavior.

In this study we used a rat model of graded perinatal asphyxia to study the long-term consequences of this manipulation on rat maternal behavior at adulthood. Rats were delivered by cesarean (C) section and the pups, still in the uterus horns, were placed into a water bath at 37 degrees C for periods of 0 (controls) or 20 min (asphyxia). Subsequently, female pups were given to surrogate mothers, weaned at 21 days postnatally and then left undisturbed until adulthood, when they were mated. Once they gave birth, on postnatal days (Pnds) 1, 3, 5, 7, 9, 11 and 13 they were observed in the home cage five times per day to assess their maternal behavior in an undisturbed condition. In addition, maternal behavior was observed for 30 min in a novel cage on Pnds 4 and 8. Perinatal asphyxia affected maternal behavior in the home cage, hypoxic females being more often found outside the nest area and performing more often behaviors such as self-grooming. Principal component analysis confirmed a more 'active' behavioral profile for hypoxic females. Hypoxic mothers were characterized by a longer latency to perform on-nest behavior and by a reduced frequency of pup retrieval and licking in the novel cage. No significant differences in corticosterone secretion in response to an acute stressor were found in dams belonging to the different treatments or in the body weights of the offspring. These results are suggestive of an arousal deficit due to perinatal hypoxia and point to the dopaminergic system as a potential neurochemical target for an early hypoxic insult.

Prenatal AZT or 3TC and mouse development of locomotor activity and hot-plate responding upon administration of the GABA(A) receptor agonist muscimol.

RATIONALE: Zidovudine (AZT) and lamivudine (3TC) are nucleoside analogues administered prenatally in clinical practice, separately or in combination, as antiretroviral drugs to prevent HIV mother-to-child transmission by inhibiting viral reverse transcriptase. In animal studies pre- and/or perinatal exposure to AZT and 3TC induce age- and sex-dependent neurobehavioural alterations in the offspring. OBJECTIVE: Investigation of short- and medium-term effects of in utero exposure to AZT or 3TC on development of the GABAergic system. METHODS: Pregnant CD-1 mice were given orally twice daily AZT (160 mg/kg), 3TC (500 mg/kg) or vehicle solution (NaCl 0.9%) from pregnancy day 10 to delivery. Offspring locomotion and nociceptive sensitivity were examined on postnatal day (pnd) 8, 14, and 28 after administration of two doses of GABAergic agonist muscimol (pnd 8 and 14: 0.05 and 0.2 mg/kg; pnd 28: 0.2 and 1.0 mg/kg). A 30-min locomotor activity test and a 60 s hot-plate test (50+/-1 degrees C) were used. RESULTS: AZT and 3TC treated mice showed a mild increase of locomotor activity after administration of the high dose muscimol on pnd 8. On pnd 14 the low muscimol dose enhanced locomotor activity in vehicle and 3TC, but not in AZT pups, whereas no prenatal treatment effect was evident on pnd 28. AZT increased nociceptive sensitivity at all ages considered. CONCLUSIONS: Prenatal AZT effects on locomotor activity appear clearly detectable after GABAergic challenge and seem to be transient. AZT effects on pain sensitivity did not appear to be dependent on GABA regulated nociceptive mechanisms. Prenatal 3TC exposure had rather limited effects on locomotor activity development, and no effect on nociception.

Prenatal exposure to anti-HIV drugs: neurobehavioral effects of zidovudine (AZT) + lamivudine (3TC) treatment in mice.

BACKGROUND: The new antiretroviral treatments that combine the zidovudine (AZT) regimen with lamivudine (3TC) appear as a cost-effective alternative to the current AZT monotherapy to prevent mother-to-fetus transmission of the HIV-1 virus. Recent evidence in uninfected children raised concern about the long-term effects of perinatal exposure to AZT and 3TC, especially when used in combination. Animal studies indicated behavioral changes in offspring exposed perinatally to both AZT and 3TC, whereas no animal data are available on the effects of the perinatal exposure to the AZT + 3TC combination on neurodevelopment. METHODS: Pregnant CD-1 mice received p.o. AZT + 3TC (160 and 500 mg/kg, respectively) or vehicle solution (NaCl 0.9%) twice daily from gestational day 10 to delivery. Maternal reproductive endpoints such as pregnancy length, abortion, litter size, sex ratio, and offspring viability were assessed. Pups were scored for different somatic and behavioral endpoints, including sensorimotor development, homing performance on postnatal day (PND) 10, passive-avoidance testing (PND 22-23), locomotor activity (PND 23), and social interaction (PND 35). RESULTS: While no effects were observed on maternal reproductive endpoints, treated pups showed a long-lasting reduction of body weight and a slightly delayed maturation of placing and grasping reflexes and pole grasping. No effects on passive-avoidance or locomotor activity were found. AZT + 3TC-treated mice showed selective alterations in the social interaction test; the treated female offspring also displayed a significant reduction of affiliative interactions. CONCLUSIONS: The combination of AZT and 3TC (1) induced small, but more marked, effects on somatic and sensorimotor development than either of these drugs administered separately; and (2) affected juvenile social behavior.

[Animal models of hypoxic-ischemic encephalopathy]. / I modelli animali di encefalopatia ipossico-ischemica.

This review presents the animal models more widely used to study neurological and neuropathological outcomes of perinatal asphyxia. Methods used to induce hypoxia/ischemia in fetal and newborn non-human primates, lambs, piglets and rodents are concisely described, reporting the more relevant neuropathological and behavioural findings. In line with human observational data, experimental studies indicate that motor behaviour and attentional/cognitive abilities are among those behavioural regulations more significantly affected by perinatal asphyxia.

Prenatal exposure to anti-HIV drugs. long-term neurobehavioral effects of lamivudine (3TC) in CD-1 mice.

The present study was aimed at investigating the long-term effects of prenatal exposure to lamivudine (3TC), an antiretroviral drug used in clinical practice alone or in combination with zidovudine (AZT) to prevent mother-to-child transmission of the HIV virus. Pregnant CD-1 mice were given per os twice daily either 3TC at different doses (125, 250, or 500 mg/kg) or vehicle solution (NaCl 0. 9%) from pregnancy day 10 to delivery. Offspring behavior was examined on postnatal day 35 in a 20-min social interaction test. At adulthood different behavioral endpoints were analyzed, including locomotor activity and exploration in an open field following administration of the muscarinic antagonist scopolamine (2 mg/kg), spatial learning in either radial arm or Morris water maze, virgin female behavior in a maternal induction test, and pain sensitivity in a hot-plate test (52 +/- 0.1 degrees C). Our findings confirm the low neurotoxicity of 3TC in comparison to AZT. However some significant behavioral alterations were found, namely (1) a decrease in immobility in the open field test, (2) an increase in the responsiveness to scopolamine shown by the 500-mg/kg 3TC mice (sniffing behavior) in the open field, and (3) a longer escape latency in the first day of the reversal phase in the Morris task (particularly marked in the 250-mg/kg treatment group). No significant changes in either pain sensitivity, social/affiliative, or maternal behavior were found, although a higher occurrence of aggressive behavior toward foster pups was noted in both 125- and 500-mg/kg 3TC females.

Effects of prenatal AZT+3TC treatment on open field behavior and responsiveness to scopolamine in adult mice.

Treatment of pregnant seropositive women and their neonates with the nucleoside analogs (reverse transcriptase inhibitors) zidovudine (AZT), lamivudine (3TC) and their combination has become a standard of care in industrialized countries to prevent transmission of the HIV-1 virus. Animal studies indicated limited but significant behavioral changes in AZT or 3TC-prenatally exposed offspring, whereas data on the potential neurobehavioral outcomes of AZT+3TC combination are still lacking. The aim of the present study was to assess in mice prenatally exposed to AZT+3TC the functional state of cholinergic muscarinic neuroregulation at adulthood. Pregnant CD-1 mice received per orem twice daily AZT+3TC (160 and 500 mg/kg, respectively) or vehicle solution (NaCl 0.9%) from gestational day (GD) 10 to delivery (GD 19). Locomotor activity, exploratory behavior and responsiveness to the muscarinic cholinergic blocker scopolamine (2 mg/kg) were analyzed at adulthood (PND 70) in offspring of both sexes in an open field test. Results indicated that prenatal AZT+3TC exposure does not influence responsiveness to the muscarinic cholinergic antagonist as measured by analysis of the drug's effects on locomotor and exploratory activity and different behavioral items. However, AZT+3TC-treated mice displayed higher frequency of rearing, and lower frequency and duration of self-grooming behavior, consistent with an effect on dopaminergic neurotransmission. However, this would need confirmatory experiments.

Neurobehavioral effects of prenatal lamivudine (3TC) exposure in preweaning mice.

The present study provides a characterization of the behavioral changes induced in preweaning mice by prenatal exposure to lamivudine (3TC), an antiviral drug recently entered in the clinical practice to treat HIV patients. Pregnant CD1 mice were given per os bidaily either 3TC at different doses (125, 250, or 500 mg/kg) or vehicle solution (saline 0.9%) from pregnancy day 10 to delivery. Data on reproductive performance, such as gestation length, litter size, and offspring viability, were collected. Offspring were then examined for a series of different somatic and behavioral end points, including sensorimotor development, ontogenetic pattern of ultrasonic vocalization, passive avoidance learning, and locomotor activity. In the absence of gross changes in somatic and sensorimotor development, a slight change in ultrasound emission was found on postnatal day (PND) 3, with 125 and 500 mg/kg 3TC-treated offspring emitting a lower number of ultrasounds. Learning and retention performances of a passive-avoidance task on PND 20-21 were unaffected by 3TC treatment, while decreased habituation in an automated locomotor activity test was evident in male offspring exposed to 250 and 500 mg/kg 3TC.

Neonatal 192 IgG-saporin lesions of basal forebrain cholinergic neurons selectively impair response to spatial novelty in adult rats.

The role of the developing cholinergic basal forebrain system on cognitive behaviors was examined in 7 day-old rats by giving lesions with intraventricular injections of 192 IgG-saporin or saline. Rats were subjected to passive avoidance on postnatal days (PND) 22-23, water maze testing on PND 50-60, and a open-field test (in which reactions to spatial and object novelty were measured) on PND 54. Behavioral effects of the lesions were evident only in the open-field test with 5 objects. Unlike controls, the lesioned rats did not detect a spatial change after a displacement of 2 of the 5 objects. Control and lesioned rats, however, showed comparable novelty responses to an unfamiliar object. Lesion effectiveness was confirmed by 75% and 84% decreases in choline acetyltransferase activity in cortex and hippocampus. These results suggest that the developing cholinergic system may be involved in spatial information processing or attention to spatial modifications.

Scopolamine impairs memory recall in Octopus vulgaris.

The involvement of the central cholinergic system in predatory performance, and on the recall of individual and observational memory in Octopus vulgaris was studied by treating the animals with the muscarinic antagonist scopolamine (2 mg/kg). The absence of the effects of the injection of scopolamine on blood circulation was also checked. Scopolamine did not affect the ability of octopuses to prey on live crabs. However, it interfered significantly with memory recall. In fact, the ability to solve the jar problem was impaired within the first hour after injection (short-term effects) and was only partially recovered after 24 h (long-term). Moreover, both individual and observational learning of a visual discrimination were significantly reduced at the short- and long-term testing. These results support a role of the cholinergic system in the processes of memory recall of O. vulgaris.

Neonatal cocaine alters behavioural responsiveness to scopolamine and cholinergic development in mice.

CD-1 mice received daily subcutaneous injections of either cocaine (20 mg/kg or 40 mg/kg) or saline solution (0.9% NaCl) from postnatal days 2 to 15. Pups were tested on days 16-17 for learning and 24-h retention of a passive avoidance task, where entering a dark compartment was punished with a mild foot shock. Locomotor activity and general behaviour in an open field arena were assessed on day 21, following administration of either the muscarinic blocker scopolamine (0.8 mg/kg) or saline solution. In addition, immunostaining for the enzyme choline acetyltransferase (ChAT) was measured in different basal forebrain areas (medial septum, striatum, and nucleus basalis) on day 30. Cocaine treatment failed to affect either learning or retention capabilities. Nonetheless, neophobic behaviour during the learning session was enhanced in control nonpunished mice exposed to the 20-mg/kg dose. In the open field test, although baseline activity levels were unaffected by cocaine exposure, the 40-mg/kg cocaine-treated pups showed decreased sensitivity to the hyperkinetic effects of scopolamine. ChAT immunocytochemistry revealed a significant reduction of the number of ChAT-immunopositive neurons in the nucleus basalis but not in the other cholinergic basal forebrain regions.

Systemic administration of anti-NGF antibodies to neonatal mice impairs 24-h retention of an inhibitory avoidance task while increasing ChAT immunoreactivity in the medial septum.

Neonatal mice received subcutaneous injections of either antibody against murine NGF raised in goat (3 mg, injection volume 50 microliters) or preimmune serum on postnatal days 2, 4, 6, 8, 10, and 12. They were tested on postnatal days 15-16 or 20-21 for learning and 24-h retention of a passive avoidance step-through task. Immunostaining for choline acetyltransferase (ChAT) was measured in two cholinergic forebrain areas (septum and caudate-putamen) on postnatal day 16 or 21. Locomotor activity and exploratory behavior in an open-field test were also assessed on day 17 or 22, following a single administration of either scopolamine (2 mg/kg) or saline solution. While anti-NGF treatment did not affect acquisition on day 15, impairment in retention was evident on day 16. On days 20-21, no effects were found either on acquisition or on retention capabilities. Analysis of ChAT immunostaining revealed a significant increase of ChAT-immunopositive cells in the medial septal area in 16-day-old but not in 21-day-old mice. Behavior in the open-field test and age-typical response to scopolamine were not altered by anti-NGF at either of the two ages considered. These data support the view that immunological neutralization of endogenous NGF specifically affects the maturation of retention capabilities in altricial rodents, and confirm the involvement of forebrain cholinergic mechanisms in early memory processes.

Neonatal exposure to anti-nerve growth factor antibodies affects exploratory behavior of developing mice in the hole board.

The aim of this study was to assess in developing mice whether the neutralization of endogenous NGF following ICV administration of anti-NGF antibodies (50 micrograms/2 microliters) on postnatal days 3, 6, 9, and 12 affected locomotor activity, exploratory behavior, and response to the cholinergic blocker scopolamine. In Experiments 1 and 2 activity and age-typical scopolamine effects were evaluated on PND 13 or 17 in an automated apparatus. No significant main effect of anti-NGF treatment was found at either age. On day 13 scopolamine (0.2, 1, or 2 mg/kg) decreased locomotion in both anti-NGF and control animals. In Experiment 3, locomotion and exploratory behavior were analyzed in an open field arena or in a hole board apparatus on PND 16. No significant effects of anti-NGF treatment on general motor activity and investigation of a novel object in the open field was found, though anti-NGF animals tended to be less active than controls. In the hole board anti-NGF pups showed a different pattern of head dipping behavior from controls, exploring mainly the holes located in the periphery of the apparatus.

Age-dependent effects of NGF and scopolamine on suckling behavior of neonatal mice.

Nerve growth factor (NGF) influences the neurochemical differentiation of central cholinergic neurons of developing rodents. In this study, NGF was given intracerebrally to mice on different postnatal days (days 5 and 7, or days 8 and 10). Pups were tested for suckling behavior 24 h after the second NGF injection, following systemic administration of either the muscarinic cholinergic antagonist scopolamine or saline solution. Scopolamine significantly impaired nipple attachment on day 11 but not on day 8, and decreased locomotor activity in 11-day pups. NGF given on days 5 and 7 increased paddling and treading on day 8, and this effect was more pronounced in scopolamine injected pups. Pretreatment with NGF on days 8 and 10 decreased activity levels in 11-day pups. The differences in the effects of scopolamine at successive ages suggest that distinct portions of the cholinergic system mature at different rates and that sensitivity to NGF is age dependent. NGF appears to influence functional maturation of that portion of the cholinergic system involved in the regulation of locomotor activity.

Growth factors in behavioral teratology.

Polypeptide Growth Factors are protein molecules which regulate cell proliferation and/or differentiation. A number of different Growth Factors (GFs) have been identified and characterized in recent years, and they have been shown to control several physiological processes, such as growth, repair, differentiation, and development of specific cell populations. In particular Nerve Growth Factor, the best characterized among the about 30 GF molecules, is endowed with specific activities on cholinergic and peptidergic CNS neurons. Several GFs originally named according to their biological activity (Epidermal Growth Factor, EGF; Fibroblast Growth Factor: FGF; Transforming Growth Factor: TGF; Insulin-like Growth Factor: IGF) have been recently found in the central nervous system. The effects of in vivo GF administration on the ontogenesis of altricial rodents are reported. Indexes of neurobehavioral maturation are accelerated upon neonatal NGF and bFGF exposure, while a similar treatment with EGF exerts both growth-promoting and growth-inhibiting effects on mouse somatic and behavioral development. Administration of IGF appears to affect ultrasonic vocalization in mouse pups. Moreover, NGF given intracerebroventricularly to newborn mice anticipates both the appearance of the scopolamine-induced hyperactivity and the maturation of behaviours under cholinergic control. The present findings are in agreement with a model where different GFs can "switch on" developmental events leading sometimes to dramatic changes in the normal ontogenetic pattern.

NGF and cholinergic control of behavior: anticipation and enhancement of scopolamine effects in neonatal mice.

Male mouse pups of the Swiss-CD1 strain received on postnatal days 2 and 4 either an intracerebroventricular (i.c.v.) administration of 30 micrograms murine nerve growth factor (NGF) or cytochrome c. Pups were then tested for suckling behavior on their anesthetized multiparous dam on day 5, following intraperitoneal (i.p.) administration of either the muscarinic cholinergic antagonist scopolamine (2 mg/kg) or saline solution (0.9%). Scopolamine produced a significant increase in latency time to suckle, while reducing the time pups spent attached to the nipple. NGF exposure enhanced scopolamine effects on latency to suckle as well as on time spent attached to the nipple. More striking, NGF pups showed a marked hyperactivity after scopolamine, an effect which normally appears only around weaning time. These results support the hypothesis that NGF plays a crucial role in the functional maturation of central cholinergic mechanisms involved in the control of behavior.