Susan Barron

Neonatal Alcohol Exposure Alters Suckling Behavior in Neonatal Rat Pups

Prenatal alcohol exposure has been associated with a variety of suckling deficits in both humans and animals. In this study, the effect of neonatal alcohol exposure on suckling performance was examined in 15-day-old rat pups. Neonatal alcohol exposure has been used as a model to study the effects of alcohol exposure during a period equivalent to the human third trimester with respect to brain growth. Subjects were Long-Evans rats which had been artificially reared (AR) and fed through gastrostomy tubes from postnatal day (PN) 4-PN 12. The AR groups included two groups given ethanol doses of 6 g/kg/day or 4 g/kg/day and an isocaloric maltose-dextrin control group. A suckled control group raised by their natural mothers was also included to control for artificial rearing. Fifteen-day-old pups were individually placed with an anesthetized dam for a 1-h videotaped test session. Pups in the 6 g/kg alcohol group took longer to attach to the nipple and spent less time suckling than pups from all other treatment groups. Nipple-shifting behavior was disrupted in all artificially reared groups, but it was most severely affected in the 6 g/kg group. These findings suggest that neonatal alcohol exposure interferes with suckling performance and these altered behaviors may contribute to the postnatal growth deficits that have been reported following alcohol exposure in utero.

The effects of prenatal alcohol exposure on odor associative learning in rats

Alcohol was administered to pregnant females via a liquid diet that contained either 35% ethanol-derived calories (35% EDC) or 0% EDC on gestation days 6-20. An ad lib lab chow group (LC) was also included. In Experiment 1, odor-aversion learning was examined in 10-day-old offspring. While both the 0% EDC and LC groups displayed odor aversions, the 35% EDC offspring did not. In Experiment 2, learning was assessed in an appetitive paradigm in three-day-old offspring. Once again, the 35% EDC offspring showed no evidence of learning. Experiment 3 examined odor-aversion learning in adults. Both alcohol-exposed offspring and controls learned the odor association equally well. These findings suggest that odor associative learning is a sensitive indicator for alcohol-related learning deficits in rat pups although these deficits may dissipate as the offspring matures. Since odor associations play a critical role in neonatal behaviors, these deficits may help explain other behavioral anomalies noted following prenatal alcohol exposure.

Agmatine reduces balance deficits in a rat model of third trimester binge-like ethanol exposure

This study examined the effects of binge-like ethanol (ETOH) exposure in neonatal rats on a cerebellar-mediated balance task, and the ability of agmatine, an n-methyl-d-aspartate receptor (NMDAR) modulator, to reverse such effects. Five neonatal treatments groups were used, including ETOH (6.0 g/kg/day), AG (20 mg/kg), ETOH plus AG (6.0 g/kg/day and 20 mg/kg), a maltose control, and a non-treated control. Ethanol was administered via oral intubation twice daily for eight days, (AG was administered with the last ETOH intubation only). Two exposure periods were used; PND 1-8 or PND 8-15. On PND 31-33, balance performance on a single dowel was tested. Treatment with AG during withdrawal in ETOH exposed animals improved performance relative to ETOH alone among the PND 1-8 exposure period. ETOH exposure during the 2nd postnatal week did not impair balance. These findings provide further support that exposure to ETOH during critical developmental periods can impair performance on a cerebellar-dependent balance task. Of perhaps greater significance, co-administration of agmatine reduced these deficits suggesting that NMDA modulation via polyamine blockade may provide a novel approach to attenuating damage associated with binge-like ETOH consumption.

Neural grafting reverses prenatal drug-induced alterations in hippocampal PKC and related behavioral deficits

Administration of heroin or phenobarbital to pregnant mice evokes neurochemical and behavioral deficits consequent to disruption of septohippocampal cholinergic innervation. The present study evaluates the relationship between the drug-induced biochemical changes and the behavioral deficits, applying two different approaches: neural grafting and within-individual correlations of biochemistry and behavior. Mice were exposed transplacentally to phenobarbital or heroin on gestational days 9-18 and tested in adulthood. Drug-exposed mice displayed impaired radial arm maze performance, increases in presynaptic choline transporter sites (monitored with [(3)H]hemicholinium-3 binding), upregulation of membrane-associated protein kinase C (PKC) activity, and desensitization of the PKC response to a cholinergic agonist, carbachol. Grafting of cholinergic cells to the impaired hippocampus reversed the behavioral deficits nearly completely and restored basal PKC activity and the PKC response to carbachol to normal; the drug effects on hemicholinium-3 binding were also slightly obtunded by neural grafting, but nevertheless remained significantly elevated. There were significant correlations between the performance in the eight-arm maze and both basal PKC activity and PKC desensitization, and to a lesser extent, between behavioral performance and hemicholinium-3 binding. Taken together, these findings indicate an inextricable link between the biochemical effects of prenatal drug exposure on the PKC signaling cascade and adverse behavioral outcomes. The ability of neural grafting to reverse both the drug-induced changes in PKC and behaviors linked to septohippocampal cholinergic function suggest a mechanistic link between this signaling pathway and neurobehavioral teratology caused by heroin or phenobarbital.

Polyaminergic agents modulate contextual fear extinction in rats

Polyamines, such as spermidine and spermine, have been reported to improve memory retention through the activation of N-methyl-d-aspartate receptors (NMDAr). However whether polyamine agonists and antagonists alter extinction remains unclear. In the current study, we investigated whether spermidine and polyamine antagonists that selectively block the NR2B subunit at the NMDAr alter the extinction of contextual conditioned fear in male Wistar rats. The bilateral intra-hippocampal administration of exogenous spermidine (2 nmol/site) immediately after, but not 6h after extinction training, facilitated the extinction of fear conditioning. The injection of the NMDAr antagonists arcaine (0.2 nmol/site), ifenprodil (20 nmol/site) and traxoprodil (0.2 nmol/site), disrupted fear extinction and, at doses that had no effect per se, reversed the facilitatory effect of spermidine on fear extinction. These results suggest that exogenous and endogenous polyamines facilitate the extinction of contextual conditioned fear through activation of NR2B subunit-containing NMDAr in the hippocampus. Since extinction-based exposure therapy is widely used as treatment for a number of anxiety-related disorders, including phobias and post-traumatic stress, the currently reported facilitation of extinction by polyaminergic agents suggest these compounds as putative candidates for drug development.

Hyperactivity in preweanling rats following postnatal alcohol exposure

Neonatal alcohol exposure in rats has been used as a model to study the effects of third-trimester alcohol consumption in humans. In the present research, male and female rat pups were artificially reared (AR) and received condensed alcohol exposure (6 g/kg/day or 4 g/kg/day) on postnatal days (PNs) 4 through 9. Controls consisted of both artificially reared animals receiving maltose-dextrin substituted for alcohol and normally reared animals. These rats were tested for open-field activity at 18 days of age for four days. Both male and female rat pups that received the high dose of alcohol (6 g/kg/day) evidenced overactivity relative to pups in both control groups. These findings extend previous work and indicate that neonatal alcohol exposure can induce hyperactivity in young male rats.

Alcohol withdrawal-induced hippocampal neurotoxicity in vitro and seizures in vivo are both reduced by memantine.

BACKGROUND The ethanol withdrawal (EWD) syndrome is typically treated using benzodiazepines such as diazepam. However there is concern that benzodiazepines may not prevent neurotoxicity associated with EWD. Antagonists of glutamate/N-Methyl-D-Aspartate receptors (NMDARs) such as MK801 have been shown to be effective against both EWD-induced neurotoxicity in vitro and seizures in vivo. However, most of these agents have adverse side effects. An exception is the moderate affinity NMDAR channel blocker memantine, used in Alzheimers dementia. The present studies examined the ability of memantine to protect against EWD-related toxicity in vitro and seizures in vivo. METHODS Organotypic hippocampal slice cultures from neonatal rat pups were treated starting at 15 days in vitro with 100 mM ethanol for 10 days followed by a 24-hour EWD period. During the 24-hour EWD period cultures were treated with memantine (15 or 30 microM). MK801 (10 microM) was utilized as a positive control. For the in vivo studies, the ability of memantine (2, 5, 10, and 15 mg/kg) to reduce convulsions was analyzed in Swiss-Webster mice using the handling induced convulsion test paradigm. RESULTS In vitro studies demonstrated that memantine is effective at blocking EWD-induced neurotoxicity. In vivo experiments showed that memantine also significantly reduced convulsions induced by EWD in mice. CONCLUSIONS Memantine may be of therapeutic value during alcohol detoxification by virtue of its having neuroprotective effects in addition to anti-seizure activity. The potential role of memantine in treatment of alcoholism is deserving of further study.

Neonatal ethanol and nicotine exposure causes locomotor activity changes in preweanling animals

Sprague-Dawley rats were used to investigate the effects of neonatal ethanol (ETOH) and nicotine (NIC) exposure on activity levels in preweanling offspring. Male and female pups received daily oral intubations of ethanol ((ETOH) 5 g/kg/day), nicotine ((NIC) 12 mg/kg/day), ethanol and nicotine ((ETOH+NIC) 5 g/kg/day+12 mg/kg/day) or isocaloric maltose (control) on either postnatal days (PND) 1-7 or PND 8-14. A non-treated control group was also included. Peak blood ethanol concentrations (BECs) measured in a separate subset of animals ranged from 167 and 344 mg/dl depending upon neonatal treatment and period of exposure. Subjects were tested in an open field apparatus on PND 19-21. Animals exposed to ETOH or ETOH+NIC on PND 1-7 were hyperactive relative to the other treatment groups. In contrast, animals exposed to NIC or ETOH+NIC during PND 8-14 were hypoactive relative to other treatment groups. Males appeared more sensitive than females on measures of anxiety (distance traveled in the center of the open field) but this also varied dependent on neonatal treatment and period of exposure. These findings suggest that the third trimester is a critical period for ETOH and NIC effects on offspring activity although the pattern of effects on activity are different depending on when drug exposure occurred during the neonatal period.

The effects of prenatal alcohol exposure on behavioral and neuroanatomical components of olfaction

Prenatal alcohol exposure is associated with deficits in odor-associative learning in very young rat pups. One alternative explanation for these findings is that rather than a learning deficit per se, alcohol-exposed pups may display a sensory deficit. The present study was designed to examine the effects of prenatal alcohol exposure on behavioral and neuroanatomical components involved in olfaction. The subjects in this study were pups exposed to 35% ethanol-derived calorie (EDC) liquid diet from gestation days (GD) 6-20. Two control groups were included, a 0% EDC pair-fed and an ad lib lab chow group. In Experiment 1, respiratory response to a novel odor was examined in pups tested at either 3, 4, or 10 days of age. The 35% EDC offspring clearly detected the odor. Furthermore, there was an apparent alcohol-related development delay in respiratory rate as shown by a lower baseline respiratory rate at PN 3 relative to controls which was no longer apparent by PN 4. Experiment 2 examined the volume of two neuroanatomical structures involved in olfaction, the main olfactory bulb (MOB) and the vomeronasal organ (VNO) in 3-day-old pups. Prenatal alcohol exposure was associated with a decreased volume of the MOB although the VNO was unaffected.

The Behavioral and Neuroanatomical Effects of Prenatal Alcohol Exposure in Animals

One of the most devastating and obvious long-term effects of prenatal alcohol exposure is its impact on the developing nervous system. In fact, one criterion for a diagnosis of fetal alcohol syndrome (FAS) is some evidence of a central nervous system (CNS) dysfunction such as microcephaly or mental retardation.’ Even in the absence of the full FAS, learning and behavioral deficits such as shortened attention span and overactivity are frequently reported in children with heavy maternal alcohol histories?.’ Rodent models have proved useful in the study of the behavioral effects and underlying mechanisms involved in alcohol’s behavioral teratogenic effects. In this paper, we discuss some behavioral data collected following prenatal and neonatal alcohol exposure using rodent models concentrating on data gathered in our laboratory. We have tried to relate these results to clinical findings in children exposed to alcohol in utero and also in terms of potential brain areas that might be involved in these dysfunctions. In our laboratory, alcohol is administered in a liquid diet with vitamin and mineral supplements. This diet is designed to provide 35% of the total caloric content as ethanol (35% ethanol-derived calories (EDC)). An isocaloric 0% EDC pair-fed liquid diet group and a nontreated ad libitum water and chow group are included in all of our studies in order to rule out possible nutritional effects. Recently, we have also begun investigating neonatal alcohol exposure in rats. While the pattern of development of the CNS is relatively similar across species, the timing in which birth occurs in relation to CNS development differs In rats, early postnatal life coincides with a period of rapid CNS growth and proliferation that occurs in humans during the third trimester of de~elopment.’.~ Therefore, it has been argued that administration of alcohol to neonatal rats is a useful model to examine the effects of alcohol during the equivalent of the human third trimester “brain growth spurt. ’ I h To deliver alcohol neonatally, we use the “pup-in-the-cup” technique, in which pups are surgically implanted with an intragastric cannula and artificially A milk formula containing alcohol or maltose/dextrin in an isocaloric concentration is delivered via an infusion pump at scheduled periods throughout the day. We also include a sham-surgery, suckled control that is kept with its dam to control for possible effects of the artificial rearing procedure.