Neonatal Brain Growth Spurt Research Articles

Characterization of motor function in mice developmentally exposed to ethanol using the Catwalk system: Comparison with the triple horizontal bar and rotarod tests

Studies with human subjects indicate that ethanol exposure during fetal development causes long-lasting alterations in motor coordination that are, in part, a consequence of cerebellar damage. Studies with rats exposed to ethanol during the neonatal brain growth spurt have consistently recapitulated these deficits. However, studies with mice have yielded mixed results. We hypothesized that the use of highly sensitive motor function tests, such as the Catwalk test, would reliably detect motor function deficits in mice developmentally exposed to ethanol. Venus-vesicular GABA transporter transgenic mice were ethanol exposed during postnatal days 4–9 using vapor inhalation chambers and then subjected to the Catwalk test during adolescence. Catwalk data were rigorously analyzed using an innovative multistep statistical approach. For comparison, motor coordination and strength were assessed with the triple horizontal bar and rotarod tests. Unexpectedly, we found that out of 186 parameters analyzed in the Catwalk test, only one was affected by ethanol exposure (i.e., reduced coupling between left front paw and the right hind paw). In the triple horizontal bar test, ethanol-exposed mice were able to hold to the bars for less time than controls. Surprisingly, ethanol-exposed mice performed better in the rotarod test than controls. These data indicate that neonatal ethanol exposure of mice causes mixed effects on motor function during adolescence. The Catwalk test suggests that gait is generally preserved in these mice, whereas the triple horizontal bar test revealed deficits on motor strength and the rotarod test an increase in motor coordination.

Exposure to Polybrominated Diphenyl Ethers 203 and 206 during the Neonatal Brain Growth Spurt Affects Proteins Important for Normal Neurodevelopment in Mice

The period of rapid brain growth and development (BGS) is postnatal in mice and rats, spanning the first 3-4 weeks of life, reaching its peak around postnatal day 10, whereas in humans, the BGS is perinatal. CaMKII, GAP-43, synaptophysin, and tau play important roles during the BGS. One class of flame retardants, polybrominated diphenyl ethers (PBDEs), is present and increasing in the environment and in human milk. The only congener still in use, decabrominated diphenyl ether (PBDE 209), is thought to be debrominated into lower brominated congeners. In the present study, nona- and octabrominated PBDEs were examined. Neonatal mice were exposed to 21 mumol PBDE 203 or 206/kg bodyweight on postnatal day 10, and different brain regions were analyzed for CaMKII, GAP-43, synaptophysin, and tau, 24 h after exposure. The protein analysis showed that CaMKII and synaptophysin increased significantly in the hippocampus, but not in the cerebral cortex, after neonatal exposure to PBDE 203 or 206. Furthermore, there were no significant changes in the levels of GAP-43 and tau in the cerebral cortex or hippocampus after neonatal exposure to PBDE 203 or 206. This shows that PBDE 203 and 206 affect important proteins involved in normal maturation of the brain and strengthens our findings that highly brominated PBDEs cause developmental neurotoxicity. In addition, the increases in CaMKII and synaptophysin are the same changes seen after neonatal PBDE 209 exposure; supporting the suggestion that PBDE 209 must be metabolized, likely debrominated into lower brominated PBDEs, to exert its neurotoxic effects.

Update on Neonatal Anesthetic Neurotoxicity

A fundamental premise of general anesthesia is that anesthetics produce a reversible state of unconsciousness and unresponsiveness. Implicit in this premise is that the brain and spinal cord are neurophysiologically the same before and after anesthesia. Recent experimental data has questioned the complete reversibility of anesthesia. In certain circumstances, anesthetic exposure in neonatal animals leads to neuronal death. Given the large number of neonates and infants that undergo surgery and anesthesia, the implications of these data for anesthesia in humans are readily apparent. Although the relevance of these findings to humans is a subject of heated debate, the unequivocal demonstration of neuronal death in animals exposed to clinically relevant concentrations of anesthetics has provoked significant concern amongst anesthesia care providers and patients. This editorial reviews new laboratory and clinical research presented at the Anesthesiology/Foundation for Anesthesia Education and Research Session at the Annual Meeting of the American Society of Anesthesiologists, Oct 21, 2008 which advances our understanding in this area.

Voluntary exercise influences behavioral development in rats exposed to alcohol during the neonatal brain growth spurt.

Children exposed to alcohol prenatally may suffer from severe brain damage, expressed as a variety of behavioral problems, including hyperactivity and learning deficits. There is a critical need to identify effective treatments for fetal alcohol effects. Physical exercise enhances cognitive ability and increases neurogenesis in the hippocampus, a brain area important for learning and memory. Thus, the present study examined whether physical exercise might reduce the severity of alcohol-induced behavioral alterations. Sprague-Dawley rats were intubated with 5.25 g/kg/day ethanol during the third trimester equivalent (postnatal days [PDs] 4-9). Intubated sham control and nontreated controls were included. From PD 21 to PD 51, half of the subjects were given access to running wheels. On PD 52, subjects were tested on the Morris water maze, and on PD 60, open field activity levels were measured. Morris maze performance was significantly impaired among ethanol-exposed subjects; exercise significantly improved performance of all groups. Similarly, ethanol-exposed subjects were overactive in the open field, an effect attenuated with exercise. In sum, these data suggest that exercise may increase neuronal plasticity not only in controls, but also in subjects exposed to alcohol during development.

Choline supplementation following third-trimester-equivalent alcohol exposure attenuates behavioral alterations in rats.

Despite the known adverse consequences of prenatal alcohol exposure, some pregnant women continue to drink alcohol, making it imperative to identify treatments for children with fetal alcohol spectrum disorders. The authors recently reported that perinatal choline supplementation can reduce some fetal alcohol effects (J. D. Thomas, M. Garrison, & T. M. O'Neill, 2004), and the present study examined whether choline supplementation is effective when administered after third-trimester-equivalent ethanol treatment. Rat pups were exposed to 6.0 g/kg/day ethanol during the neonatal brain growth spurt (Postnatal Days [PD] 4-9) and treated with choline chloride (0, 10, 50, or 100 mg/kg) from PD 10-30. Behavioral testing occurred after choline treatment had ceased. Female subjects exposed to ethanol were overactive and exhibited spatial learning deficits, effects that were attenuated with all doses of choline supplementation. These data indicate that choline supplementation can alter brain development following a developmental insult. Moreover, the data suggest that early dietary interventions may reduce the severity of some fetal alcohol effects, even when administered after birth.

Proteomic Insights into Brain Development: Neurotoxic Effects of PBDE-99 in Mice

The amounts of polybrominated diphenyl ethers (PBDEs) found in human tissues, particularly breast milk, are rising steadily. PBDEs are flame retardants that reduce fire risks when added to electronic equipment and household products. Epidemiologic studies have yet to show conclusive evidence of PBDE toxicity in humans. However, studies in animals have shown these chemicals to produce effects such as liver damage, altered thyroid hormone levels, developmental changes, and neurotoxicity. In the present study, a Swedish team uses proteomic methods to investigate the early-stage neurotoxic effects of PBDE-99 on two parts of the developing mouse brain: the striatum and the hippocampus [EHP 114:254–259]. The findings help deconstruct the mysterious mechanisms that underlie PBDE-99 neurotoxicity. The striatum and the hippocampus are both part of the cholinergic and monaminergic systems, which play roles in neurotransmitter functioning and other aspects of cognition. The authors sought specifically to uncover PBDE-induced cellular events leading from the neonatal brain growth spurt to permanent neurological problems in adult animals. PBDE-99 is among the most common PBDE congeners found in environmental and human tissue samples. This pentabrominated compound impairs spontaneous behavior and habituation among adult mice exposed neonatally to moderate doses. These effects are progressive, worsening with age. During the study, 10-day-old mice of both sexes were given a single dose of 12 milligrams PBDE-99 per kilogram body weight. After 24 hours, the mice were sacrificed, their brains dissected, and the striatum and hippocampus isolated for study. The team used 2D-DIGE to compare protein expression patterns between treatment and control groups. The analysis illuminated 40 proteins in the striatum and 56 proteins in the hippocampus whose expression was altered by PBDE-99 exposure. From this initial grouping, nine striatal proteins and ten hippocampal proteins were selected for identification using MALDI-ToF-MS. Among the striatal proteins affected by PBDE-99 were several (including Gap-43/neuromodulin and stathmin) that participate in neurode-generation and neuroplasticity. The affected hippocampal proteins (including α-enolase, γ-enolase, Atp5b, and α-synuclein) tend to participate in metabolism and energy production. Many of these proteins are linked to protein kinase C, a signaling molecule whose role in development and function, as well as in learning and memory, has been intensively studied. Based on these findings, the authors conclude they have identified potential protein biomarkers that reflect the immediate consequences of early-stage PBDE-99 toxicity, in addition to the processes that drive its neurological effects in older animals. The researchers suggest that protein kinase C signaling is a target of PBDE-99 toxicity in the developing mouse brain. Moreover, the authors propose that neonatal cell stress induced by PBDE-99 exposure, in addition to related neurodegenerative processes and aberrant neuroplasticity, may contribute to the latter-stage behavioral effects observed in adult mice. The responses within the striatum and hippocampus differ, however, reflecting the underlying heterogeneity between different brain parts and cell populations.

Selective and Enduring Deficits in Spatial Learning After Limited Neonatal Binge Alcohol Exposure in Male Rats

In rats, heavy bingelike alcohol exposure during the neonatal brain growth spurt [postnatal days (PD) 4-9] can impair development of spatial learning. This study tested whether binge exposure limited to the latter half of this period (PD 7-9) produced selective spatial learning deficits that endured into adulthood. On PD 7 to 9, Long-Evans rats were given intubations of alcohol (5.25 g/kg/day), sham intubations, or no intubations. Rats were tested as adults (>or=PD 70) in the Morris water maze under one of three different conditions: place (submerged escape platform in the same location each trial), random (submerged platform in a different, random location each trial), or redundant (visible platform that protruded above the water, in the same location for each trial). A 60-sec probe trial (with no platform present) followed the last acquisition trial. The mean peak blood alcohol concentration was 401 mg/dl on PD 7. Neonatal alcohol treatment significantly impaired acquisition and reduced place biases on the probe trial in place-trained males, but not females. Neonatal alcohol treatment had no significant effects on acquisition performance of the random or redundant groups. Redundant training yielded rapid acquisition for all groups. The visible cue overshadowed place cues for all treatment groups, but small place biases were evident in controls. After random training, no group showed place biases. Heavy alcohol exposure in rats limited to the last half of the neonatal brain growth spurt caused enduring deficits in spatial learning, but only in males. The deficits were specific to place learning in the Morris water maze and emerged only when learning and performance depended on the use of distal place cues.

Nicotine exposure during the neonatal brain growth spurt produces hyperactivity in preweanling rats

Despite warning labels and increases in evidence of the adverse effects of tobacco use, women continue to use tobacco products during pregnancy. Cigarette smoking has been linked to increased prenatal mortality, increased incidence of SIDS, reductions in birth weight, and disruptions in CNS and behavioral development. Animal model systems have critically established the causal relationship between nicotine and adverse developmental outcome. The present study examines the behavioral effects of nicotine exposure in the rat during the third trimester equivalent of the human brain growth spurt, a period of rapid development of the cholinergic systems and a period during which the CNS is particularly vulnerable to a number of insults. Sprague–Dawley rat pups were exposed to nicotine (6.0 mg/kg/day) from postnatal days (PD) 4–9 via an artificial rearing procedure. This procedure ensures that observed effects are not due to nutritional deficits. Two control groups were employed, an artificially reared control group and a normally reared control group. Activity level was measured on PD 18–19. Nicotine-exposed subjects were significantly overactive compared to both control groups, which did not differ significantly from one another. This behavioral alteration was observed in the absence of nicotine-induced body weight deficits. These results suggest that women who use tobacco products during late gestation may place their fetuses at risk for hyperactivity later in life, particularly during early adolescence.

Impairment in Eyeblink Classical Conditioning in Adult Rats Exposed to Ethanol as Neonates

Eyeblink classical conditioning is a learning task that engages well-defined neural circuitry in the cerebellum and brainstem. Binge-like exposure to alcohol during the neonatal brain growth spurt in rats produces neurotoxic effects on both the cerebellum and the brainstem. The precise localization of the neural substrates of eyeblink conditioning makes it an ideal task to study functional disruptions in the cerebellum and brainstem caused by early exposure to alcohol. The purpose of this study was to determine whether impairments in eyeblink conditioning caused by neonatal binge exposure to alcohol persist into adulthood, indicative of long-lasting abnormalities in cerebellar and brainstem function. Group Ethanol received alcohol doses of 5.25 g/kg/day via intragastric intubation on postnatal days 4-9. Group Sham Intubated underwent the intragastric intubation procedures on postnatal days 4-9 but did not receive any infusions. Group Nonintubated did not receive any intubations. When all rats were at least 3 months old, they were tested in either paired or unpaired eyeblink conditioning. Group Ethanol showed impaired eyeblink conditioning and some abnormalities in conditioned response timing. Control groups did not differ from each other. The present data indicate that early exposure to alcohol has long-term effects on eyeblink conditioning, perhaps through enduring effects associated with alcohol-induced loss of Purkinje cells of the cerebellum.

Sex Differences in Vulnerability to Developmental Spatial Learning Deficits Induced by Limited Binge Alcohol Exposure in Neonatal Rats

The two main objectives of this study were (1) to replicate previous findings that 6 days of binge-like exposure to alcohol during the neonatal brain growth spurt induces significant place learning deficits in juvenile rats and (2) to determine whether more limited (3-day) binge-like exposure during the neonatal period induces place learning deficits and whether the effects depend on the developmental timing of the exposure. Using artificial rearing methods and a split-litter experimental design, groups of male and female neonatal rats were given binge-like exposure to 4.5 g/kg/day of ethanol in milk formula either on Postnatal Days (PD) 4–6, PD 7–9, or PD 4–9, which yielded mean peak blood alcohol concentrations of 230–260 mg/dl. Controls included an artificially reared gastro- stomy control group (GC) given an isocaloric milk formula diet on PD 4–9 and a suckle control group reared normally by lactating dams. Acquisition of place learning in the Morris spatial navigation task was trained for 6 consecutive days beginning on PD 26; a probe trial was given at the end of the sixth day. As expected, both males and females given alcohol on PD 4–9 had significant deficits in acquisition and probe trial performance relative to SC and GC groups. Males given the PD 7–9 exposure had significant place learning deficits which were as severe as with the full 6-day exposure. The PD 4–6 exposure in males produced only a nonsignificant trend toward slower acquisition. Females were not significantly affected by either 3-day exposure. The latter phase of the neonatal brain growth spurt appears to constitute a sex-specific period of enhanced vulnerability to alcohol-induced developmental spatial learning deficits.

Transient cortical astrogliosis induced by alcohol exposure during the neonatal brain growth spurt in rats

The astrocyte response to central nervous system injury induced by neonatal alcohol exposure was evaluated using radioimmunoassay and immunocytochemistry of glial fibrillary acidic protein (GFAP). Rat pups were exposed to alcohol on postnatal days 4 through 9 via artificial rearing. Alcohol solutions were administered as one of the following treatments: 10.2% (v/v) in two feedings (4.5 g/kg/day), 5.1% (v/v) in four feedings (4.5 g/kg/day), or 2.5% (v/v) in 12 feedings (6.6 g/kg/day), producing mean blood alcohol concentrations (BACs) of approximately 300, 180, and 50 mg/dl, respectively. Littermates were included as gastrostomy controls (GC) and suckle controls (SC). On postnatal day 10, GFAP concentration increased as a function of BAC, and the 10.2% alcohol treatment significantly and dramatically increased GFAP in the cortex (325% of SC). GFAP immunocytochemistry revealed frequent loci of heavily labeled reactive astrocytes surrounding small cortical blood vessels in the 10.2% group. In addition, a generalized increase in GFAP immunoreactivity was present in the deep layers of the cortex in all alcohol groups, marked by astrocytic fibrillary hypertrophy and increased density. Three-dimensional counts in layer V of parietal cortex using confocal microscopy indicated that the density of GFAP-labeled astrocytes of the 10.2% group was twice that of controls. The layer V gliosis was observable even at low BACs, while gliosis around the vasculature occurred only with high BACs. By postnatal day 15, the astroglial effects were no longer evident. These transient astroglial reactions likely constitute an important aspect of cortical pathophysiology resulting from binge alcohol exposure during the brain growth spurt of the third trimester equivalent.

Long-term deficits in cerebellar growth and rotarod performance of rats following “binge-like” alcohol exposure during the neonatal brain growth spurt

The cerebellum is vulnerable to growth restriction and neuronal depletion induced by alcohol exposure during the brain growth spurt of neonatal rats. This study examined whether neonatal alcohol exposure permanently restricted brain growth and induced motor performance deficits in adults. Two groups of rats were given 4.5 g/kg of alcohol per day during postnatal days 4 through 9, using artificial-rearing procedures. One group was given the alcohol as a 10.2% (v/v) solution in two of the 12 daily feedings, producing peak BACs of 361 mg/dl. The second group was given the alcohol as a 5.1% (v/v) solution in four of the feedings, producing peak BACs of 187 mg/dl. Controls included an artificially reared group and a normally reared group. All rats were tested on a rotarod at approximately 405 days of age, then perfused 1–2 weeks later. The 10.2% group was significantly impaired in acquiring the task and had significant reductions in whole brain and cerebellar weight, compared to controls. The 5.1% treatment also significantly restricted whole brain and cerebellar growth, and rotarod performance of that group was intermediate between the control groups and the 10.2% group. The cerebellar reductions and deficits in motor performance in adulthood demonstrate permanent structural and functional consequences of binge-like alcohol exposure during the brain growth spurt.