Louise C. Abbott

Smoking during pregnancy: lessons learned from epidemiological studies and experimental studies using animal models.

Numerous epidemiological studies in the human population clearly indicate that smoking while pregnant has deleterious effects on fetal development as well as long-term adverse consequences on postnatal development and maturation of several organ systems. Low birth weight, sudden infant death syndrome (SIDS), behavioral disorders including attention deficit hyperactivity disorder (ADHD), externalizing and internalizing behavioral problems and conduct disorders in children have all been linked to prenatal exposure to tobacco smoke. The major pharmacologically active chemical found in tobacco smoke is nicotine, and prenatal exposure to nicotine has been shown to have significant effect on the development of multiple organ systems, including the nervous, respiratory, and cardiovascular systems. In this review, we define mainstream and sidestream smoke, summarize the major classes of compounds found in cigarette smoke, and describe how use of laboratory animal models can be used to assess mechanisms of toxicity and risk in the human population in general. We then discuss the association with smoking during pregnancy and the occurrence of reduced lung function, low birth weight, the incidence of congenital structural malformations, SIDS, ADHD, cognitive impairment, and mood disorders in children, and review pertinent experimental studies using a variety of animal models of developmental nicotine exposure, including, rats, mice, monkeys, lambs, and pigs that have increased our understanding of the pathophysiology of these disorders.

An ultrastructural study of granule cell/Purkinje cell synapses in tottering (tg/tg), leaner (tgla/tgla) and compound heterozygous tottering/leaner (tg/tgla) mice

Homozygous tottering (tg/tg) and compound heterozygous tottering/leaner (tg/tg(la)) mutant mice exhibit juvenile onset of three abnormal neurological phenotypes: (i) petit mal-like epilepsy; (ii) ataxia; and (iii) an intermittent myoclonus-like movement disorder. Homozygous leaner mice (tg(la)/tg(la)) exhibit early onset of ataxia (postnatal days 10-12), and also exhibit the myoclonus-like movement disorder and evidence of absence seizure activity; the myoclonus-like disorder is most evident in the first month of life, then diminishes in severity and frequency. The ultrastructure of the cerebellar molecular layer was examined in adult (six to eight months) and juvenile (20-25 days) mice of all three mutant genotypes. In mice of all three genotypes and both ages, Purkinje cell dendritic spines were observed to make multiple contacts with individual parallel fiber varicosities in all sections analysed. These multiple synaptic units were observed in both anterior and posterior vermis and hemispheres of the cerebellum, and ranged from two to nine spines/parallel fiber varicosity. Occasionally, one of the postsynaptic spines belonged to an ectopic spine emerging from the proximal region of a Purkinje cell dendrite. This increase in the multiple synaptic index of some parallel fiber varicosities was observed in juvenile tottering mice before the onset of the symptoms of the neurological disorders. This is highly suggestive that the onset of the neurological phenotype is not a primary cause of multiple Purkinje cell dendritic spines synapsing with single parallel fiber varicosities in these mice, but on the contrary, that it could be the cause of the ataxic symptoms.

Knife-edge scanning microscopy for imaging and reconstruction of three-dimensional anatomical structures of the mouse brain

Anatomical information at the cellular level is important in many fields, including organ systems development, computational biology and informatics. Creating data sets at resolutions that provide enough detail to reconstruct cellular structures across tissue volumes from 1 to 100 mm3 has proven to be difficult and time‐consuming. In this paper, we describe a new method for staining and imaging large volumes of tissue at sub‐micron resolutions. Serial sections are cut using an automated ultra‐microtome, whereas concurrently each section is imaged through a light microscope with a high‐speed line‐scan camera. This technique, knife‐edge scanning microscopy, allows us to view and record large volumes of tissue in a relatively small amount of time (approximately 7 mm2 s−1).

Chronic, low-dose prenatal exposure to methylmercury impairs motor and mnemonic function in adult C57/B6 mice.

Methylmercury (MeHg) has cytotoxic effects on animals and humans, and a major target organ for MeHg is the central nervous system (CNS). It is well known that the developing CNS is extremely vulnerable to MeHg-induced changes in comparison to the mature brain. Most studies have concentrated on the direct effects of high levels of prenatal MeHg exposure. Surprisingly, behavioral outcomes found in adult offspring exposed developmentally to the neurotoxic effects of chronic, low-dose mercury more akin to ingestion in humans are not well characterized. The objective of this study was to determine whether such exposure produces deleterious effects on behavior in adult mice, including motor/coordination abilities, overall activity and mnemonic function. Developing mouse fetuses were exposed in utero during gestational days 8-18 by giving pregnant C57Bl/6J female mice food containing MeHg at a daily dose of 0.01 mg/kg body weight. Adult mice prenatally exposed to MeHg exhibited significant deficits in motor abilities, coordination, and overall activity, as measured by rotarod, footprint analysis and open field. In addition, MeHg-exposed mice were impaired with respect to reference memory but not in a visible, cued version of the Morris water maze task. These results indicate that prenatal exposure to the lowest dose of MeHg examined to date can have long-lasting motor and cognitive consequences on adult offspring. These findings have far reaching implications related to putative safe levels of MeHg ingestion, particularly during pregnancy, and increasing rates of cognitive and psychological disorders (e.g. attention hyperactivity deficit disorder, autism) in our society.

Morphologic investigation of rolling mouse Nagoya (tgrol/tgrol) cerebellar Purkinje cells: an ataxic mutant, revisited

Rolling mouse Nagoya (rolling: tg(rol)) is a neurologic mutant mouse exhibiting severe ataxia. Two alleles of the rolling mutation, tottering (tg) and leaner(tg(la)), have been identified as mutations in the voltage-dependent calcium channel alpha1A subunit. No specific light and electron microscopic findings have been reported for the rolling mouse cerebellum except a decreased number of granule cells, while altered Purkinje cell/parallel fiber synapses have been observed in tottering and leaner cerebella. Rolling mouse cerebella were analyzed using anti-calbindin-D immunohistochemistry and transmission electron microscopy to investigate Purkinje cell morphology and synaptic contacts between Purkinje cell dendritic spines and parallel fiber varicosities. Multiple Purkinje cell dendritic spines synapsing with single parallel fiber varicosities were frequently observed in rolling cerebella. The correlation between the presence of altered Purkinje cell synapses and ataxia in rolling mice warrants further investigation.

Expression of calcium channel α1A mRNA and protein in the leaner mouse (tgla/tgla) cerebellum

Homozygous leaner mice carry an autosomal recessive mutation in the Ca2+ channel subunit gene, alpha1A, causing them to exhibit severe ataxia, petit-mal-like epilepsy and a myoclonus-like movement disorder. Expression of alpha1A mRNA in cerebella from 20-day-old homozygous leaner mice was compared to control mice, using in situ hybridization histochemistry. Expression of alpha1A protein was examined in cerebella from 20-day-old homozygous leaner and control mice using immunocytochemistry. No differences in either mRNA or protein expression of the alpha1A subunit were observed when homozygous leaner mice were compared to age-matched controls. Therefore, functional alterations in P/Q-Type Ca2+ channels containing the alpha1A subunit need to be explored to further understand the relationship of mutations in the alpha1A gene to the pathogenesis of the neurologic disorders occurring in leaner mice.

Effects of chronic neonatal nicotine exposure on nicotinic acetylcholine receptor binding, cell death and morphology in hippocampus and cerebellum

Nicotine, the major psychoactive ingredient in tobacco interacting with nicotinic acetylcholine receptors (nAChR), is believed to have neuroprotective and neurotoxic effects on the developing brain. Neurotoxicity has been attributed to activation of homomeric alpha7 nAChRs, neuroprotection to heteromeric alpha4beta2 nAChRs. Thus, developmental nicotine could have opposite effects in different brain regions, depending on nAChR subtype expression. Here, we determined if chronic neonatal nicotine exposure (CNN), during a period of brain growth corresponding to the third human trimester, differentially regulates nAChR expression, cell death, and morphological properties in hippocampus and cerebellum, two structures maturing postnatally. Rat pups were orally treated with 6 mg/kg/day nicotine from postnatal day (P)1 to P7. On P8, expression for alpha4, alpha7 and beta2 mRNA was determined by in situ hybridization; nAChR binding sites by receptor autoradiography, dying neurons by TUNEL and Fluoro-Jade staining and morphological properties by analysis of Cresyl Violet-stained sections. In control cerebellum, strong expression of alpha4, beta2 mRNA and heteromeric nAChRs labeled with [125I]-epibatidine was found in granule cells, and alpha7 mRNA and homomeric nAChRs labeled with [125I]-alpha-bungarotoxin were in the external germinal layer. In control hippocampus, low expression of alpha4 mRNA and heteromeric nAChRs and high expression of alpha7 mRNA and homomeric nAChRs were detected. CNN increased heteromeric nAChR binding in hippocampus but not cerebellum and significantly decreased neuronal soma size and increased packing density in hippocampal principal cells but not in cerebellum. CNN did not increase the number of dying cells in any area, but significantly fewer TUNEL-labeled cells were found in CA3 strata oriens and radiatum and cerebellar granule layer. Thus, the hippocampus seems to be more sensitive than the cerebellum to CNN which could result from different nAChR subtype expression and might explain long-lasting altered cognitive functions correlated with gestational nicotine exposure due to changes in hippocampal cell morphology.

The effect of methylmercury exposure on early central nervous system development in the zebrafish (Danio rerio) embryo

Much attention is focused on environmental contamination by heavy metals. The heavy metal mercury is found worldwide and is ranked number 3 on the Comprehensive Environmental Response, Compensation and Liability Act substance list. We examined the effect of low‐level methylmercury exposure on central nervous system development of wild‐type zebrafish embryos (ZFEs) of the AB strain because methylmercury is the most common form of mercury to which humans are exposed in the environment. ZFEs were exposed to nine different concentrations of methylmercury [0 (negative control), 5, 10, 50, 80, 100, 200, 500 and 1000 parts per billion (μg l−1)] starting at 6 h post‐fertilization, which is the time the neural tube is first beginning to form. ZFEs were exposed to 2% ethanol as positive controls (100% embryonic death). ZFEs were assessed at 30, 54, 72 and 96 h post‐fertilization for changes in embryonic development, mortality, time of hatching and morphological deformities. No abnormalities were observed in ZFEs exposed to 5 μg l−1 methylmercury. The time of hatching from the chorion was delayed in ZFEs exposed to methylmercury concentrations of 50 μg l−1 or higher. Significantly more ZFEs exposed to 0, 5 or 10 μg l−1 methylmercury successfully completed hatching compared with ZFEs exposed to 50 μg l−1 or higher methylmercury. ZFEs exposed to more than 200 μg l−1 methylmercury exhibited 100% embryonic mortality. The rate of cell proliferation within the neural tube was significantly decreased in embryos exposed to 10, 50 and 80 μg l−1 methylmercury and there were no differences between these doses. Copyright

Initial characterization of mice null for Lphn3, a gene implicated in ADHD and addiction

The LPHN3 gene has been associated with both attention deficit-hyperactivity disorder (ADHD) and addiction, suggesting that it may play a role in the etiology of these disorders. Unfortunately, almost nothing is known about the normal functions of this gene, which has hampered understanding of its potential pathogenic role. To begin to characterize such normal functions, we utilized a gene-trap embryonic stem cell line to generate mice mutant for the Lphn3 gene. We evaluated differential gene expression in whole mouse brain between mutant and wild type male littermates at postnatal day 0 using TaqMan gene expression assays. Most notably, we found changes in dopamine and serotonin receptors and transporters (Dat1, Drd4, 5Htt, 5Ht2a), changes in neurotransmitter metabolism genes (Th, Gad1), as well as changes in neural developmental genes (Nurr, Ncam). When mice were examined at 4-6 weeks of age, null mutants showed increased levels of dopamine and serotonin in the dorsal striatum. Finally, null mutant mice had a hyperactive phenotype in the open field test, independent of sex, and were more sensitive to the locomotor stimulant effects of cocaine. Considered together, these results suggest that Lphn3 plays a role in development and/or regulation of monoamine signaling. Given the central role for monoamines in ADHD and addiction, it seems likely that the influence of LPHN3 genotype on these disorders is mediated through alterations in monoamine signaling.

Fluoro-jade identification of cerebellar granule cell and purkinje cell death in the α1A calcium ion channel mutant mouse, leaner

Cell death is a critical component of normal nervous system development; too little or too much results in abnormal development and function of the nervous system. The leaner mouse exhibits excessive, abnormal cerebellar granule cell and Purkinje cell death during postnatal development, which is a consequence of a mutated calcium ion channel subunit, alpha(1A). Previous studies have shown that leaner cerebellar Purkinje cells die in a specific pattern that appears to be influenced by functional and anatomical boundaries of the cerebellum. However, the mechanism of Purkinje cell death and the specific timing of the spatial pattern of cell death remain unclear. By double labeling both leaner and wild-type cerebella with Fluoro-Jade and terminal deoxynucleotide transferase-mediated, deoxyuridine triphosphate nick-end labeling or Fluoro-Jade and tyrosine hydroxylase immunohistochemistry we demonstrated that the relatively new stain, Fluoro-Jade, will label neurons that are dying secondary to a genetic mutation. Then, by staining leaner and wild-type cerebella between postnatal days 20 and 80 with Fluoro-Jade, we were able to show that Purkinje cell death begins at approximately postnatal day 25, peaks in the vermis about postnatal day 40 and in the hemispheres at postnatal day 50 and persists at a low level at postnatal day 80. In addition, we showed that there is a significant difference in the amount of cerebellar Purkinje cell death between rostral and caudal divisions of the leaner cerebellum, and that there is little to no Purkinje cell death in the wild type cerebellum at the ages we examined. This is the first report of the use of Fluoro-Jade to identify dying neurons in a genetic model for neuronal cell death. By using Fluoro-Jade, we have specifically defined the temporospatial pattern of postnatal Purkinje cell death in the leaner mouse. This information can be used to gain insight into the dynamic mechanisms controlling Purkinje cell death in the leaner cerebellum.