Critical evaluation of transcription factor Atf2 as a candidate modulator of alcohol preference in mouse and human populations.

In prior work, congenic strains carrying the DBA/2Igb (D2) region of chromosome 2 (Chr2) for alcohol preference were bred onto a C57BL/6Ibg (B6) background and as predicted were found to reduce voluntary consumption. Subsequently, interval-specific congenic recombinant strains (ISCRS) were generated and also tested. These ISCRS strains reduced the quantitative trait loci (QTL) interval to a comparatively small 3.4 Mb region. Here, we have exploited an integrative approach using both murine and human populations to critically evaluate candidate genes within this region. First, we used bioinformatics tools to search for genes relevant to alcohol preference within the QTL region. Second, we searched for single nucleotide polymorphisms (SNPs) within exons of every gene in this region. Third, we conducted follow-up microarray analyses to identify differentially expressed genes between the B6 and ISCRS strains in mice from each group. Fourth, we analyzed correlations between the expression level of candidate genes and phenotypes of alcohol preference in a large family of BXD recombinant inbred strains derived from B6 and D2. Finally, we evaluated SNP segregation in both BXD mouse strains and in humans who were heavy alcohol drinkers or non-drinkers. Among several potential candidate genes in this region, we identified activating transcription factor 2 (Atf2) as the most plausible gene that would influence alcohol preference. However, the candidacy of Atf2 was only weakly supported when we used a genetic network approach and by focused reanalysis of genome-wide association study data from European-American and African-American populations. Thus, we cannot conclude that Atf2 plays a role in the regulation of the QTL of mouse Chr2.

High-throughput behavioral phenotyping in the expanded panel of BXD recombinant inbred strains.

Genetic reference populations, particularly the BXD recombinant inbred (BXD RI) strains derived from C57BL/6J and DBA/2J mice, are a valuable resource for the discovery of the bio-molecular substrates and genetic drivers responsible for trait variation and covariation. This approach can be profitably applied in the analysis of susceptibility and mechanisms of drug and alcohol use disorders for which many predisposing behaviors may predict the occurrence and manifestation of increased preference for these substances. Many of these traits are modeled by common mouse behavioral assays, facilitating the detection of patterns and sources of genetic coregulation of predisposing phenotypes and substance consumption. Members of the Tennessee Mouse Genome Consortium (TMGC) have obtained phenotype data from over 250 measures related to multiple behavioral assays across several batteries: response to, and withdrawal from cocaine, 3,4-methylenedioxymethamphetamine; "ecstasy" (MDMA), morphine and alcohol; novelty seeking; behavioral despair and related neurological phenomena; pain sensitivity; stress sensitivity; anxiety; hyperactivity and sleep/wake cycles. All traits have been measured in both sexes in approximately 70 strains of the recently expanded panel of BXD RI strains. Sex differences and heritability estimates were obtained for each trait, and a comparison of early (N = 32) and recent (N = 37) BXD RI lines was performed. Primary data are publicly available for heritability, sex difference and genetic analyses using the MouseTrack database, and are also available in GeneNetwork.org for quantitative trait locus (QTL) detection and genetic analysis of gene expression. Together with the results of related studies, these data form a public resource for integrative systems genetic analysis of neurobehavioral traits.

Increased cell death in the developing vestibulocochlear ganglion complex of the mouse after prenatal ethanol exposure.

BACKGROUND: Previous studies have demonstrated that excessive prenatal alcohol exposure can damage the auditory and vestibular systems, in particular, cochlear hair cells. However, the direct effect of ethanol on the peripheral neurons in these pathways has not been examined. To study the effects of prenatal ethanol exposure on the developing vestibulocochlear ganglion (VCG) complex and the peripheral sensory organs, we exposed pregnant mice to ethanol and examined the levels of cell death in the inner ear. METHODS: Pregnant C57BL/6J mice were administered one of three doses of either ethanol (3.0, 4.5, and 5.5 g/kg) or isocaloric maltose/dextrin via intragastric intubation on gestational day (GD) 12.5. Embryos were dissected out of the uterus 8 hr after the intubation. Dying cells in the inner ear were stained with Nissl stain and labeled by in situ terminal dUTP nick-end labeling (TUNEL), and the percentage of dying cells was quantified. RESULTS: Ethanol exposure produced region-specific effects, with ethanol-exposed embryos exhibiting enhanced cell death only in the VCG complex, and not in the primitive saccule, cochlea, semicircular canal, or endolymphatic sac. The effects of ethanol on cell death in the VCG are dose dependent, with a significant increase in the level of cell death found only at the higher doses. CONCLUSIONS: Ethanol has a selective cytotoxic dose-dependent effect on the VCG at GD 12.5 suggesting that loss of VCG neurons may contribute to hearing and /or vestibular abnormalities in FAS children. Furthermore, the presence of TUNEL-positive cells and DNA laddering is consistent with the cells undergoing apoptotic cell death.

Granule cells and cerebellar boundaries: analysis of Unc5h3 mutant chimeras.

Mutations in the Unc5h3 gene, a receptor for the netrin 1 ligand, result in abnormal migrations of both Purkinje and granule cells to regions outside the cerebellum and of granule cells to regions within the cerebellum. Because both Purkinje and granule cells express this molecule, we sought to determine whether one or both of these cell types are the primary target of the mutation. Chimeric mice were made between wild-type ROSA26 transgenic mouse embryos (whose cells express beta-galactosidase) and Unc5h3 mutant embryos. The resulting chimeric brains exhibited a range of phenotypes. Chimeras that had a limited expression of the extracerebellar phenotype (movement of cerebellar cells into the colliculus and midbrain tegmentum) and the intracerebellar phenotype (migration of granule cells into white matter) had a normal-appearing cerebellum, whereas chimeras that had more ectopic cells had attenuated anterior cerebellar lobules. Furthermore, the colonization of colliculus and midbrain tegmentum by cerebellar cells was not equivalent in all chimeras, suggesting different origins for extracerebellar ectopias in these regions. The granule cells of the extracerebellar ectopias were almost entirely derived from Unc5h3/Unc5h3 mutant embryos, whereas the ectopic Purkinje cells were a mixture of both mutant and wild-type cells. Intracerebellar ectopias in the chimera were composed exclusively of mutant granule cells. These findings demonstrate that both inside and outside the cerebellum, the granule cell is the key cell type to demarcate the boundaries of the cerebellum.

meander tail acts intrinsic to granule cell precursors to disrupt cerebellar development: analysis of meander tail chimeric mice.

The murine mutation meander tail (gene symbol: mea) causes a near-total depletion of granule cells in the anterior lobe of the cerebellum, as well as aberrantly located Purkinje cells with misoriented dendrites and radial glia with stunted processes. Whether one, two or all three of these cell types is the primary cellular target(s) of the mutant gene is unknown. This issue is addressed by examining cerebella from adult chimeras in which both the genotype and phenotype of individual cells are marked and examined. From this analysis, three novel observations are made. First, genotypically mea/mea Purkinje cells and glial cells exhibit normal morphologies in the cerebella of chimeric mice indicating that the mea gene acts extrinsically to these two cell populations. Second, few genotypically mea/mea granule cells are present in the anterior lobe or, unexpectedly, in the posterior lobe. These findings indicate that the mea gene acts intrinsically to the granule cell or its precursors to perturb their development. Third, there are near-normal numbers of cerebellar granule cells in the chimeric cerebellum. This result suggests that mea/mea cells are out-competed and subsequently replaced by an increased cohort of wild-type granule cells resulting from an upregulation of wild-type granule cells in the chimeric environment. We propose that the wild-type allele of the mea gene is critical for the developmental progression of the early granule cell neuroblast.

Cerebellar mutant mice and chimeras revisited.

Neurological mutant mice have yielded an early and continuously rich resource for studying the role of genes in the developing cerebellum. Experimentally produced chimeric mice, containing mixtures of genetically normal and mutant cells, provided a means of deducing the primary site of gene action and studying cell interactions in these mutant cerebella. Recently, three mutant genes, reeler, weaver, and staggerer, have been cloned and their gene products identified. These three genes have been examined earlier by the chimera technology. Here, we review the chimera studies in the light of what we now know to be the products of these mutant genes.

Analysis of gene action in the meander tail mutant mouse: examination of cerebellar phenotype and mitotic activity of granule cell neuroblasts.

The meander tail (mea) gene results in a stereotypic pattern of cerebellar abnormalities, most notably the virtual depletion of granule cells in the anterior lobe of the cerebellum. The causal basis of this mutation is unknown. In this paper we have taken a three-part approach to the analysis of mea gene action. First, we quantitatively determined the effect of the mea gene on granule cell and Purkinje cell number. We found, in addition to the marked depletion of anterior lobe granule cells ( > 90%), there were also significantly fewer granule cells in the posterior lobe (20-30%) without a concomitant loss of Purkinje cells. Second, we explored the relationship between granule cell depletion caused by the mea gene and by the mitotic poison, 5-fluoro-2'-deoxyuridine (FdU). Prenatal and postnatal ICR mice were treated with FdU to ascertain the regimen that best produces a meander tail-like cerebellar phenotype. The similarity of the effects of the mea gene and injections of FdU at E17 and PO suggests the hypothesis that the mea gene acts to disrupt the cell cycle of cerebellar granule cell precursors. Thus, the third part of this study was to test this hypothesis by using injections of either BrdU (5-bromo-2'-deoxyuridine) or 3H-thymidine into homozygous and heterozygous meander tail littermates at E17 or PO. After processing the tissue for BrdU immunocytochemistry or 3H-thymidine autoradiography, counts were made of the number of labeled and unlabeled external granule layer (EGL) cells to determine the percentage that had incorporated the mitotic label (labeling index). No difference in the labeling index was found between homozygous meander tail mice and normal, heterozygous littermate controls. Therefore, the mitotic activity of the EGL neuroblasts is not disrupted by the mea gene. Furthermore, while a mitotic poison can produce a phenotype similar to the action of the mea gene, mea is phenomenologically different from FdU treatment.

The annexins: specific markers of midline structures and sensory neurons in the developing murine central nervous system.

The annexins are a family of cytoplasmic proteins that have been shown to have numerous actions within a cell. Recent evidence suggests that at least one of these proteins plays a role in the development of the central nervous system (CNS). The present study examines the temporal expression and spatial distribution of annexins I, II, IV, V, and VI during development and at maturity in the murine CNS by immunocytochemical analysis. The results demonstrate that annexins I, II and IV exhibit clear immunolabeling in the murine CNS with distinct patterns of temporal and spatial expression. Annexin IV is the first annexin to be expressed on embryonic day (E) 9.5 while annexin I is the last to be expressed (E11.5). Annexins I, II and IV are found in the floor plate region, but to differing rostrocaudal extents. Annexin I has a very restricted distribution, only present in the midline raphe of the brainstem. Annexin II is present in the spinal cord, brainstem and mesencephalon. Annexin IV has the widest midline distribution, being observed in the floor and roof plates of the developing CNS. Additionally, antibodies against annexin II and IV immunolabel most dorsal root and sensory ganglion cells and their axons. During early postnatal development, immunolabeling with each antibody gradually disappears in many structures, and only first order sensory neurons and their fibers are immunopositive for annexins II and IV at weaning. Three functions of the annexins are suggested by the present findings: (1) to help establish the midline structures of the floor and roof plates, (2) to help direct the decussation of sensory fibers, and (3) to regulate some aspect of sensory neuron processing, such as signal transduction.

The effects of the timing of ethanol exposure during the brain growth spurt on the number of cerebellar Purkinje and granule cell nuclear profiles.

Ethanol exposure during development is particularly deleterious to cerebellar Purkinje cells and granule cells, but the mechanism(s) underlying this sensitivity and the variables which affect it remain unknown. One important variable that has not been fully investigated, is the timing of the ethanol exposure. Ethanol exposure during the brain growth spurt causes a differential loss of Purkinje cells across the 10 lobules of the vermal cerebellum. However, whether or not changing the timing of the ethanol exposure during the brain growth spurt alters the extent and location of the loss of Purkinje cells within the cerebellar vermis has not been investigated. Moreover, the loss of cerebellar granule cells has been shown to parallel the loss of Purkinje cells, leading to the conclusion that the loss of granule cells occurred as a function of the loss of their targets, the Purkinje cells. The purpose of this study was to address both issues. Male rat pups were exposed to ethanol, via an artificial-rearing method, during one of the following 2-day time periods: postnatal days (PD) 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, or 12-13. Gastrostomy control (GC) and suckle control (SC) groups also were included. All pups were sacrificed on PD21. The number of Purkinje cell nuclear profiles from three vermal sections were counted in all groups, while the number of granule cell nuclear profiles in the ten lobules was estimated from pups in selected groups. No loss of Purkinje cells was observed in pups exposed to ethanol on PD7-8 or at any of the later exposure times. Additionally, among the three exposure groups in which significant Purkinje cell loss was observed (PD4-5, PD5-6 and PD6-7), seven lobules exhibited significant differences particularly between the PD4-5 and PD6-7 groups. The group with the greatest loss of Purkinje cells (PD4-5) also was the group with the greatest loss of granule cells. A significant loss of granule cells did not occur without a corresponding loss of Purkinje cells. The loss of both the Purkinje and granule cells was affected by the timing of the ethanol exposure, and that the extent and the location of Purkinje cell loss were extremely sensitive to the effects of the timing of the ethanol exposure.

Dissociation of spatial navigation and visual guidance performance in Purkinje cell degeneration (pcd) mutant mice.

Spatial learning in rodents requires normal functioning of hippocampal and cortical structures. Recent data suggest that the cerebellum may also be essential. Neurological mutant mice with dysgenesis of the cerebellum provide useful models to examine the effects of abnormal cerebellar function. Mice with one such mutation, Purkinje cell degeneration (pcd), in which Purkinje cells degenerate between the third and fourth postnatal weeks, were evaluated for performance of spatial navigation learning and visual guidance learning in the Morris maze swim-escape task. Unaffected littermates and C57BL/6J mice served as controls. Separate groups of pcd and control mice were tested at 30, 50 and 110 days of age. At all ages, pcd mice had severe deficits in distal-cue (spatial) navigation, failing to decrease path lengths over training and failing to express appropriate spatial biases on probe trials. On the proximal-cue (visual guidance) task, whenever performance differences between groups did occur, they were limited to the initial trials. The ability of the pcd mice to perform the proximal-cue but not the distal-cue task indicates that the massive spatial navigation deficit was not due simply to motor dysfunction. Histological evaluations confirmed that the pcd mutation resulted in Purkinje cell loss without significant depletion of cells in the hippocampal formation. These data provide further evidence that the cerebellum is vital for the expression of behavior directed by spatial cognitive processes.

Cell population depletion associated with fetal alcohol brain damage: mechanisms of BAC-dependent cell loss.

Neuronal death is one of the most serious consequences of alcohol exposure during development. Studies described in this paper used a neonatal rat model to address factors affecting neuronal death following alcohol exposure during the period of rapid brain growth, and relate them to possible mechanisms of damage. The profile of blood alcohol concentrations (BACs) is an important variable influencing both brain growth deficits and neuronal death--a smaller daily dose of alcohol can be more damaging than a larger daily dose, if it is consumed in a binge-like pattern that produces relatively higher BACs. Alcohol exposure for a single day also can be damaging, producing both brain growth deficits and neuron loss, if high BACs are obtained. Various brain regions and different neuronal populations within a given brain area exhibit different degrees of vulnerability. Some neuronal loss clearly is a function of cell death due to direct effects of alcohol, while other deficits may be due to either primary or secondary effects of the alcohol insult. In the cerebellum, a maturational or metabolic factor also appears to be involved with alcohol-induced neuronal death. Immunocytochemical studies using a monoclonal antibody against microtubule-associated protein 2 (MAP2) indicated that cerebellar lobules containing Purkinje cells that are in the process of extending dendrites are ones that are more vulnerable to alcohol than lobules containing Purkinje cells that mature later. Alcohol exposure during brain development may be producing neuron attrition in multiple ways, including disruption of membrane integrity, inhibition of protein synthesis or other alterations such as lipid solubility, or by disruption of cytoskeletal elements.

Regional differences in the timing of dendritic outgrowth of Purkinje cells in the vermal cerebellum demonstrated by MAP2 immunocytochemistry.

Detailed, within-subjects Golgi analyses of regional differences in cerebellar Purkinje cell dendritic development are impractical due to the capriciousness of that technique. Immunocytochemical labeling of microtubule-associated protein 2 (MAP2) was used to reveal the dendritic development of Purkinje cells, and indicated marked differences in the timing of initial outgrowth of Purkinje cell dendrites for different lobules in the developing rat cerebellar vermis. In particular, an early maturing region of Purkinje cell dendritic outgrowth (lobules I, II, IX and X and along the primary fissure), and a late maturing region (distal lobule VI, lobule VII and dorsal lobule VIII) were documented.

The development of laminar staining for neuron-specific enolase in the rat somatosensory cortex.

The expression of the enzyme neuron-specific enolase (NSE) in the central nervous system (CNS) has been used as a developmental marker based on observations that it is expressed shortly after the arrival of afferent inputs. The immunostaining pattern of NSE was examined in the laminae of the somatosensory cortex of the rat and the relationship of this staining pattern with previous data on the timing of afferent and efferent arrival was determined. Male rat pups were sacrificed on postnatal days 1 (24 h after birth), 3, 5, 8, 10, 12, 15 and 20, and as an adult (over 90 days of age). Sections were stained with an anti-NSE antibody using the avidin-biotin immunocytochemical method. Sections from day 1 animals revealed stained cells in the subplate layer and cortical plate, presumably in cells destined to form layers VI and V. By day 8 there was staining in layers II, III, V and VI, the same layers that exhibited staining in the adult rat. This appears consistent with the arrival of afferents and efferents which is completed by approximately postnatal day 7. On day 10, there was a change in the staining pattern: cell staining in layer VI was decreased and then increased gradually up to adult levels by day 20. A stable pattern of NSE staining was not observed previous to day 20. These results suggest that changes in NSE expression following the initial arrival of afferents may relate to maturation of the neurons.

Alz-50 immunoreactivity in the neonatal rat: changes in development and co-distribution with MAP-2 immunoreactivity.

Alz-50 is a monoclonal antibody that recognizes pathological alterations in Alzheimer's disease. It has recently been noted also to mark some subplate neurons in human infants under the age of 2 years. We now report that Alz-50 recognizes many neurons in the normal neonatal rat in a pattern that changes with development. Immunoreactivity decreases substantially in intensity as the rat matures. This immunoreactivity co-distributes with microtubule-associated protein-2 (MAP-2) immunoreactivity in terms of topography, cellular localization and changes over the developmental time-course. This observation raises the possibility of exploring cytologic triggers that may lead to re-expression of Alz-50 immunoreactivity in aging and in pathological conditions.

Effects of ethanol exposure during the third trimester equivalent on neuron number in rat hippocampus and dentate gyrus.

An artificial rearing procedure was used to expose neonatal rats to a formula containing 3.74% ethanol during postnatal days 4 through 10. This treatment produced a mean blood ethanol concentration of 379.8 +/- 17.3 mg/dl. When the pups were killed on the afternoon of postnatal day 10, brain weight to body weight ratio in the ethanol-exposed rats was reduced 22.4% and 21.5% compared to suckle and pair-fed controls, respectively. Ethanol exposure also resulted in a 16% reduction of neurons in hippocampal field CA4, compared to controls, but did not produce deficits in fields CA1 or CA3. There was also a 10% increase in the number of neurons (a population of cells in the midst of a proliferative phase at the time of the exposure) in the granule cell layer of the dentate gyrus. The ethanol exposure did not affect cell size in any of the four neuron populations measured. These results suggest, that within the dose and timing parameters examined, ethanol exposure during the third trimester equivalent appears to be preferentially harmful to specific populations of developing neurons.

Effects of alcohol exposure during different periods of development: changes in hippocampal mossy fibers.

Exposure to 10-12 g/kg/day of alcohol either during days 1-10 or 11-21 of gestation had no detectable effect on hippocampal mossy fiber development. Exposing artificially reared rat pups to 7.0-7.5 g/kg/day of alcohol during days 1-10 postpartum dramatically altered the organization of the Timm-stained mossy fiber terminal field when the animals were examined as adults, suggesting that alcohol exposure during a period equivalent to the human third trimester is more deleterious to brain development than exposure during periods equivalent to either the first or second trimesters.

Delay in brain growth induced by alcohol in artificially reared rat pups.

The effect of alcohol on body and brain growth of the neonatal rat was examined. An artificial rearing procedure was used to administer a milk formula containing 2.8% alcohol to rat pups during days 4-10 postpartum. Mean blood alcohol levels taken at hourly intervals between feelings at the end of the second day of exposure ranged between 151 and 163 mg/dl. Body growth in both groups of artificially reared pups was similar to that of the suckle control pups. Gross measurements indicated that while alcohol exposure did not arrest body growth, it did arrest several parameters of brain growth. There were deficits in brain weight and volume and in the brain weight to body weight ratio. Furthermore, there were sex-related differences. The brain weight to body weight ratio was significantly decreased in females and there was also a trend toward a greater deficit in brain volume as well. However, deficits in gross measures were not reflected in the development of the hippocampal formation. Areal measurements of the hippocampus and dentate gyrus failed to indicate any differential effects on the growth of the pyramidal and granule cell layers, or their dendritic fields and corresponding Timm-stained sublaminae, due to the alcohol exposure. These data suggest that the blood alcohol concentrations reached in the present study may be near the threshold dose for producing deficits in brain growth, and that the females have a lower threshold than the males.