Cynthia A. Dlugos

Ethanol effects on three strains of zebrafish: model system for genetic investigations

The effects of acute and chronic ethanol administration on the wild-type (WT), long-fin striped (LFS), and blue long-fin (BLF) strains of zebrafish were investigated. In the LFS strain, acute exposure to 0.25% (v/v) ethanol inhibited the startle reaction and increased both the area occupied by a group of subjects and the average distance between each fish and its nearest neighbor. Similar effects were found in the WT fish although higher concentrations of ethanol were required. No effects on the behavior of the BLF fish were observed with up to 1.0% (v/v) ethanol. Brain alcohol levels were comparable among the three strains precluding a pharmacokinetic explanation for the behavioral results. In LFS zebrafish, behavioral tolerance was observed after 1 week of continual exposure to ethanol. Conversely, chronic ethanol exposure of the WT fish for up to 2 weeks did not result in the development of tolerance, but rather appeared to increase the disruptive action of the drug. The present results suggest the observed strain differences in the effects of ethanol reflect genotypic differences in both the response of the central nervous system (CNS) to ethanol as well as the ability of the CNS to adapt to ethanol exposure. Although preliminary, the present study indicates that the zebrafish is an excellent model system to investigate the genetic determinants involved in regulating the responses to ethanol.

Alcohol‐Induced Neurodegeneration: When, Where and Why?

This manuscript reviews the proceedings of a symposium organized by Drs. Antonio Noronha and Fulton Crews presented at the 2003 Research Society on Alcoholism meeting. The purpose of the symposium was to examine recent findings on when alcohol induced brain damage occurs, e.g., during intoxication and/or during alcohol withdrawal. Further studies investigate specific brain regions (where) and the mechanisms (why) of alcoholic neurodegeneration. The presentations were (1) Characterization of Synaptic Loss in Cerebella of Mature and Senescent Rats after Lengthy Chronic Ethanol Consumption, (2) Ethanol Withdrawal Both Causes Neurotoxicity and Inhibits Neuronal Recovery Processes in Rat Organotypic Hippocampal Cultures, (3) Binge Drinking-Induced Brain Damage: Genetic and Age Related Effects, (4) Binge Ethanol-Induced Brain Damage: Involvement of Edema, Arachidonic Acid and Tissue Necrosis Factor alpha (TNFalpha), and (5) Cyclic AMP Cascade, Stem Cells and Ethanol. Taken together these studies suggest that alcoholic neurodegeneration occurs through multiple mechanisms and in multiple brain regions both during intoxication and withdrawal.

Ocular deficits associated with alcohol exposure during zebrafish development

Approximately 90% of fetal alcohol syndrome cases are accompanied by ocular abnormalities. The zebrafish (Danio rerio) is a well‐known developmental model that provides an opportunity for better understanding the histological and cytological effects of developmental exposure to ethanol on the vertebrate eye. The purpose of the present study was to determine the gross, microscopic, and ultrastructual effects of developmental exposure to ethanol in the zebrafish model. Eggs were obtained from WT outbred zebrafish and exposed to 0%, 0.1%, 0.2%, 0.4%, 0.5%, or 1.0% (v/v) ethanol to assess viability and the effect of dose and duration of exposure on eye size. Light and electron microscopy were performed on ethanol‐treated and control larvae. Results showed that ethanol treatment decreased viability by about 20% at concentrations of 0.1–0.5% ethanol and by 50% at 1.0% ethanol. Ethanol‐related decreases in eye size were recorded at 6 days postfertilization (dpf) and were dose dependent. There were significant decreases in the volumes of the photoreceptor, inner nuclear, and ganglionic layers and in the lens of 9 dpf ethanol‐exposed compared with control larvae. Ultrastructural examination showed signs of developmental lags in the ethanol‐treated fish as well as abnormal retinal apoptosis in the 6 dpf ethanol‐treated larvae compared with their controls. These results demonstrate that the developing zebrafish eye is sensitive to perturbation with ethanol and displays some of the eye deficits present in fetal alcohol syndrome. J. Comp. Neurol. 502:497–506, 2007.

Morphometric analyses of purkinje and granule cells in aging F344 rats

In this study, the Purkinje neurons and granule neurons in the cerebellar cortex were studied in male Fischer 344 rats at 3, 9, 18, and 27 months of age. The numbers of Purkinje cells (PC) and granule cells (GC) in folia IV, VII, and X of the vermis were quantitated with the disector stereological technique. The numbers of PC and GC and the ratio of PC to GC were stable with advancing age. Measurements of the molecular layer, however, showed that this layer, the site of synaptic contact between GC and PC, decreased in volume with age.

Quantitative immunocytochemistry of glia in the cerebellar cortex of old ethanol-fed rats

It is clear from results of studies in this laboratory that chronic ethanol consumption causes regression of the extensive Purkinje neuron (PN) dendritic arbor. There are, however, a paucity of studies on the effects of chronic ethanol consumption on glia cells that reside in the molecular layer of the cerebellar cortex with PN dendrites. The purpose of the present study was to investigate the possibility that chronic ethanol consumption in old F344 rats results in gliosis within the molecular layer of the cerebellar cortex. Ten 12-month-old, male, F344 rats received a liquid diet containing 35% ethanol for 40 weeks. Pair-fed controls (n=10) received a liquid diet in which maltose dextrins were substituted for ethanol. Chow-fed rats (n=10) served as controls for age. At the end of the treatment period, rats were euthanized and perfused through the aorta, and cerebella were prepared for immunocytochemistry. Free floating sections were stained with (1) glial fibrillary acidic protein antibody for labeling of Bergmann glial cells and fibers, (2) OX-42 antibody for labeling of microglia, and (3) 0.5% cresyl violet for estimates of molecular layer volume. Results indicate that the densities of Bergmann glial cell processes and microglia within the cerebellar molecular layer are not altered by ethanol consumption.

Ethanol-related Increases in Degenerating Bodies in the Purkinje Neuron Dendrites of Aging Rats

Chronic ethanol consumption in aging rats results in regression of Purkinje neuron (PN) dendritic arbors ([Pentney, 1995 Measurements of dendritic pathlengths provide evidence that ethanol-induced lengthening of terminal dendritic segments may result from dendritic regression. Alcohol Alcohol. 30, 87-96]), loss of synapses (Dlugos and Pentney, 1997), dilation of the smooth endoplasmic reticulum (SER), and the formation of degenerating bodies within PN dendrites ([Dlugos, C.A., 2006a. Ethanol-Related Smooth Endoplasmic Reticulum Dilation in Purkinje Dendrites of Aging Rats. Alcohol., Clin. Exp. Res. 30, 883-891,Dlugos, C.A., 2006b. Smooth endoplasmic reticulum dilation and degeneration in Purkinje neuron dendrites of aging ethanol-fed female rats. Cerebellum. 5, 155-162]). Dilation of the SER and the formation of degenerating bodies may be a predictor of dendritic regression. Ethanol-induced effects on mitochondria may be involved as mitochondria cooperate with the SER to maintain calcium homeostasis. The purpose of this study was to determine whether degenerating body number and mitochondrial density and structure are altered by chronic ethanol treatment in PN dendrites. Male, Fischer 344 rats, 12 months of age, were fed an ethanol or pair-fed liquid diet, or rat chow for a period of 10, 20, or 40 weeks (15 rats/treatment; 45 rats/treatment duration). Ethanol-fed rats received 35% of their calories as ethanol. At the end of treatment, all animals were euthanized, perfused, and tissue prepared for electron microscopy. The densities of degenerating bodies and mitochondria, mitochondrial areas, and the distance between the SER and the mitochondria were measured. Results showed that there was an ethanol-related increase in degenerating bodies compared to controls at 40 weeks. Ethanol-induced alterations to mitochondria were absent. Correlation of the present results with those of previous studies suggest that degenerating bodies may be formed from membrane reabsorption during dendritic regression or from degenerating SER whose function has been compromised by dilation.

Effects of chronic ethanol consumption on SER of Purkinje neurons in old F344 rats

The purpose of this study was to determine whether the observed swelling of smooth endoplasmic reticulum (SER) profiles in Purkinje dendrites in our old ethanol-fed F344 rats: (1) represented measurable dilatation, (2) was present in dendritic shafts and spines, and (3) was reversed following recovery from ethanol. Of the 45 rats in 3 treatment groups (chow-fed, pair-fed, and ethanol-fed), 30 rats were euthanized after 40 weeks, and 15 were maintained on rat chow for an additional 20-week recovery period. Electron microscopy of cerebellar preparations was used to analyze morphological alterations in SER profile size within the dendritic shafts and spines of Purkinje neurons. Results showed significant SER dilatation following 40 weeks of ethanol consumption, which disappeared after ethanol withdrawal.

Structural and functional effects of developmental exposure to ethanol on the zebrafish heart.

BACKGROUND Fetal alcohol exposure during development results in a host of cardiac abnormalities including atrial and ventricular septal defects, teratology of Fallot, d-transposition of the great arteries, truncus arteriosus communis, and aortico-pulmonary window. The mechanisms behind these ethanol-induced deficits are unknown. The purpose of this study was to determine whether the zebrafish, a simple model in which heart development and the sequence of gene expression is well elucidated and comparable to that in higher vertebrates, is sensitive to developmental exposure of pharmacologically relevant concentrations of ethanol. METHODS Zebrafish eggs of the AB strain were raised in egg water or in 0.5% (v/v) ethanol solution for either 54 hpf (hours postfertilization) or 72 hpf. Heart pathology and volumes were evaluated on the latter group at 5 dpf (days postfertilization) on tissue sections from fixed larvae embedded in glycolmethacrylate. Heart rates were determined in embryos of 54 hpf and larvae of 5 dpf. The functional maturity of the hearts conducting system was measured by determining the response of ethanol-treated and control embryos and larvae to the adrenergic agonist, isoproterenol, and the cholinergic agonist, carbachol. RESULTS Ethanol-induced alterations occurred in heart morphology and heart volume. A developmental lag in the isoproterenol response and the absence of carbachol-mediated bradycardia were also observed following ethanol treatment. CONCLUSIONS These results show that exposure of the zebrafish to ethanol during development results in structural and functional changes in the heart that mimic malformations that occur in patients with fetal alcohol syndrome (FAS). These findings promote the zebrafish heart as a future model for investigating the mechanisms responsible for ethanols adverse effects on vertebrate heart development.

The Number of Granule Cells and Spine Density on Purkinje Cells in Aged, Ethanol-fed Rats

The purpose of this study was to determine whether chronic intake of ethanol by aged F344 rats was associated with a reduction in parallel fiber input to cerebellar Purkinje neurons (PN). Previous results from this laboratory provided direct evidence that synaptic density in PN dendritic arbors was significantly decreased and indirect evidence that terminal dendritic segments of PN were deleted during chronic ethanol treatment. From these results, it was hypothesized that an ethanol-related deletion of PN terminal dendritic segments might result from 1) a reduction in parallel fiber input to PN from cerebellar granule neurons or 2) a reduction in dendritic spines, the postsynaptic sites for parallel fiber input to PN dendrites. Measurements of the total number of cerebellar granule neurons (GN) and the volume of the GN layer, and measurements of the density of spines on PN terminal dendritic segments were made in separate groups of aged, ethanol-treated and control rats. There were no significant ethanol-related changes in these parameters after 40-48 weeks of ethanol treatment.

Gender differences in ethanol-induced behavioral sensitivity in zebrafish

Gender-related differential sensitivity to ethanol has long been recognized. Our previous studies have demonstrated that the zebrafish, an animal model used currently to study genetics and development related to a variety of human diseases, is also sensitive to pharmacologically relevant concentrations of ethanol. Sensitivity to ethanol in the zebrafish can be easily gauged with a simple nonintrusive behavioral test that measures ethanol-related alterations in schooling by determining the distance between each fish and its nearest neighbor. The purpose of this study was to determine the influence of gender on the strain-specific ethanol sensitivity that we had observed previously. One hundred and sixty zebrafish of the wild-type (WT) and the long fin striped (LFS) strains were equally divided by gender for use in this study. For acute ethanol treatment, the fish were separated by gender and strain and exposed to 0.0, 0.125, 0.25 0.50, or 1.0% (vol/vol) ethanol. In the chronic study, eight fish of each strain and gender were exposed to 0.5% (vol/vol) ethanol for a period of 10 weeks and the swimming behavior tested before treatment and after each week of treatment. Results showed that female WT zebrafish displayed enhanced sensitivity to the effects of chronic ethanol exposure of increased nearest neighbor distances, whereas male and female LFS fish were not significantly affected by chronic ethanol exposure. Results of the acute ethanol study showed a dose-dependent effect in both strains and a gender effect that needs to be further investigated before enhanced female sensitivity to acute ethanol can be verified.