lcoholism and other diseases affect the structure and function of many organs in the human body, includ­ ing the brain. To understand the causes and to diagnose and track the progress of such diseases, scientists must understand the struc­ tural and functional changes in the affected organs. For many studies, particularly those involving the brain, researchers rely on non­ invasive methods to visualize the organ under investigation. One technique com­ monly used to obtain images of a patient’s brain or other area of the body is magnetic resonance imaging (MRI). This technique exposes the subject to radio waves in the presence of a strong magnetic field and then measures how the atoms that make up the body’s tissues absorb the radio waves’ en­ ergy. The manner of absorption varies, de­ pending on the composition of the tissues (e.g., their water or fat content); with the help of computers, these variations can be converted into images. This article describes how MRI tech­ nology can be used not only on human patients but also in studies of laboratory ani­ mals that frequently serve as experimental models for human diseases. Also presented are findings of MRI studies that analyzed

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.

This article represents a symposium of the 2002 joint meeting of RSA and ISBRA held in San Francisco. Presentations were Neuropathology of alcohol-related cerebellar damage in humans, by Antony J. Harding; Neuropathological evidence of cerebellar damage in an animal model of alcoholism, by Roberta Pentney and Cynthia Dlugos; Understanding cortical-cerebellar circuits through neuroimaging study of chronic alcoholics, by Peter R. Martin and Mitchell H. Parks; and Functional reorganization of the brain in alcoholism: neuroimaging evidence, by John E. Desmond, S.H. Annabel Chen, Michelle R. Pryor, Eve De Rosa, Adolf Pfefferbaum, and Edith V. Sullivan.

Background Ethanol-related synaptic loss, Purkinje neuron dendritic regression, and parallel fiber degeneration have been reported in the molecular layer of the adult cerebellar cortex. The known plasticity of the cerebellar cortex suggests that this region may respond to ethanol-related losses by compensatory remodeling of cerebellar circuitry. Stellate and basket interneurons may play an essential role in the remodeling process. Little is known about ethanol-related effects on cerebellar interneurons or on the GABAergic synapses that they form despite the fact that ethanol-related alterations in these components may contribute to the sensitivity of the cerebellum to ethanol. The paucity of data on GABAergic synapses extends to other synaptic components as well including synaptophysin, a glycoprotein component of synaptic vesicles and a synaptic marker. Methods Thirty 12-month-old F 344 rats were divided into ethanol-fed, pair-fed, and chow-fed groups (10/group). Ethanol rats were treated for 40 weeks with a liquid diet in which 35% of the calories were derived from ethanol. At the end of treatment, rats were perfused, and tissue processed for quantitative immunohistochemistry of GABA and synaptophysin labels. Results Levels of GABA within inhibitory synapses formed by stellate and basket neurons and levels of synaptophysin were not altered by long-term ethanol treatment. Conclusions Stable levels of GABA within GABAergic basket and stellate interneuron synapses suggest that interneurons in the molecular layer of the cerebellar cortex may not play a major role in remodeling of cerebellar circuitry following long-term ethanol consumption. The lack of ethanol-related alterations in synaptophysin levels reported here suggests that synaptic vesicles may be relatively insensitive to ethanol and that known ethanol-related effects on synapse number are due to other mechanisms.

It is generally accepted that long term chronic ethanol consumption by young rats will lead to significant losses of cerebellar granule neurons (GN). A recent study in this laboratory showed, however, that 40 weeks of chronic ethanol consumption had no effect on the total numbers of GN in aged Fischer 344 rats (F344). The goals of the present study were to determine whether F344 GN were resistant to ethanol toxicity only in aged rats and whether resistance of GN in aged rats to ethanol toxicity occurred only in the F344 strain. To accomplish those goals, young and aged adult F344 and Wistar-Kyoto (WKY) rats were treated chronically with ethanol for 40 weeks during the first or second half of their life span. In each rat the total numbers of GN were estimated with the optical fractionator and the volumes of the GN layer were estimated according to Cavalieri's theorem. After the 40 weeks of ethanol, there were significant age-related differences in the total numbers of GN in the F344 rats. There were also significant strain-related differences in the total numbers of GN and volumes of the GN layer. There were no significant ethanol-related differences, however, in numbers of cerebellar GN or volumes of the GN layer in F344 rats or WKY rats. The results presented here show that consumption of ethanol over long periods of time had no effect on the total numbers of cerebellar GN or the granular layer volumes in young or aged F344 or WKY 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.

Previous studies from this Laboratory have shown that cerebellar Purkinje neurons (PN) in ageing, ethanol-fed Fischer 344 rats may have terminal dendritic segments that are longer than in control rats. They also showed that the longer terminal segments represented a toxic effect of ethanol on PN, because their increase in length resulted from an ethanol-induced deletion of other terminal dendritic segments and not from dendritic growth. The purpose of the present study was to determine whether this effect of ethanol was localized to specific lobules or was widely distributed within all lobules of the cerebellar vermis. Twelve-month-old male Fischer 344 rats were treated with a liquid ethanol diet for 48 weeks. Age- and weight-matched controls were pair-fed with an isocaloric control diet. Terminal dendritic segments in Golgi-Cox-stained PN in four groups of lobules in the cerebellar vermis of control and ethanol-fed rats were measured for treatment-related changes in length. Results from these measurements showed that ethanol-exposed PN with significantly longer terminal segments were present in all groups of lobules, i.e. they were widely distributed and not confined to specific lobules. Results from these measurements also confirmed in a large sample of neurons (40 neurons per rat) that the topologically distinct unpaired terminal segments were characteristically longer than the paired terminal segments in PN of control and ethanol-fed rats and that both types of terminal segments were longer in the ethanol-fed rats than in the controls.

The present investigation assessed the chronic toxicity of ibogaine on cerebellar Purkinje cells in male Fischer 344 rats. A behaviorally active dose of ibogaine (10 mg/kg, i.p.) was administered to a group of six subjects every other day for 60 days while the control group received an equivalent volume of saline (1 ml/kg). Estimates of Purkinje cell number were determined using the optical dissector/fractionator technique. No significant differences in Purkinje cell number were observed between the ibogaine (243764[+/-32766]) and control groups (230813[+/-16670]).

Clinical symptoms of alcohol abuse may be confused with symptoms of age-related neuro-pathologies in human patients. It is important, therefore, to determine the relationships between alcohol abuse and changes in brain structures in well-controlled studies of ageing subjects. Currently there is little well-documented information of this type available. The purpose of this study was to determine whether long-term ethanol treatment during ageing would lead to reductions in synaptic input to cerebellar Purkinje neurons (PN) of old F344 rats that; (1) were more severe than those attributable to ageing alone; (2) might be responsible for an ethanol-related deletion of dendritic segments of PN in old F344 rats shown previously in this laboratory. The total number of synapses per PN dendritic arbor was determined after ethanol treatment of old F344 rats for 40 weeks between 12 and 22 months of age and in similarly treated rats given a subsequent 20-week period of recovery between 22 and 27 months of age. Groups of age-matched rats fed a chow diet and water and rats pair-fed an isocaloric liquid diet lacking ethanol served as controls. The volume of the molecular layer per PN arbor and the numerical density of synapses in the molecular layer was determined from light microscopic preparations of a fixed volume of the cerebellar cortex. Photographic montages of the ultrastructure of the molecular layer of the cerebellum were also prepared from each rat for measurements of synaptic numerical densities. From the volume of the molecular layer per PN arbor and the numerical density of synapses in the molecular layer, the total number of synapses per PN arbor was estimated for each rat. There was a significant reduction in synapses in the old ethanol-treated rats relative to age-matched chow-fed rats. There were also significant interactions between recovery and treatment prior to recovery During recovery, synaptic numbers in the old, ethanol-treated rats were restored to pre-recovery control levels whereas synaptic numbers in the old, chow-fed rats were significantly reduced during the same period of time. There were no significant diet- or age-related changes in synaptic numbers in the pair-fed control rats during treatment or recovery. The pattern of reduction in synaptic numbers during ethanol treatment and restoration of synaptic numbers during recovery from treatment paralleled the pattern of ethanol-related segment loss and recovery-related segment regrowth noted earlier in PN arbors of old F344 rats, suggesting that reductions in the numbers of synapses and deletion of terminal dendritic segments were causally related in this strain.

KEY WORDS: animal model; magnetic resonance imaging; thiamine deficiency; brain damage; AOD abstinence; AODE (alcohol and other drug effects) AIcoholism and other diseases affect the structure and function of many organs in the human body, including the brain. To understand the causes and to diagnose and track the progress of such diseases, scientists must understand the structural and functional changes in the affected organs. For many studies, particularly those involving the brain, researchers rely on noninvasive methods to visualize the organ under investigation. One technique commonly used to obtain images of a patient's brain or other area of the body is magnetic resonance imaging (MRI). This technique exposes the subject to radio waves in the presence of a strong magnetic field and then measures how the atoms that make up the body's tissues absorb the radio waves' energy. The manner of absorption varies, depending on the composition of the tissues (e.g., their water or fat content); with the help of computers, these variations can be converted into images. This article describes how MRI technology can be used not only on human patients but also in studies of laboratory animals that frequently serve as experimental models for human diseases. Also presented are findings of MRI studies that analyzed the effects of thiamine deficiency--the lack of a vitamin required for normal brain functioning--on the brain structure of rats. Preliminary findings indicate that similar studies can be used to assess the effects of long-term alcohol consumption and withdrawal on the brain. Although MRI has been used successfully in humans to diagnose diseases and study alterations in organ structures, its applications in elucidating the cause of a pathology or functional abnormality are more limited. To investigate the cause of a disease, researchers ideally would compare images of patients taken before and after they develop the disorder. Only rarely, however, do patients have images available for comparison that were taken prior to an illness. Alternatively, investigators can compare one group of subjects with a disease (e.g., alcoholics) with another group of people without the disease (e.g., nonalcoholics) to identify consistent differences between the two groups that might shed light on the cause of alcoholism. The subjects in such studies, however, will have different characteristics in addition to their alcohol intake (e.g., their medical and drinking histories and their genetic makeup). To make valid observations and to control for all possible variations among subjects, researchers must examine a large number of patients before attributing specific differences between the two study groups to alcohol use. Because of these limitations of human studies, researchers frequently rely on animal models, often using rats and other small animals. In general, animal studies provide more consistent information with fewer subjects than do human studies. The animals can be examined both before and after they develop a certain disease or undergo a particular treatment. Furthermore, animal studies can be rigidly controlled so that a particular change in organ structure or function can be directly attributed to the modification of a single experimental variable. Differences between animals can be minimized by using animals from one family (i.e., strain), a method that in humans would be feasible only with identical twins. And in contrast to studies on humans, studies using animals can be conducted across an animal's life span (about 2.5 years for rats), allowing the investigation of age-related effects. Conventional Techniques Histological techniques (i.e., studies that determine the structure of organs by examining thin tissue slices under a microscope) are commonly used to follow disease development in animals. Although these techniques allow researchers to view directly the tissue under investigation, they have several drawbacks, especially in studies of the brain. …

The present study proposed to determine the significance of previously reported ethanol-induced dendritic lengthening in mature cerebellar Purkinje neurons (PN). An analysis of dendritic path lengths (PL) was used to make this determination. A PL is the curvilinear length from the origin of the dendritic root segment to the free tip of a dendritic terminal. It was hypothesized that treatment-induced dendritic lengthening resulted from dendritic growth, and that growth should produce an upward shift in the value of the median PL. The PL and the segment length (SL) for each dendritic terminal were measured in PN of 24 month old male F344 rats, previously fed a liquid diet containing 35% ethanol-derived calories for 48 weeks, to test that hypothesis. Because there are large numbers of terminal dendrites in PN networks a shift in the value of their median PL is a sensitive measure of change in these networks. It was found that almost 30% of the ethanol-exposed cell sample had median PL that were shorter than expected and that the median SL in these same neurons tended to be longer than expected. At the end of the ethanol treatment, half of these rats were weaned from the ethanol diet for a subsequent 8 weeks to test for reversibility of these changes. Following the period of abstinence from dietary ethanol, the significant ethanol-related differences previously found in dendritic lengths were no longer present. A consideration of two models of dendritic regression suggested that elongation of surviving terminal dendritic segments in the ethanol-exposed neurons occurred through deletion of other terminal segments at their vertices (branch points). It was shown that deletions of entire terminal dendritic segments at their vertices would produce increases in the lengths of associated terminal segments in the absence of real growth. Deletions of dendritic segments would also entail loss of PL unique to the deleted segments, thereby causing a shift in the value of the median PL of affected networks.

This series of experiments measured ethanol-induced changes in levels of free intracellular calcium. Cerebellar macroneurons, harvested from rat embryos on embryonic day 17, were cultured in the presence of 75 mM ethanol for 24, 48, or 96 hr. Intracellular calcium concentrations in control and ethanol-exposed neurons did not differ after 24 hr, but they were significantly elevated in the neurons exposed to ethanol for 48 or 96 hr. Similarly, increases in intracellular calcium elicited by stimulation with 50 microM NMDA were not significantly different in control and ethanol-exposed neurons after 24 hr. After 48 and 96 hr, however, NMDA-stimulated increases in intracellular calcium levels in control neurons were significantly greater than in the ethanol-exposed neurons. These results showed that, when calcium levels were elevated by prolonged exposure to ethanol, the neurons were significantly less responsive to NMDA stimulation. Increases in intracellular calcium elicited by stimulation with 30 mM KCI were not significantly different in the control and treated neurons after 24 and 48 hr of ethanol exposure. After 96 hr of exposure to ethanol, however, there was a significant increase in intracellular calcium levels in control neurons following KCI stimulation, but not in the ethanol-exposed neurons. The fact that neuronal responses to KCI stimulation were depressed only following 96 hr of exposure to ethanol makes it unlikely that voltage-regulated channels were the primary mediators of the ethanol-induced elevations in intracellular calcium in chronically exposed neurons.

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.

Effects of ethanol on neurite outgrowth and morphometry were investigated in primary cultures of rat cerebella. Cell cultures were prepared from cerebella on embryonic day 17 (E17) for treatment with a series of ethanol concentrations (50, 75, 100, 150 and 200 mM). Ethanol did not reduce neuronal survival or attachment to the substrate at any of the concentrations that were used. Treatment with 75 mM ethanol significantly enhanced neurite outgrowth. Measurements from dissociated cultures exposed to 75 mM ethanol immediately after plating showed a significant increase in the percentage of neurite-bearing cells after 8 and 24 h in vitro. Measurements of the area and perimeter of neuronal cell bodies in dissociated cell cultures showed that the cell bodies of ethanol-treated neurons were also larger than those of control neurons. Ethanol was also associated with significant increases in the total neuritic length per cell and in the length of the longest neurite in each cell. The mean number of neurite branches was also greater in the ethanol-treated neurons. Measurements from suspension cell cultures, in which dissociated cells were suspended overnight in the presence of 75 mM ethanol prior to plating, corroborated these results. These findings suggest that ethanol may have distinct effects on neurite initiation and outgrowth and branching. The cellular mechanisms involved and the functional significance of these effects are currently not known. The present results also indicated that high concentrations of ethanol (150–200 mM) and long periods of exposure (4–7 days) were required to produce toxic effects on neurons and glial cells in this system.

Small animal magnetic resonance imaging (SAMRI) was developed to detect structural tissue changes associated with disease states in animal models. The disease state of particular interest here is that associated with long-term alcohol abuse. The small animal model used for this study was the thiamine-deficient Sprague-Dawley rat, a model that provides a relatively rapid means of mimicking the ventriculomegaly frequently found in human chronic alcohol abusers. A custom-designed coil tuned to the magnetic field of a 1.5 Tesla clinical magnetic resonance imager provided the technology necessary to delineate discreet regions of the rat brain with clarity. Adult, male rats were imaged, placed on a thiamine-deficient pellet diet for approximately 6 weeks, and then reimaged. Treatment associated enlargement of the lateral ventricles identified in the images was verified by posttreatment histological analysis of the brains of these rats. The results demonstrated that SAMRI is capable of providing dramatic and reliable visual evidence of pathological structural changes in small tissue volumes with high resolution and reproducibility. Furthermore, the noninvasiveness of SAMRI allowed for imaging of the same animals over time, thereby reducing the numbers of animals needed for convincing documentation of the changes in ventricular size.

Fischer 344 male rats were fed a liquid diet containing either sucrose (Sucrose group) or ethanol (Ethanol group) in equicaloric amounts or chow ad lib (Chow group). Treatment was begun at three months of age and continued for approximately 26 weeks. At the end of this time one rat from each group was subjected to magnetic resonance imaging (MRI). All rats were weighed weekly; and once each week 24 hour urine volumes were collected along with tail vein blood for measurement of renal function. After euthanasia brains and kidneys were removed, kidneys dissected into cortex, outer medulla and inner medulla and sorbitol and inositol measured in these tissues. The results demonstrated that the Sucrose group gained weight at a significantly greater rate than either of the other two groups. This was associated with large accumulation of fat when visualized by MRI. The major effect on renal function was a continual osmotic diuresis resulting from the liquid diet and accordingly plasma osmolalities were significantly greater in the Sucrose and Ethanol groups than in the Chow group. Small but significant decreases in sodium and potassium excretion were found for the Ethanol group when compared to the Sucrose Group at 25 to 26 weeks. Prolonged exposure to high plasma osmolalities was also associated with an apparent down regulation of intracellular sorbitol and inositol concentrations, since these polyols were significantly lower (less than half the concentration) in the brain and kidneys of the Sucrose and Ethanol groups than in the Chow group.

The topological structure of a binary tree is characterized by a measure called tree asymmetry, defined as the mean value of the asymmetry of its partitions. The statistical properties of this tree-asymmetry measure have been studied using a growth model for binary trees. The tree-asymmetry measure appears to be sensitive for topological differences and the tree-asymmetry expectation for the growth model that we used appears to be almost independent of the size of the trees. These properties and the simple definition make the measure suitable for practical use, for instance for characterizing, comparing and interpreting sets of branching patterns. Examples are given of the analysis of three sets of neuronal branching patterns. It is shown that the variance in tree-asymmetry values for these observed branching patterns corresponds perfectly with the variance predicted by the used growth model.