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.

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.

Cerebellar Purkinje neurons with altered terminal dendritic segments are present in all lobules of the cerebellar vermis of ageing, ethanol-treated F344 rats.

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.

Effects of chronic ibogaine treatment on cerebellar Purkinje cells in the rat.

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]).

Morphometric evidence that the total number of synapses on Purkinje neurons of old F344 rats is reduced after long-term ethanol treatment and restored to control levels after recovery.

Clinical symptoms of alcohol abuse may be confused with symptoms of age-related neuropathologies 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.

Continuous exposure of cultured rat cerebellar macroneurons to ethanol-depressed NMDA and KCl-stimulated elevations of intracellular calcium.

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.

Measurements of dendritic path lengths provide evidence that ethanol-induced lengthening of terminal dendritic segments may result from dendritic regression.

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.

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.

Small animal magnetic resonance imaging: a means of studying the development of structural pathologies in the rat brain.

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.

Models for growth, decline and regrowth of the dendrites of rat Purkinje cells induced from magnitude and link-length analysis.

This study examines Purkinje neurons of rats aged 1, 10, 18 and 28 months to investigate growth and decline in the magnitude of the dendritic tree, i.e. the number of exterior links (terminal segments) per cell. Growth in the mean number of exterior links was observed from 1 to 10 months, decline at 18 months and regrowth at 28 months. At 10, 28, and especially at 18 months, the cell size frequency distribution indicates two groups of cells, one of small and the other of large sized cells. The study also examines the relationship of age to lengths of topologically defined links of various types. For each age group we find that exterior links are longer than interior links (non-terminal or intermediate segments). Analysis of the geometric mean lengths of subtypes of exterior and interior links at maturity (10 months) indicates that they follow a Fibonacci series of link lengths, such that mean lengths of topologically defined types of mean exterior links are either about 13 or 8 microns long, while interior links are about 5 microns long. A sequential growth model for adding exterior links is suggested to illustrate a style of growth which could account for the various mean link lengths and the Fibonacci ratio (1.618) between their lengths. Interior link lengths are also dependent on the generation of exterior links from the sides of pre-existing interior links. If the Strahler branching ratio, Rb, should increase owing to growth of terminals from interior links, then mean interior link length would decline. During a period of regression, mean exterior link lengths become shorter and mean interior link lengths become longer. Changes in mean interior link length are much less affected by changes in Rb during regression than is the situation during growth. Finally, the changes in link lengths dictate that the ratio of mean exterior to mean interior link length increases during growth phases from 1 to 10 and 18 to 28 months, and declines during regression from 10 to 28 months. The lowest values of the ratio of mean exterior to mean interior lengths are found at 1 month. This is the period of most intense growth. During this period, the rate of development of new exterior links outbalances the rate at which the links increase in length.

Ethanol enhances neurite outgrowth in primary cultures of rat cerebellar macroneurons.

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.

Tree asymmetry--a sensitive and practical measure for binary topological trees.

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.

Effects of long durations of ethanol treatment during aging on dendritic plasticity in Fischer 344 rats.

Twelve-month-old Fischer 344 rats were fed a liquid diet containing 35% ethanol until they were 18 or 24 months old. Pair-fed and chow-fed control rats were matched to each ethanol-fed rat for concurrent treatment. Cerebellar Purkinje cell networks were measured in half of the rats at the end of the ethanol treatment and in the remaining rats after a subsequent 2-month recovery period. Chronic ethanol consumption resulted in significant elongation of terminal segments in the networks, and the unpaired terminal segments were the predominant sites of this growth. An increase in the duration of ethanol consumption from 24 to 48 weeks caused significantly greater segment elongation in the ethanol-fed rats in spite of the fact that circulating blood levels of ethanol declined markedly with the increased duration of treatment. During the same period of time, a pattern of terminal segment regression followed by terminal segment regrowth characterized age-induced changes in these networks. Thus the effects of long-term ethanol consumption were distinct from effects of concurrent aging processes in the Purkinje cell networks. There were significant interactions between the diets and the longer duration of treatment, such that as segments elongated in the ethanol-fed rats, they shortened in the pair-fed rats, and between the diets and the recovery period, such that as segments elongated during recovery in the pair-fed rats, they shortened in the ethanol-fed rats.(ABSTRACT TRUNCATED AT 250 WORDS)

MR imaging of normal rat brain at 0.35 T and correlated histology.

A custom-built small-animal transceiver was used for in vivo imaging of normal rat brain at 0.35 T, with the objective of identifying anatomic components by comparison of images with corresponding histologic sections. The cerebrum, cerebellum, brain stem, ventricles, hippocampus, and subarachnoid space were identified and cerebrospinal fluid (CSF) was differentiated from gray matter and white matter on coronal and transaxial magnetic resonance (MR) images. These images compare favorably with those obtained by others at higher field strengths in regard to delineating major neuroanatomic structures. It is concluded that this technique will be useful for investigating small-animal models of human neurologic disease involving morphologic and morphometric changes in gray matter, white matter, and CSF-filled spaces.

Effects of local delta and mu opioid receptor activation on basal and stimulated dopamine release in striatum and nucleus accumbens of rat: an in vivo electrochemical study.

The magnitude and duration of spontaneous and of potassium-stimulated dopamine release were electrochemically measured in striatum and nucleus accumbens of chloral hydrate-anesthetized rats following [D-Pen2-D-Pen5]enkephalin, a delta opioid receptor agonist, or [Tyr-D-Ala-MePhe-Gly-ol], a mu opioid receptor agonist, microinjected directly into the voltammetric recording sites. The data show that delta receptor activation potentiated potassium-stimulated dopamine efflux in striatum and in nucleus accumbens but had no effect on spontaneous dopamine release in either region, whereas mu receptor activation produced unreliable effects in both regions, either having no effect or inhibiting stimulated dopamine efflux without affecting basal levels of extracellular dopamine in either region. The data suggest that some delta opioid receptors in the caudate-putamen and in the nucleus accumbens presynaptically enhance impulse-dependent dopamine release from nigrostriatal and mesolimbic dopamine terminals.

Remodeling of neuronal dendritic networks with aging and alcohol.

Chronic ethanol treatment and aging processes produce significant changes in neuronal dendritic morphometry, but the precise patterns of change are different for these two factors. Furthermore, the morphometric effects of long term ethanol treatment on neuronal structure may be expressed in different ways at different times during the life span. When rats were treated with ethanol chronically from 12 months of age for 24 or 48 weeks, the predominant morphometric change in surviving cerebellar Purkinje neurons was a nonrandom elongation of terminal segments in the dendritic arbors. The results suggested that the synaptic circuitry in the cerebellar cortex of these rats was altered in an ethanol-specific and nonrandom pattern by selective compensatory growth of a particular category of terminal segments. During normal aging between 12 and 18 months, Purkinje cell networks in rats underwent regression of terminal dendritic segments, but after 18 months localized regrowth of proximal terminal dendritic segments coincided with regression of distal terminal segments, a pattern of dendritic change that was markedly different from that induced by ethanol.

Dendritic hypertrophy in Purkinje neurons of old Fischer 344 rats after long-term ethanol treatment.

Metric parameters of Purkinje cell dendritic networks in 24- to 26-month-old Fischer 344 rats were determined after 48 weeks of chronic ethanol intake. Measurements included the total number and length of all segments/network, the total number and length of segments within topologically defined segment categories, and the mean length of segments in each category. A main effect of ethanol was expressed as a significant increase in cumulative length within one category of terminal segments. This increase was the result of a significant increase in mean length/segment in that category. Metric changes in other segment categories were not significant, showing that changes in the networks during ethanol treatment were not distributed randomly. Recovery after ethanol treatment was associated with further nonrandom remodeling of these networks. Significant differences in lengths of terminal segments were no longer present, but internal segments in networks of both pair-fed and ethanol-fed rats were significantly longer. Only one category of internal segments showed this change during recovery. The data showed that long term ethanol treatment in old rats, at a time when effects of aging processes were prominent in Purkinje cell networks, was associated with remodeling of those networks through dendritic extension. This effect was interpreted as compensatory growth in surviving networks following ethanol-related neuronal loss and/or damage to the surrounding neuropil. Recovery from dietary treatment produced further internal remodeling of those networks that was not related specifically to ethanol. It could be shown, however, that the restructuring processes that resulted in longer internal segments after dietary recovery were different in the pair-fed and the ethanolfed rats.

Evidence for nonrandom regression of dendrites of Purkinje neurons during aging.

This study examined dendritic networks of Purkinje neurons for randomness of age-related changes in cerebella from Fischer 344 rats at three ages. Terminal dendritic segments were studied in relation to their distance from the neuronal soma (path length). The data indicated that the terminal dendritic segments furthest from the cell body were preferentially affected during aging and there was a redistribution of the remaining terminal segments in cells from rats of the intermediate age group. The data also suggested that dendritic regrowth occurred in proximal regions of networks from rats in the oldest group.

Length changes in dendritic networks of cerebellar Purkinje cells of old rats after chronic ethanol treatment.

Dendritic networks of cerebellar Purkinje neurons from aging ethanol-treated Fisher 344 rats were analyzed for metric changes in terminal and internal segments of the networks. Mean lengths of three categories of dendritic segments were determined. No significant metric changes in segment lengths were detectable immediately at the termination of 24 weeks of ethanol treatment, but significant changes were apparent after 8 weeks of recovery from ethanol treatment. Return to a diet of standard laboratory chow was associated with a period of dendritic extension in networks from pair-fed control rats but not in those from the ethanol-treated rats. The resulting significant differences in mean segment length were restricted to the paired terminal segments at the peripheral tips of the bifurcating networks. Unpaired terminal segments and internal segments of the networks showed no significant changes in length during the recovery period.

Morphometric parameters of Purkinje dendritic networks after ethanol treatment during aging.

Spine densities on terminal branches of Purkinje cell dendrites of Fischer 344 rats were significantly altered by ethanol treatment and aging processes. An effect of the control liquid diet on the lengths of terminal branches and an interaction effect of this diet with age on the numbers of terminal branches/cell also occurred, but there were no detectable interaction effects of ethanol with age on dendritic parameters. Changes in spine density on terminal branches, which accounted for 63-67% of the total dendritic length/cell, represented a major quantitative modulation of synaptic input to these neurons during age and following ethanol treatment.