Department of Anatomy and Cell Biology, 317 Farber Hall, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, NY 14214-3000, USA
Received 22 April 1999; in revised form 26 July 1999; accepted 17 August 1999
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ABSTRACT |
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INTRODUCTION |
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Several of the above studies utilized procedures based on quantitative data from entire dendritic arbors. Two major drawbacks of that type of analysis result from the stringent criteria that must be used for selection of entire dendritic arbors. Those criteria yield only a limited number of PN for study in each subject, and the lobular subdivisions of the cerebellar vermis are not represented equally or extensively. An important conclusion, that all of the ethanol-induced modifications in PN of ageing F344 rats involved terminal segments of the PN arbors, did emerge from those early studies, however. Furthermore, even in the restricted cell samples in those studies, only 30% of the PN had ethanol-modified dendritic arbors (Pentney, 1995), suggesting that the effects of ethanol might be localized, rather than widespread throughout the cerebellar vermis. It has been known for some time that, during perinatal development, rodent PN demonstrate a regional sensitivity to ethanol's effects related to their maturational status (Ward and West, 1992
). The question of regional differences in sensitivity of PN to ethanol within the cerebellar vermis has not been addressed previously in the ageing cerebellum.
The study presented here used only terminal segments of a large number of PN dendritic arbors in each subject for quantitative analysis. The exclusion of all dendritic segments, except terminal segments, offered the advantage of increasing the number of cells that could be sampled in each subject. The sampling procedure was also designed to obtain equal samples of PN from all subdivisions of the cerebellar vermis in order to address the question of PN regional sensitivity to ethanol. The main purpose of this analysis was to determine whether the effect of ethanol on PN terminal dendritic segment lengths was expressed broadly throughout the vermis or was localized to specific lobules of the vermis. A secondary outcome of this study was that two key results from previous studies, i.e. a consistent significant difference between the mean lengths of paired and unpaired terminal dendritic segments in the arbors and consistent ethanol-related increases in terminal segment lengths (Pentney et al., 1989), were also corroborated.
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METHODS |
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Treatment conditions were identical for the control and ethanol-treated rats. They were housed in a room dedicated to this study in the Laboratory Animal Facilities of the School of Medicine and Biomedical Sciences at the University of Buffalo. Lighting was on a 12 h light/12 h dark cycle (lights on at 06:00), and temperature was maintained at 22 ± 2°C. The rats were housed individually in wire cages suspended from racks. They were weighed weekly to monitor their health during treatment with the liquid diets. The Institutional Animal Care and Use Committee (IACUC) of the School of Medicine and Biomedical Sciences approved all procedures used for treatment and care of the animals.
Blood alcohol concentrations
After 6 weeks of ethanol treatment, blood samples for measurements of circulating blood alcohol levels (BAL) were collected from tail veins at 23:00, 13 h after fresh diet was placed on the cages and 5 h after the start of the dark period. Blood samples were also collected from three control rats. The samples were analysed by gas chromatography with n-propanol as an internal standard.
Tissue preparations
Following 48 weeks of dietary treatment, each rat was given a lethal dose of chloral hydrate (400 mg/kg, intraperitoneal). Once the rat was deeply anaesthetized, the cervical spinal cord was severed, and the brain was freed gently from the skull. The cerebellum was then isolated from the brainstem, and the vermis was separated from the cerebellar hemispheres and placed immediately in the GolgiCox fixative. Details of the fixation and block-staining procedures have been described in detail previously (Pentney, 1986). The blocks of tissue were subsequently dehydrated, embedded in 10% celloidin, and sectioned parasagittally at 120 µm. All tissue blocks were coded prior to the embedding step to avoid investigator bias during selection of cells for analysis.
Quantitative procedures
PN selected for measurements of dendritic segment lengths were completely filled with the GolgiCox precipitate, had unbroken terminal dendritic segments (Figs 1 and 2A), and could be brought into sharp focus with a x100 oil immersion objective lens. In a bifurcating arbor, such as that of PN, two-thirds of the spiny terminal segments will be at the peripheral tips of the dendritic branches. These segments were arranged in pairs, or rarely as triplets, at the ends of the bifurcating branches (Fig. 2A
, arrowheads). The remaining third of the spiny terminal segments, the unpaired terminal segments, branched singly from second- or higher-order segments within the arbors (Fig. 2A
, arrow). It was shown previously that the mean lengths of paired and unpaired terminal dendritic segments were significantly different (Pentney et al., 1989
). For that reason, the paired and unpaired terminal segments were measured and analysed separately.
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Statistical analysis
Mean values for the lengths of paired and unpaired terminal dendritic segments were initially determined for each PN. Subsequently the data were grouped in two different ways. First, the mean lengths of all paired terminal segments and of all unpaired terminal segments in each PN arbor were determined. From the mean values in each cell, means for the group of 40 PN arbors in each rat were then determined, yielding a single mean value for each type of segment in each rat. Mean values for each treatment group of eight rats were then determined. A split-plot analysis of variance (ANOVA) was used to test for significant differences between the mean values for the lengths of the two types of terminal dendritic segments (paired and unpaired) in the control and ethanol-fed rats. This modified ANOVA adjusted for the fact that measurements of paired and unpaired terminal segment lengths were from the same rats.
Second, the data from the 40 PN in each rat were separated according to the lobular location of the cells to determine mean values for the ten cells in each of the four groups of lobules. From the values determined for each lobular group in each rat, mean values were subsequently determined for each treatment group. Two-way ANOVA was used to determine whether there were significant differences in the lengths of terminal segments in the four groups of lobules in the vermis of the control and ethanol-fed rats. Separate tests were performed for the paired and unpaired terminal segment values. Main effects identified in the two-way ANOVA were analysed further by a simple ANOVA and localized with a post hoc Duncan multiple range test, as necessary. In all tests an alpha level less that 0.05 was accepted as significant.
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RESULTS |
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Measurements of BAL
The mean BAL of the ethanol-treated rats was 90.3 ± 34.3 mg/dl, confirming that intoxicating levels of circulating ethanol were present in many of the rats when the blood samples were collected. The volume of the ethanol diet consumed by each rat prior to blood collection was recorded, but the time between blood collection and the last feeding by each rat was not known. It could not be determined, therefore, whether the BAL measured at 23:00 h in individual rats actually corresponded to peak BAL in these rats. Alcohol levels from metabolic sources in blood samples from the control rats ranged from 0 to 3 mg/dl.
Lengths of terminal segments in control and ethanol-fed rats
The mean length of the unpaired terminal segments in all rats was significantly longer than the corresponding mean for the paired terminal segments (F1,28 = 98.765, P < 0.001, Fig. 4). There was also a main effect of chronically consumed ethanol on the mean lengths of all terminal dendritic segments in PN, regardless of terminal type. The mean length of all terminal segments in PN of the ethanol-treated rats was significantly longer than the corresponding mean in the controls (F1,28 = 17.274, P < 0.005, Fig. 5
).
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DISCUSSION |
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The use of randomly selected dendritic terminal segments for measurements of dendritic lengths was unquestionably a more efficient approach to data collection than was the use of entire dendritic arbors for measurements in our earlier studies (Pentney et al., 1989; Pentney and Quackenbush, 1990
, 1991
; Pentney, 1995
). It must be acknowledged, however, that justification for the use of a random selection of only terminal dendritic segments for measurements was tied directly to results from measurements of entire dendritic arbors. The latter measurements indicated strongly that terminal dendritic segments were the primary targets of the actions of ethanol.
Whereas PN that are sensitive to ethanol were shown here to be distributed widely within all lobules of the cerebellar vermis, it would not be correct to deduce that terminal segments in all PN were equally sensitive to ethanol. In fact, this appears not to be true from the previous study in which 30% of randomly sampled PN in the ethanol-fed rats were markedly sensitive to the actions of ethanol, with the remaining 70% appearing to be insensitive (Pentney, 1995). Those results had suggested that PN sensitive to ethanol might be distributed within the vermis in a localized, rather than a widespread, pattern but results presented above refute this suggestion. The full extent of dendritic damage that can be induced by ethanol in affected PN before that damage becomes lethal for the cell is not known. In our studies, the dendritic damage could be completely reversed by a sufficiently lengthy withdrawal period (Pentney and Quackenbush, 1991
; Pentney, 1995
; Dlugos and Pentney, 1997
), suggesting that the ethanol-induced dendritic damage detected in our model remained sublethal for lengthy periods of time. Other investigators have reported that, in Sprague Dawley rats, chronic ethanol intake during 80% of their life span was required to produce a significant loss of PN (Tavares et al., 1987a
). It may be that PN have such expansive dendritic arbors that they may sustain large amounts of dendritic damage before that damage proves to be lethal.
As shown above, PN with longer terminal dendritic segments were present in all lobules of the vermis in ethanol-fed rats. Given that ~90% of the vermis was sampled in the present study, we conclude that PN sensitive to ethanol were distributed widely throughout the vermis. It appears likely, therefore, that the sensitivity of mature PN to ethanol may be independent of particular sources of afferent input to PN or particular efferent targets of PN, since it is independent of particular lobular locations. PN sensitive to ethanol may also be widely distributed within the cerebellar hemispheres, but the hemispheres were not included in the present study.
It is important to note that there is a potential caveat to the above conclusion that could not be addressed in this study. It is possible that PN sensitive to ethanol might be restricted to specific cerebellar microzones, functionally organized units of the cerebellum (Oscarsson, 1979). PN within specific sagittally oriented microzones might share a sensitivity to ethanol, whereas PN in adjacent microzones might be insensitive. It appears unlikely that sensitivity to ethanol itself would be a characteristic feature of microzonal organization, but the possibility that certain biochemical characteristics of PN that are features of microzonal organization may predispose PN to be sensitive to elevated levels of ethanol cannot be ruled out at present. To our knowledge, the sensitivity of PN to ethanol has not been studied in relation to microzonal organization, and microzones of the vermis cannot be identified in GolgiCox preparations.
Some regional differences have been shown, e.g. PN in the posterior lobe of the cerebellum were shown to have significantly reduced synaptic input in rats that consumed an ethanol diet for long periods of time (Dlugos and Pentney, 1997). Earlier studies of synaptic organization in the molecular layer of ethanol-treated rats had not examined differences in the numbers of synapses in different lobes (Phillips, 1985
; Tavares et al., 1987b
). The only studies in which PN that were sensitive to ethanol were found to be limited to specific lobules of the cerebellar vermis were studies of the effects of ethanol on the developing cerebellum during the immediate postnatal period (Bauer-Moffett and Altman, 1975
, 1977
; Pierce et al., 1989
; Bonthius and West, 1990
). In these studies, a lobule-related sensitivity was shown to stem from differences in PN maturation in different lobules at the time of exposure to ethanol.
Maturational differences between PN are not likely to be an important cause of the sensitivity of PN to ethanol in adult subjects. It could be expected, however, that age-related neuronal degeneration might be important. The normal life span of F344 rats is ~25 months (data supplied in Harlan Product Guide, effective 1 July 1999), and the rats used in the study reported here were 2425 months of age. Currently available data show that the numbers of PN in rodent cerebella are stable between 3 and 27 months of age (Druge et al., 1986; Bakalian et al., 1991
; Dlugos and Pentney, 1994
). Several other morphometric parameters of the cerebellum are altered by ageing, however. The volume of the cerebellar molecular layer (Dlugos and Pentney, 1994
), the numbers of PN terminal dendritic segments (Woldenberg et al., 1993
), and the total volume of anterior lobe PN arbors (Dlugos and Pentney, 1997
) all decline with normal ageing. It was also shown some time ago that PN dendritic terminal segments shortened significantly by 18 months of age in F344 chow control rats (Pentney, 1986
). Nonetheless, PN dendritic teminal segments of 18-month-old F344 rats treated with ethanol between 12 and 18 months of age were unchanged in length (Pentney and Quackenbush, 1991
). We cannot dismiss the possibility that an age-related shortening of terminal dendritic segments and an ethanol-related lengthening of terminal dendritic segments (through deletion events) may fortuitously balance out to produce normal mean values for terminal segment lengths in 18-month-old ethanol-fed rats. Only after an additional lengthy period of alcohol consumption, however, was there significant evidence of an effect of the ethanol on dendritic terminal segment lengths. These results suggest that the ageing process per se did not sensitize PN dendritic arbors to the effects of ethanol. Previous studies have shown that there are interactions between dietary treatment and treatment duration and between dietary treatment and recovery (Pentney and Quackenbush, 1991
), but not between dietary treatment and ageing. It can be presumed that rats that survive to 24 months of age represent naturally healthier subjects in our treatment groups, yet PN dendritic terminals in 24-month-old rats were not immune to the effects of a sufficiently lengthy ethanol treatment (Pentney and Quackenbush, 1991
).
There was a difference between weights of ageing, control and ethanol-treated F344 rats after extended treatment with liquid diets in this study, as was also noted previously (Pentney and Quigley, 1987; Pentney and Quackenbush, 1991
; Dlugos and Pentney, 1997
). A possible influence of body weight on terminal dendritic segment lengths was considered in several earlier studies, but none was detected. It was also shown previously that there were no direct relationships between body weights and brain weights (Dlugos and Pentney, 1997
) or between diet-induced changes in body weights and ethanol-induced changes in dendritic length (Pentney and Quigley, 1987
; Pentney et al., 1989
; Pentney and Quackenbush, 1991
).
Considering that during ageing PN dendritic terminal segments shortened by dying back from their free tips and that following ethanol intake some terminal dendritic segments lengthened as others lost contact with their junctions, we conclude that different mechanisms were probably responsible for the age-related and the ethanol-related changes in terminal segment length. Predictably, each age-related or ethanol-induced event must result eventually in the loss of entire terminal segments, but they do so within different time frames, as noted above.
There is abundant evidence that ethanol alters membrane protein functions, and that it does so at blood concentrations measured in this study (Fadda and Rosetti, 1998). The actions of ethanol on membrane proteins commonly disrupt membrane regulation of calcium. A major and extensive organelle involved in calcium regulation within PN dendrites is the smooth endoplasmic reticulum (SER), and there are important indications that structural and functional features of the SER may be altered by ethanol. It is known, for example, that the average relative volume of the SER in dendrites of PN is greatly increased in young Wistar rats after only 3 months of ethanol treatment (Lewandowska et al., 1994
). It has been reported further that mRNA levels of inositol 1,4,5-triphosphate receptor 1, a major component of SER membranes, were reduced in C57BL/6J mice following ethanol treatment for 4 weeks (Simonyi et al., 1996
). More recently, preliminary data from this laboratory showed that many SER profiles in PN dendritic shafts and spines of ageing, ethanol-treated F344 rats were significantly enlarged after 40 weeks of ethanol treatment (Dlugos and Pentney, 1998
). A frequent association between excessive calcium influx into neurons and unusual expansion of the endoplasmic lumen (Garthwaite et al., 1992
) suggested that dilatation of SER profiles results from imbalances in intracellular calcium that are produced by the effects of ethanol on membrane proteins and receptors. As indicated above, the ethanol-induced dilatation of SER profiles was present in dendritic shafts and spines, but the effect was more robust in dendritic shafts than in dendritic spines. The link between SER dilatation in dendritic shafts and selective vulnerability of dendritic branch points involving terminal segments has yet to be delineated, however. A number of vital pieces of information relative to effects of ethanol on specific membrane proteins, receptors, signal-transducing proteins, and calcium-binding proteins of the SER may all be needed to complete such delineation.
There is no reason to suppose that age-related and ethanol-related dendritic degeneration cannot progress simultaneously in the same PN, but, as alluded to above, we have not obtained evidence of interactions between age-related and ethanol-related dendritic degeneration in our model. It appears from our studies that some PN are more sensitive to ethanol, and therefore sustain more dendritic damage, than others (Pentney, 1995; Dlugos and Pentney, 1997
). Nonetheless, the distribution of PN that have been altered morphometrically by ethanol-induced mechanisms does not appear to be determined by their level of sensitivity to ethanol.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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