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Effects of chronic ethanol consumption on SER of Purkinje neurons in old F344 rats
Alcohol 20 (2000) 125–132
Effects of chronic ethanol consumption on SER of Purkinje neurons in old F344 rats Cynthia A. Dlugos*, Roberta J. Pentney Department of Anatomy and Cell Biology, 317 Farber Hall, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214-3000, USA Received 14 June, 1999; received in revised form 23 August, 1999; accepted 24 August, 1999
Abstract 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. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ethanol-related dilatation; Purkinje neurons; Smooth endoplasmic reticulum; Calcium homeostasis; Aged F344 rats; Electron microscopy
1. Introduction The neurotoxic effects of alcohol consumption on dendritic morphology and synaptic input in human alcoholism are well recognized (Jones, 1988) and result in impairments in body temperature regulation, short-term memory, and coordination (Oscar-Berman et al., 1997). Ethanol-related alterations in dendritic morphology have been analyzed with the Golgi-Cox method during development (Abel et al., 1983; Davies & Smith, 1981; Shetty et al., 1993), young adulthood (Berman et al., 1996; Kozlowski et al., 1997; Tavares et al., 1983), and old age (Pentney, 1982; Pentney & Quackenbush, 1990, 1991; Pentney, 1995; Pentney & Dlugos, in press), and the information gained from these morphometric studies presents further proof that ethanol adversely affects dendritic area, length, and spine number. Although convincing evidence of the effect of ethanol on dendrites exists, little is known about the mechanism(s) through which ethanol affects dendritic structure and function. The Purkinje neuron (PN) of the cerebellar cortex is an excellent model for studying ethanol induced dendritic regression because it has the most extensive dendritic arbor of * Corresponding author. Tel.: 716-829-2545; Fax: 716-829-2915. E-mail address: [email protected] (C.A. Dlugos)
any neuron and alcoholism severely affects the human cerebellum (Jones, 1988). The PN dendritic arbor lies within the molecular layer of the cerebellar cortex and has been the target in this laboratory of multiple studies concerning the effects of chronic alcohol consumption on old F344 rats (Pentney, 1982; Pentney & Quackenbush, 1990, 1991; Pentney, 1995; Pentney & Dlugos, in press; Dlugos & Pentney, 1997; Tabbaa et al., 1999). A major and repetitive result of these ongoing investigations is that chronic ethanol intake results in the lengthening of PN terminal dendritic segments (Pentney & Quackenbush, 1990, 1991; Pentney & Dlugos, in press) without a subsequent increase in dendritic path lengths (Pentney & Quackenbush, 1990, 1991). This apparent conflict can best be resolved by a model of ethanol-related dendritic regression (Pentney, 1995) in which deletion of one member of a pair of terminal dendritic segments occurs at a branching point from the parent dendritic segment. Quantitation of segment and path lengths in PN of ethanol-fed rats support this model (Pentney, 1995) as does the ethanol-related decrease in synaptic input to the PN (Dlugos & Pentney, 1997). The latter evidence is indirect and could be attributed to a decline in afferent input to the PN; however, a recent study from this laboratory showed that chronic ethanol treatment does not alter this input (Tabbaa et al., 1999). Another unique attribute of the PN is an extensive, con-
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tinuous, and three- dimensional network of dendritic smooth endoplasmic reticulum (SER) consisting of an interconnected network of endomembranes of different conformations including hypolemmal cisternae adjacent to the neurolemma, cisterns of the dendritic shaft present throughout the shaft in both lamellar and tubular forms, and single or lamellar profiles within the spines (Martone et al., 1993). The extensive SER in PN stores intracellular calcium and functions in the complex calcium signalling inherent to this neuron due to its extensive dendritic surface and synaptic inputs (Eilers et al., 1996). The presence of SER in PN dendritic spines has recently been correlated with the role of the cerebellum in motor coordination since mutant rats who lack SER within PN spines but are otherwise normal are also ataxic (Dekker-Ohno et al., 1996). Qualitative studies have reported that SER profiles in PN of ethanol-fed rats were dilated (Lewandowska et al., 1994), but this has not been confirmed quantitatively. In our own preparations, SER profiles within PN dendrites of 22-month old F344 rats appeared to be swollen following 40 weeks of ethanol treatment (unpublished data). The purpose of the present study was to (1) confirm the presence of ethanolrelated dilatation of the SER in PN dendrites, (2) determine whether ethanol-related dilatation of the SER in the PN dendrite occurred similarly in the dendritic shafts and in the dendritic spines, and (3) determine whether dilatation of the SER persisted with recovery. The discussion arising from the data presented here provides the basis for future experiments aimed at elucidation of a mechanism through which chronic ethanol consumption contributes to dendritic regression and the role of SER dilatation in that regression.
2. Methods 2.1. Animal model Forty-five male F344 rats (NIA, Harlan Sprague-Dawley, Inc.) were used in this study. Rats were obtained at 12 months of age and randomly assigned to one of three treatment groups (n ⫽ 15/group). Rats in the ethanol group received a liquid diet (Bio-Serv, Frenchtown, NJ) in which 35% of the dietary calories were derived from ethanol. Rats in the pair-fed control group received the Bio-Serv control liquid diet, which was made isocaloric with the ethanol diet by substitution of maltose/dextrins for ethanol. Rats in the chow-fed control group received standard laboratory rat chow (RMH 1000 diet, Agway Inc., Syracuse, NY) and water ad libitum. The chow control rats served as controls for the effects of the liquid diet. Pair-fed and ethanol-fed rats were matched by weight. The intake of a pair-fed rat was determined daily by the intake of its ethanol partner to insure that the pair-fed and ethanol-fed rats received the same volumes of food during the treatment period. At the termination of the 40-week treatment period, 10 22-month-old rats from each treatment group were euthanized. There was no recovery period from ethanol in this
group. The remaining 15 rats were maintained in the laboratory animal facility for an additional 20 weeks. At the beginning of the recovery period, the ethanol-fed and pair-fed groups were weaned from ethanol and control diets and returned to standard laboratory rat chow (RMH 1000 diet). The chow controls were retained as age-matched controls. The recovered rats were 27 months old when euthanized at the end of the 20-week recovery period. All dietary treatments, animal care and use, and euthanasia procedures were approved by the IACUC committee of the State University of New York at Buffalo. 2.2. Blood alcohol concentrations Blood alcohol levels (BAL) were monitored in randomly selected ethanol-fed rats after 3 days (n ⫽ 8), 12 weeks (n ⫽ 3), and 33 weeks of treatment (n ⫽ 3). Blood samples were collected for enzymatic measurements (Sigma Diagnostic Kit #322A) from the tail veins 23 h after fresh diet had been placed in the cages. The mean circulating levels of ethanol at these intervals were 66 ⫾ 43 mg/dl, 91 ⫾ 26 mg/dl, and 56 ⫾ 43 mg/dl, respectively. Measurements presented here are not considered to be the peak circulating levels because the interval between ethanol intake and the collection of blood samples is not known. 2.3. Tissue preparation Rats were anesthetized with sodium pentobarbitol (i.p., 65 mg/kg). Tissue perfusion was performed through the aorta with 40 ml of heparinized physiological saline (50 units heparin/ml) followed by 400 ml of 1% paraformaldehyde, 1% glutaraldehyde solution dissolved in 0.1 M phosphate buffer (pH ⫽ 7.4). The brains were removed and coded to prevent investigator bias. Each brain was weighed, the cerebellum was separated from the cerebrum, and the cerebellar vermis was separated from the cerebellar hemispheres. The vermis was hemisected, and the posterior lobe was separated from the anterior lobe. The posterior lobe in each half of the vermis was divided into three subunits containing (1) lobules VI and VII, (2) lobule VIII, and (3) lobule IX. Lobule X was excluded from the study. The remaining subunits were washed in buffer, postfixed in 1% osmium tetroxide, dehydrated, oriented in flat embedding molds for parasagittal sectioning, and embedded in an Araldite-Epon 812 mixture (Electron Microscopy Sciences, Fort Washington, PA). Tissue blocks were sectioned at 65 nm and section thickness was confirmed with the fold method of Small (Small, 1968; De Groot, 1988). Sections were collected onto Formvar coated, single slot grids, and stained with uranyl acetate and lead citrate. Sections were viewed with a JEOL 100CX electron microscope at 14000⫻. 2.4. Quantitative procedures Montages of the molecular layer were constructed for measurements. Each montage represented a complete parasag-
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ittal strip of molecular layer which consisted of a series of overlapping fields that were photographed at ⫻14,000 and printed at a final magnification of ⫻37,500. Each montage extended from the pial surface to the PN layer. Two montages representing different lobular subunits within the posterior lobe were constructed per rat. Each montage consisted of over 30 photographic prints and represented approximately 750 m2 of the molecular layer. The minimun and maximum diameters of SER profiles were measured in the dendritic shafts and spines. In the shafts, hypolemmal, lamellar, and tubular conformations were measured. Measurements were made by superimposing a 16 cm ⫻ 16 cm square counting frame, representing 18.27 m2, in a sequential linear pattern from the pial surface to the PN layer. SER profiles within the counting frame were temporarily marked and standard rules for exclusion and inclusion of structures were followed (Gundersen, 1977). Maximum and minimum diameters of SER profiles in shafts and spines were determined with a digitizing tablet. The mean number of SER profiles measured in each rat was 55 in dendritic shafts and 36 in dendritic spines. A mean was determined for measurements of the maximum and minimum diameters in each montage, and results from two montages were averaged to determine mean maximum and minimum diameters of SER profiles in dendritic shafts and spines of each rat. Group means were determined for chow-fed, pair-fed, and ethanol-fed rats in the nonrecovered 22-month-old group and for the recovered 27-month-old group.
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2.5. Statistical analysis Coefficients of error and the means and standard deviations were determined for all SER measurements from each montage and treatment group, respectively. Main effects of treatment on the diameters of SER profiles in the nonrecovered 22-month-old and recovered 27-month-old rats were tested with one-way ANOVA, and the Duncan range test was used to localize significance. Two-way ANOVA (general factorial model) was used to analyze the main effects of treatment and length of treatment on measurements of diameters of SER profiles and was followed by a posthoc Scheffé test for localization of significance. The independent variable, length of treatment, divided the rats into two groups; the 30 nonrecovered rats (22 months of age) and the 15 recovered rats (27 months of age). An alpha level of less than 0.05 was accepted as significant in all analyses and the SPSS/PC⫹ program was used throughout. 3. Results 3.1. Qualtitative observations Purkinje cell dendrites were easily recognized within montages and were found to contain abundant mitochondria, microtubules, and SER. Several conformations of SER profiles were present within the dendritic shaft including hypolemmal cisternae adjacent to the plasma membrane, and lamellar and tubular cisternae within the shafts (Fig. 1). SER within the shafts appeared singly or in clusters and
Fig. 1. Various conformations of SER profiles in chow-fed nonrecovered rats. (A) Dendrite of a Purkinje neuron (PN) with visible SER profiles (dsh, dendritic shaft). (B) Hypolemmal cisternae of SER profiles (arrow). (C) Tubular conformations of SER profiles (arrowheads) in the dendritic shaft of PN. (D) Small PN dendritic shaft with associated spines. Arrowhead in inset shows a SER profile in a dendritic spine (dsp, dendritic spine).
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Fig. 2. Dilatation of SER profiles in nonrecovered ethanol-fed rats. (A) Dilatation of lamellar SER profiles in the PN dendritic shaft with asterisks in the cisterns (dsh, dendritic shaft). (B) Dilatation of an SER profile at the branch point of a dendritic shaft is indicated by an asterisk (bd, branching dendrites). (C) Dilatation of an SER cistern indicated by the asterisk. (D) Dilated SER profiles within dendritic spines indicated by arrowheads.
some interconnections between adjacent SER profiles were observed. PN dendritic spines were identified as rounded knobs containing cisternae or tubular elements of the SER (Harvey & Napper, 1991). Spinous processes usually appeared in cross-section and the slender stalk that connected the spine with the shaft was infrequently observed. SER profiles within the spines were generally single and rounded (Fig. 1), but longitudinally orientated stacks or lamellae were occasionally seen. In the ethanol-fed group following 40 weeks of treatment, SER profiles in the dendritic shafts and spines appeared dilated. Dilatation was particularly evident within the dendritic shafts in lamellar forms of SER profiles close to the neurolemma and at branch points. Within the spines, rounded or oval SER profiles appeared dilated (Fig. 2).
shafts, the maximum diameter of SER profiles [F(2, 27) ⫽ 5.181, p ⫽ 0.012] and the minimum diameter of SER profiles [F(2, 27) ⫽ 16.767, p ⬍ 0.001] were increased in the ethanol-fed rats compared to both control groups. In dendritic spines, the maximum diameter [F(2, 27) ⫽ 6.855, p ⫽ 0.004] and the minimum diameter [F(2, 27) ⫽ 5.997, p ⫽ 0.007] of SER profiles were also increased with ethanol consumption. These effects were localized in spines of the ethanol-fed rats compared to both control groups for the maximum diameter of SER profiles. Ethanol-related dilatation in the minimum diameter of SER profiles in spines was localized in the ethanol-fed group compared solely to the chow-fed group. 3.4. SER measurements in recovered rats
The maximum coefficient of error from single montages in all treatment groups and for all measurements was 0.10 and averages were 0.10, 0.07, 0.08, and 0.06 for the maximum and minimum diameter of dendritic shafts and the maximum and minimum diameter of the dendritic spines, respectively.
Following a 20-week recovery period, measurements of SER profile diameter in the 27 month old recovered rats were not significantly different from one another as indicated by analysis of the maximum [F(2, 12) ⫽ 0.855, p ⫽ 0.450] and minimum [F(2, 12) ⫽ 0.184, p ⫽ 0.834] diameters in the shafts and the maximum [F(2, 12) ⫽ 1.129, p ⫽ 0.355] and minimum [F(2, 12) ⫽ 0.767, p ⫽ 0.486] diameters in the spines (Fig. 4).
3.3. SER measurements for nonrecovered rats
3.5. Effects of treatment and length of treatment
Dilatation of SER profiles following chronic ethanol treatment was present in dendritic shafts and spines in the nonrecovered 22-month-old rats (Fig. 3.). In dendritic
Two-way ANOVA in which treatment and length of treatment were independent variables showed that diameters of SER profiles were significantly increased in the 15
3.2. Measurements of diameters of SER profiles
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Fig. 3. Graphic representation of the mean diameters (⫾ SD) of SER profiles within the PN dendritic shafts and spines of the chow-fed, pair-fed, and ethanol-fed nonrecovered rats (n ⫽ 30 ). *p ⬍ 0.05 compared to chowfed and pair-fed controls. ⫹p ⬍ 0.05 compared to chow-fed controls.
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Fig. 4. Graphic representation of the mean diameters (⫾ SD) of SER profiles within the PN dendritic shafts and spines of the chow-fed, pair-fed and ethanol-fed recovered rats.
4. Discussion recovered rats compared to the 30 nonrecovered rats. Measurements of SER profiles from the chow-fed, pair-fed, and ethanol-fed groups were pooled by length of treatment in this analysis as a nonrecovered group (22 months of age) and a recovered group (27 months of age). The maximum diameter of SER profiles in PN dendritic shafts [F(1, 44) ⫽ 6.201, p ⫽ 0.017] and the maximum diameter [F(1, 44) ⫽ 27.708, p ⬍ 0.001] and minimum diameter [F(1, 44) ⫽ 47.077, p ⬍ 0.001] of SER profiles in the spines was significantly increased with length of treatment in the recovery group compared to the non-recovered group. The minimum diameter of SER profiles in dendritic shafts approached but did not reach significance [F(1, 44) ⫽ 3.928, p ⫽ 0.055]. There were significant interactions in the two-way analysis between treatment and length of treatment in the maximum SER profile diameter of dendritic shafts [F(2, 39) ⫽ 3.743, p ⫽ 0.033] and in the minimum SER profile diameter of dendritic shafts [F(2, 39) ⫽ 4.638, p ⫽ 0.016]. These interactions were due to increased dilatation of the SER in the dendritic shaft in the control groups during the recovery period compared to a decrease in the size of the SER within the dendritic shafts in the ethanol-fed rats during recovery (Figs. 5 and 6).
The results of this study demonstrate morphometric effects of chronic ethanol consumption on SER profiles in dendritic shafts and dendritic spines of PN in old F344 rats. It was shown that chronic ethanol consumption caused dilatation of SER in PN dendritic shafts and that, following a 20-week recovery period , measurements of SER profiles did not differ between treatment groups. The present data agree with qualitative reports of SER dilatation in the dendritic shafts of PN in ethanol-fed rats (Dekker-Ohno et al., 1996) and in the sarcoplasmic reticulum of patients with alcoholic neuropathy (Mair & Tomé, 1972). Dilatation of the SER is most probably a general cellular response to toxicity because it is present in PN (Cavanagh et al., 1998), other neurons (Petrov & Lolova, 1977), and nonneuronal cells (Rodriguez-Rodriguez & Maldonado, 1996) following exposure to other toxins as well as to ethanol. Although little is known presently about the causal relationship between SER dilatation and neuronal responses to toxins, swelling of the SER precedes dendritic regression in PN (Cavanagh et al., 1998) and in other types of neurons (Petrov & Lolova, 1977). Furthermore, the ethanol-related dilatation of SER reported here occurs in a neuronal system in which we have repeatedly observed ethanol-related dendritic regression (Pentney, 1982; Pentney, 1986; Pentney & Quackenbush,
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Fig. 6. Graphic representation of the significant interaction in the minimum diameter of SER profiles within PN dendritic shafts between treatment and length of treatment during the recovery period. Fig. 5. Graphic representation of the significant interaction in the maximum diameter of SER within PN dendritic shafts between treatment and length of treatment during the recovery period.
1990, 1991; Pentney, 1995; Pentney & Dlugos, in press; Dlugos & Pentney, 1997). A repeated result of investigations in this laboratory is that chronic ethanol consumption causes a significant lengthening of dendritic terminals (Pentney & Quackenbush, 1990, 1991; Pentney & Dlugos, in press) in the absence of overall increases in dendritic path length (Pentney & Quackenbush, 1990, 1991; Pentney, 1995). This apparent dichotomy has been explained and supported by a model of ethanol-related dendritic regression in which one member of a pair of dendritic terminals is deleted at the branch point resulting in a surviving terminal with a longer segment length and an unaltered path length. This deletion would adversely affect the synaptic function of PN since deletion of dendritic terminals would be accompanied by the loss of spines present on those terminal branches. Indirect support for the ethanol-related loss of spines on PN is provided by data from this laboratory demonstrating that the number of synapses/PN decreases with long-term ethanol consumption (Dlugos & Pentney, 1997). The present data also demonstrate an ethanol-related dilatation of spinous profiles accompanying ethanol-related dilatation of SER in dendritic shafts. Ethanol-related dilatation of spinous SER most prob-
ably occurs in spines on all terminal segments that are not deleted and on internal dendritic segments as well. The impact of ethanol-related dilatation of spinous SER is unknown; however, a proportional relationship exists between the size of a spine and its component parts (Harris & Stevens, 1988), and disturbance of this relationship could adversely affect synaptic input to the PN. Following a 20-week recovery period, there was no difference in measurements of SER profiles in dendritic shafts and spines within the chow-fed, pair-fed, and ethanol-fed treatment groups. Increased dilatation of SER profiles in dendritic shafts and spines of PN occurred in the recovered rats compared to the nonrecovered group, however, when data from the chow-fed, pair-fed, and ethanol-fed groups were pooled by length of treatment. These differences were attributed to increases within the chow-fed and pair-fed groups since measurements of SER profiles in dendritic shafts of the ethanol-fed rats decreased with recovery. This bidirectional shift in SER measurements in PN dendritic shafts during the recovery period accounted for significant interactions between treatment and length of treatment. Decreases in measured SER profile diameters in the shafts of the ethanol-fed group indicated that some recovery from the effects of ethanol had occurred. Increases in SER dilatation in shafts during the extensive recovery period was highest in the chow-fed group and was linked to the normal aging process since this group alone underwent normal aging
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without a liquid dietary treatment. Age-related changes in the dendritic arbors of PN have been reported previously in this rat strain (Dlugos & Pentney, 1994, 1997; Glick & Bonderaff, 1979; Pentney, 1986). The fact that measured diameters of SER profiles within the spines increased during recovery in all treatment groups does not necessarily negate the presence of some recovery from the effects of ethanol in spinous SER of our ethanolfed recovered rats. Dilatation of SER profiles in the spines during recovery was, in fact, lowest in the ethanol group when the percent of change in measured spinous SER diameters in nonrecovered and recovered rats in each of the three treatment groups were combined and compared (47% in chow-fed rats, 20%, in pair-fed rats, and 14% in ethanol-fed rats). An explanation for this smaller increase in SER dilatation within the spines of our ethanol-fed rats relates to two observations made on two separate groups of aged rats following similar long-term ethanol treatments. These observations include the lengthening of surviving dendritic terminal segments after deletion of dendritic terminals reported previously (Pentney & Quackenbush, 1990, 1991; Pentney, 1995) and the dilatation of SER in spines of intact PN dendrites reported here. These data suggest that two populations of spines were present on PN dendrites of recovered rats including new spines with normal SER formed on regenerated terminal segments and older spines with dilated SER found on internal and external branches that had not regressed with ethanol treatment. Mean measurements of SER profile diameter would increase during recovery because the older subpopulation of spines with swollen SER would be more frequent than new spines on regenerated terminals. In fact, dilatation of SER profiles appears to be a more widespread event than terminal segment deletion which has measurable effects on only ⵑ30% of the PN (Pentney, 1995). In the chow-fed rats, the dramatic increase in dilatation of the SER in spines during recovery may be a predictor of age-related spine loss since decreasing spine density has been reported in older rats (Pentney, 1986). The occurrence of the SER dilatation reported here justifies an investigation of the function of the SER in the PN. The fact that SER within the PN has been shown to respond to calcium deficits with severe morphological alterations (Garthwaite et al., 1992) suggests that the SER plays an important role in calcium metabolism. Calcium is essential to neuronal metabolism since it functions as a second messenger in many cellular processes including development, excitability, learning and memory (Koizumi et al., 1999). Continuously high levels of cytosolic calcium , however, are toxic to the neuron and, in the PN, an important function of the extensive SER network is to sequester and store intracellular calcium (Villa et al., 1991; Sacchetto et al., 1997). Calcium concentration within the SER is dependent not only on cytosolic calcium but also on membrane components such as receptors and calcium ATPase pumps (Villa et al., 1991; Sacchetto et al., 1997). Ethanol-related alterations in cytosolic calcium levels and the membrane components
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which control these levels could contribute to the dilatation of SER reported here as well as the deletion of PN dendritic terminals we have reported previously (Pentney & Quackenbush 1990, 1991; Pentney, 1995). A further examination of the correlation between these events will serve as the basis for future experiments. The results of this study demonstrate that long-term ethanol consumption caused dilatation of SER in Purkinje neuron dendritic arborizations in old rats. This dilatation was widespread in its distribution occurring within the dendritic shafts and spines throughout the PN dendritic arbor. The ethanol-related dilation of SER was also reversible following a recovery period within the dendritic shafts.
Acknowledgments We gratefully acknowledge the technical contributions of Ann Marie Hey and William Pentney . This work was supported by a grant from NIAAA/NIH (AA05592) and by Research Development/BRSG funds from the School of Medicine and Biomedical Sciences, University of Buffalo.
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ethanol treatment during aging on dendritic plasticity in Fischer 344 rats. Alcohol Clin Exp Res 15(6),1024–1030. Pentney, R. J. (1995). Measurements of dendritic path lengths provide evidence that ethanol-induced lengthening of terminal dendritic segments may result from dendritic regression. Alcohol Alcohol 30(1), 87–96. Pentney, R. J., & Dlugos, C. A. (in press) Cerebellar Purkinje neurons with altered terminal dendritic segments are present in all lobules of the cerebellar vermis of aging, ethanol-treated F344 rats. Alcohol Alcohol. Petrov, I., & Lolova, I (1977). Electronmicroscopic investigation of rat substantia nigra after chronic amphetamine treatment 1. Changes in neurones. Acta Physiolog Pharmacol Bulgar 3(3), 24–32. Rodriguez-Rodriguez, F. A., & Maldonado, C. (1996). Brevetoxin-3 (PbTx-3) on mouse liver slices: a histological study. PRHSJ 15(4), 261–267. Sacchetto, R., Cliffer, K. D., Podini, P., Villa, A., Burgess N., Volpe C., & Volpe P. (1997). Intracellular Ca2⫹ stores in chick cerebellum Purkinje neurons: ontogenetic and functional studies. Am J Physiol 269(5):219–227. Shetty, A. K., Burrows, R. C., & Phillips, D. E. (1993). Alterations in neuronal development in the substantia nigra pars compacta following in utero ethanol exposure: immunohistochemical and Golgi studies. Neurosci 52(2), 311–322. Small, J. V. (1968). Measurement of section thickness. In: D. S. Bocciarelli (Ed.), Abstracts of Fourth Regional Conference on EM. Vol. 1 (pp. 609–610). Rome: Tipografia, Poliglotta Vatiana. Tabbaa, S., Dlugos, C., & Pentney, R. (1999). The number of granule cells and spine density on Purkinje cells in aged, ethanol-fed rats. Alcohol 17(2), 253–260. Tavares, M. A., Paula-Barbosa, M. M., & Gray, E. G. (1983). A morphometric Golgi analysis of the Purkinje cell dendritic tree after long-term, alcohol consumption in the adult rat. J Neurocytol 12(6), 939–948. Villa, A., Podini, P., Clegg, D., Pozzan T., & Meldolesi, J. (1991). Intracellular Ca2 stores in chicken Purkinje neurons: differential distribution of the low affinity-high capacity Ca2⫹ binding protein, calsequestrin, of Ca2⫹ ATPase and of the ER lumenal protein, Bip. J Cell Biol 113(4), 779–791.
Effects of chronic ethanol consumption on SER of Purkinje neurons in old F344 rats Cynthia A. Dlugos*, Roberta J. Pentney Department of Anatomy and Cell Biology, 317 Farber Hall, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, Buffalo, NY 14214-3000, USA Received 14 June, 1999; received in revised form 23 August, 1999; accepted 24 August, 1999
Abstract 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. © 2000 Elsevier Science Inc. All rights reserved. Keywords: Ethanol-related dilatation; Purkinje neurons; Smooth endoplasmic reticulum; Calcium homeostasis; Aged F344 rats; Electron microscopy
1. Introduction The neurotoxic effects of alcohol consumption on dendritic morphology and synaptic input in human alcoholism are well recognized (Jones, 1988) and result in impairments in body temperature regulation, short-term memory, and coordination (Oscar-Berman et al., 1997). Ethanol-related alterations in dendritic morphology have been analyzed with the Golgi-Cox method during development (Abel et al., 1983; Davies & Smith, 1981; Shetty et al., 1993), young adulthood (Berman et al., 1996; Kozlowski et al., 1997; Tavares et al., 1983), and old age (Pentney, 1982; Pentney & Quackenbush, 1990, 1991; Pentney, 1995; Pentney & Dlugos, in press), and the information gained from these morphometric studies presents further proof that ethanol adversely affects dendritic area, length, and spine number. Although convincing evidence of the effect of ethanol on dendrites exists, little is known about the mechanism(s) through which ethanol affects dendritic structure and function. The Purkinje neuron (PN) of the cerebellar cortex is an excellent model for studying ethanol induced dendritic regression because it has the most extensive dendritic arbor of * Corresponding author. Tel.: 716-829-2545; Fax: 716-829-2915. E-mail address: [email protected] (C.A. Dlugos)
any neuron and alcoholism severely affects the human cerebellum (Jones, 1988). The PN dendritic arbor lies within the molecular layer of the cerebellar cortex and has been the target in this laboratory of multiple studies concerning the effects of chronic alcohol consumption on old F344 rats (Pentney, 1982; Pentney & Quackenbush, 1990, 1991; Pentney, 1995; Pentney & Dlugos, in press; Dlugos & Pentney, 1997; Tabbaa et al., 1999). A major and repetitive result of these ongoing investigations is that chronic ethanol intake results in the lengthening of PN terminal dendritic segments (Pentney & Quackenbush, 1990, 1991; Pentney & Dlugos, in press) without a subsequent increase in dendritic path lengths (Pentney & Quackenbush, 1990, 1991). This apparent conflict can best be resolved by a model of ethanol-related dendritic regression (Pentney, 1995) in which deletion of one member of a pair of terminal dendritic segments occurs at a branching point from the parent dendritic segment. Quantitation of segment and path lengths in PN of ethanol-fed rats support this model (Pentney, 1995) as does the ethanol-related decrease in synaptic input to the PN (Dlugos & Pentney, 1997). The latter evidence is indirect and could be attributed to a decline in afferent input to the PN; however, a recent study from this laboratory showed that chronic ethanol treatment does not alter this input (Tabbaa et al., 1999). Another unique attribute of the PN is an extensive, con-
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tinuous, and three- dimensional network of dendritic smooth endoplasmic reticulum (SER) consisting of an interconnected network of endomembranes of different conformations including hypolemmal cisternae adjacent to the neurolemma, cisterns of the dendritic shaft present throughout the shaft in both lamellar and tubular forms, and single or lamellar profiles within the spines (Martone et al., 1993). The extensive SER in PN stores intracellular calcium and functions in the complex calcium signalling inherent to this neuron due to its extensive dendritic surface and synaptic inputs (Eilers et al., 1996). The presence of SER in PN dendritic spines has recently been correlated with the role of the cerebellum in motor coordination since mutant rats who lack SER within PN spines but are otherwise normal are also ataxic (Dekker-Ohno et al., 1996). Qualitative studies have reported that SER profiles in PN of ethanol-fed rats were dilated (Lewandowska et al., 1994), but this has not been confirmed quantitatively. In our own preparations, SER profiles within PN dendrites of 22-month old F344 rats appeared to be swollen following 40 weeks of ethanol treatment (unpublished data). The purpose of the present study was to (1) confirm the presence of ethanolrelated dilatation of the SER in PN dendrites, (2) determine whether ethanol-related dilatation of the SER in the PN dendrite occurred similarly in the dendritic shafts and in the dendritic spines, and (3) determine whether dilatation of the SER persisted with recovery. The discussion arising from the data presented here provides the basis for future experiments aimed at elucidation of a mechanism through which chronic ethanol consumption contributes to dendritic regression and the role of SER dilatation in that regression.
2. Methods 2.1. Animal model Forty-five male F344 rats (NIA, Harlan Sprague-Dawley, Inc.) were used in this study. Rats were obtained at 12 months of age and randomly assigned to one of three treatment groups (n ⫽ 15/group). Rats in the ethanol group received a liquid diet (Bio-Serv, Frenchtown, NJ) in which 35% of the dietary calories were derived from ethanol. Rats in the pair-fed control group received the Bio-Serv control liquid diet, which was made isocaloric with the ethanol diet by substitution of maltose/dextrins for ethanol. Rats in the chow-fed control group received standard laboratory rat chow (RMH 1000 diet, Agway Inc., Syracuse, NY) and water ad libitum. The chow control rats served as controls for the effects of the liquid diet. Pair-fed and ethanol-fed rats were matched by weight. The intake of a pair-fed rat was determined daily by the intake of its ethanol partner to insure that the pair-fed and ethanol-fed rats received the same volumes of food during the treatment period. At the termination of the 40-week treatment period, 10 22-month-old rats from each treatment group were euthanized. There was no recovery period from ethanol in this
group. The remaining 15 rats were maintained in the laboratory animal facility for an additional 20 weeks. At the beginning of the recovery period, the ethanol-fed and pair-fed groups were weaned from ethanol and control diets and returned to standard laboratory rat chow (RMH 1000 diet). The chow controls were retained as age-matched controls. The recovered rats were 27 months old when euthanized at the end of the 20-week recovery period. All dietary treatments, animal care and use, and euthanasia procedures were approved by the IACUC committee of the State University of New York at Buffalo. 2.2. Blood alcohol concentrations Blood alcohol levels (BAL) were monitored in randomly selected ethanol-fed rats after 3 days (n ⫽ 8), 12 weeks (n ⫽ 3), and 33 weeks of treatment (n ⫽ 3). Blood samples were collected for enzymatic measurements (Sigma Diagnostic Kit #322A) from the tail veins 23 h after fresh diet had been placed in the cages. The mean circulating levels of ethanol at these intervals were 66 ⫾ 43 mg/dl, 91 ⫾ 26 mg/dl, and 56 ⫾ 43 mg/dl, respectively. Measurements presented here are not considered to be the peak circulating levels because the interval between ethanol intake and the collection of blood samples is not known. 2.3. Tissue preparation Rats were anesthetized with sodium pentobarbitol (i.p., 65 mg/kg). Tissue perfusion was performed through the aorta with 40 ml of heparinized physiological saline (50 units heparin/ml) followed by 400 ml of 1% paraformaldehyde, 1% glutaraldehyde solution dissolved in 0.1 M phosphate buffer (pH ⫽ 7.4). The brains were removed and coded to prevent investigator bias. Each brain was weighed, the cerebellum was separated from the cerebrum, and the cerebellar vermis was separated from the cerebellar hemispheres. The vermis was hemisected, and the posterior lobe was separated from the anterior lobe. The posterior lobe in each half of the vermis was divided into three subunits containing (1) lobules VI and VII, (2) lobule VIII, and (3) lobule IX. Lobule X was excluded from the study. The remaining subunits were washed in buffer, postfixed in 1% osmium tetroxide, dehydrated, oriented in flat embedding molds for parasagittal sectioning, and embedded in an Araldite-Epon 812 mixture (Electron Microscopy Sciences, Fort Washington, PA). Tissue blocks were sectioned at 65 nm and section thickness was confirmed with the fold method of Small (Small, 1968; De Groot, 1988). Sections were collected onto Formvar coated, single slot grids, and stained with uranyl acetate and lead citrate. Sections were viewed with a JEOL 100CX electron microscope at 14000⫻. 2.4. Quantitative procedures Montages of the molecular layer were constructed for measurements. Each montage represented a complete parasag-
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ittal strip of molecular layer which consisted of a series of overlapping fields that were photographed at ⫻14,000 and printed at a final magnification of ⫻37,500. Each montage extended from the pial surface to the PN layer. Two montages representing different lobular subunits within the posterior lobe were constructed per rat. Each montage consisted of over 30 photographic prints and represented approximately 750 m2 of the molecular layer. The minimun and maximum diameters of SER profiles were measured in the dendritic shafts and spines. In the shafts, hypolemmal, lamellar, and tubular conformations were measured. Measurements were made by superimposing a 16 cm ⫻ 16 cm square counting frame, representing 18.27 m2, in a sequential linear pattern from the pial surface to the PN layer. SER profiles within the counting frame were temporarily marked and standard rules for exclusion and inclusion of structures were followed (Gundersen, 1977). Maximum and minimum diameters of SER profiles in shafts and spines were determined with a digitizing tablet. The mean number of SER profiles measured in each rat was 55 in dendritic shafts and 36 in dendritic spines. A mean was determined for measurements of the maximum and minimum diameters in each montage, and results from two montages were averaged to determine mean maximum and minimum diameters of SER profiles in dendritic shafts and spines of each rat. Group means were determined for chow-fed, pair-fed, and ethanol-fed rats in the nonrecovered 22-month-old group and for the recovered 27-month-old group.
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2.5. Statistical analysis Coefficients of error and the means and standard deviations were determined for all SER measurements from each montage and treatment group, respectively. Main effects of treatment on the diameters of SER profiles in the nonrecovered 22-month-old and recovered 27-month-old rats were tested with one-way ANOVA, and the Duncan range test was used to localize significance. Two-way ANOVA (general factorial model) was used to analyze the main effects of treatment and length of treatment on measurements of diameters of SER profiles and was followed by a posthoc Scheffé test for localization of significance. The independent variable, length of treatment, divided the rats into two groups; the 30 nonrecovered rats (22 months of age) and the 15 recovered rats (27 months of age). An alpha level of less than 0.05 was accepted as significant in all analyses and the SPSS/PC⫹ program was used throughout. 3. Results 3.1. Qualtitative observations Purkinje cell dendrites were easily recognized within montages and were found to contain abundant mitochondria, microtubules, and SER. Several conformations of SER profiles were present within the dendritic shaft including hypolemmal cisternae adjacent to the plasma membrane, and lamellar and tubular cisternae within the shafts (Fig. 1). SER within the shafts appeared singly or in clusters and
Fig. 1. Various conformations of SER profiles in chow-fed nonrecovered rats. (A) Dendrite of a Purkinje neuron (PN) with visible SER profiles (dsh, dendritic shaft). (B) Hypolemmal cisternae of SER profiles (arrow). (C) Tubular conformations of SER profiles (arrowheads) in the dendritic shaft of PN. (D) Small PN dendritic shaft with associated spines. Arrowhead in inset shows a SER profile in a dendritic spine (dsp, dendritic spine).
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Fig. 2. Dilatation of SER profiles in nonrecovered ethanol-fed rats. (A) Dilatation of lamellar SER profiles in the PN dendritic shaft with asterisks in the cisterns (dsh, dendritic shaft). (B) Dilatation of an SER profile at the branch point of a dendritic shaft is indicated by an asterisk (bd, branching dendrites). (C) Dilatation of an SER cistern indicated by the asterisk. (D) Dilated SER profiles within dendritic spines indicated by arrowheads.
some interconnections between adjacent SER profiles were observed. PN dendritic spines were identified as rounded knobs containing cisternae or tubular elements of the SER (Harvey & Napper, 1991). Spinous processes usually appeared in cross-section and the slender stalk that connected the spine with the shaft was infrequently observed. SER profiles within the spines were generally single and rounded (Fig. 1), but longitudinally orientated stacks or lamellae were occasionally seen. In the ethanol-fed group following 40 weeks of treatment, SER profiles in the dendritic shafts and spines appeared dilated. Dilatation was particularly evident within the dendritic shafts in lamellar forms of SER profiles close to the neurolemma and at branch points. Within the spines, rounded or oval SER profiles appeared dilated (Fig. 2).
shafts, the maximum diameter of SER profiles [F(2, 27) ⫽ 5.181, p ⫽ 0.012] and the minimum diameter of SER profiles [F(2, 27) ⫽ 16.767, p ⬍ 0.001] were increased in the ethanol-fed rats compared to both control groups. In dendritic spines, the maximum diameter [F(2, 27) ⫽ 6.855, p ⫽ 0.004] and the minimum diameter [F(2, 27) ⫽ 5.997, p ⫽ 0.007] of SER profiles were also increased with ethanol consumption. These effects were localized in spines of the ethanol-fed rats compared to both control groups for the maximum diameter of SER profiles. Ethanol-related dilatation in the minimum diameter of SER profiles in spines was localized in the ethanol-fed group compared solely to the chow-fed group. 3.4. SER measurements in recovered rats
The maximum coefficient of error from single montages in all treatment groups and for all measurements was 0.10 and averages were 0.10, 0.07, 0.08, and 0.06 for the maximum and minimum diameter of dendritic shafts and the maximum and minimum diameter of the dendritic spines, respectively.
Following a 20-week recovery period, measurements of SER profile diameter in the 27 month old recovered rats were not significantly different from one another as indicated by analysis of the maximum [F(2, 12) ⫽ 0.855, p ⫽ 0.450] and minimum [F(2, 12) ⫽ 0.184, p ⫽ 0.834] diameters in the shafts and the maximum [F(2, 12) ⫽ 1.129, p ⫽ 0.355] and minimum [F(2, 12) ⫽ 0.767, p ⫽ 0.486] diameters in the spines (Fig. 4).
3.3. SER measurements for nonrecovered rats
3.5. Effects of treatment and length of treatment
Dilatation of SER profiles following chronic ethanol treatment was present in dendritic shafts and spines in the nonrecovered 22-month-old rats (Fig. 3.). In dendritic
Two-way ANOVA in which treatment and length of treatment were independent variables showed that diameters of SER profiles were significantly increased in the 15
3.2. Measurements of diameters of SER profiles
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Fig. 3. Graphic representation of the mean diameters (⫾ SD) of SER profiles within the PN dendritic shafts and spines of the chow-fed, pair-fed, and ethanol-fed nonrecovered rats (n ⫽ 30 ). *p ⬍ 0.05 compared to chowfed and pair-fed controls. ⫹p ⬍ 0.05 compared to chow-fed controls.
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Fig. 4. Graphic representation of the mean diameters (⫾ SD) of SER profiles within the PN dendritic shafts and spines of the chow-fed, pair-fed and ethanol-fed recovered rats.
4. Discussion recovered rats compared to the 30 nonrecovered rats. Measurements of SER profiles from the chow-fed, pair-fed, and ethanol-fed groups were pooled by length of treatment in this analysis as a nonrecovered group (22 months of age) and a recovered group (27 months of age). The maximum diameter of SER profiles in PN dendritic shafts [F(1, 44) ⫽ 6.201, p ⫽ 0.017] and the maximum diameter [F(1, 44) ⫽ 27.708, p ⬍ 0.001] and minimum diameter [F(1, 44) ⫽ 47.077, p ⬍ 0.001] of SER profiles in the spines was significantly increased with length of treatment in the recovery group compared to the non-recovered group. The minimum diameter of SER profiles in dendritic shafts approached but did not reach significance [F(1, 44) ⫽ 3.928, p ⫽ 0.055]. There were significant interactions in the two-way analysis between treatment and length of treatment in the maximum SER profile diameter of dendritic shafts [F(2, 39) ⫽ 3.743, p ⫽ 0.033] and in the minimum SER profile diameter of dendritic shafts [F(2, 39) ⫽ 4.638, p ⫽ 0.016]. These interactions were due to increased dilatation of the SER in the dendritic shaft in the control groups during the recovery period compared to a decrease in the size of the SER within the dendritic shafts in the ethanol-fed rats during recovery (Figs. 5 and 6).
The results of this study demonstrate morphometric effects of chronic ethanol consumption on SER profiles in dendritic shafts and dendritic spines of PN in old F344 rats. It was shown that chronic ethanol consumption caused dilatation of SER in PN dendritic shafts and that, following a 20-week recovery period , measurements of SER profiles did not differ between treatment groups. The present data agree with qualitative reports of SER dilatation in the dendritic shafts of PN in ethanol-fed rats (Dekker-Ohno et al., 1996) and in the sarcoplasmic reticulum of patients with alcoholic neuropathy (Mair & Tomé, 1972). Dilatation of the SER is most probably a general cellular response to toxicity because it is present in PN (Cavanagh et al., 1998), other neurons (Petrov & Lolova, 1977), and nonneuronal cells (Rodriguez-Rodriguez & Maldonado, 1996) following exposure to other toxins as well as to ethanol. Although little is known presently about the causal relationship between SER dilatation and neuronal responses to toxins, swelling of the SER precedes dendritic regression in PN (Cavanagh et al., 1998) and in other types of neurons (Petrov & Lolova, 1977). Furthermore, the ethanol-related dilatation of SER reported here occurs in a neuronal system in which we have repeatedly observed ethanol-related dendritic regression (Pentney, 1982; Pentney, 1986; Pentney & Quackenbush,
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Fig. 6. Graphic representation of the significant interaction in the minimum diameter of SER profiles within PN dendritic shafts between treatment and length of treatment during the recovery period. Fig. 5. Graphic representation of the significant interaction in the maximum diameter of SER within PN dendritic shafts between treatment and length of treatment during the recovery period.
1990, 1991; Pentney, 1995; Pentney & Dlugos, in press; Dlugos & Pentney, 1997). A repeated result of investigations in this laboratory is that chronic ethanol consumption causes a significant lengthening of dendritic terminals (Pentney & Quackenbush, 1990, 1991; Pentney & Dlugos, in press) in the absence of overall increases in dendritic path length (Pentney & Quackenbush, 1990, 1991; Pentney, 1995). This apparent dichotomy has been explained and supported by a model of ethanol-related dendritic regression in which one member of a pair of dendritic terminals is deleted at the branch point resulting in a surviving terminal with a longer segment length and an unaltered path length. This deletion would adversely affect the synaptic function of PN since deletion of dendritic terminals would be accompanied by the loss of spines present on those terminal branches. Indirect support for the ethanol-related loss of spines on PN is provided by data from this laboratory demonstrating that the number of synapses/PN decreases with long-term ethanol consumption (Dlugos & Pentney, 1997). The present data also demonstrate an ethanol-related dilatation of spinous profiles accompanying ethanol-related dilatation of SER in dendritic shafts. Ethanol-related dilatation of spinous SER most prob-
ably occurs in spines on all terminal segments that are not deleted and on internal dendritic segments as well. The impact of ethanol-related dilatation of spinous SER is unknown; however, a proportional relationship exists between the size of a spine and its component parts (Harris & Stevens, 1988), and disturbance of this relationship could adversely affect synaptic input to the PN. Following a 20-week recovery period, there was no difference in measurements of SER profiles in dendritic shafts and spines within the chow-fed, pair-fed, and ethanol-fed treatment groups. Increased dilatation of SER profiles in dendritic shafts and spines of PN occurred in the recovered rats compared to the nonrecovered group, however, when data from the chow-fed, pair-fed, and ethanol-fed groups were pooled by length of treatment. These differences were attributed to increases within the chow-fed and pair-fed groups since measurements of SER profiles in dendritic shafts of the ethanol-fed rats decreased with recovery. This bidirectional shift in SER measurements in PN dendritic shafts during the recovery period accounted for significant interactions between treatment and length of treatment. Decreases in measured SER profile diameters in the shafts of the ethanol-fed group indicated that some recovery from the effects of ethanol had occurred. Increases in SER dilatation in shafts during the extensive recovery period was highest in the chow-fed group and was linked to the normal aging process since this group alone underwent normal aging
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without a liquid dietary treatment. Age-related changes in the dendritic arbors of PN have been reported previously in this rat strain (Dlugos & Pentney, 1994, 1997; Glick & Bonderaff, 1979; Pentney, 1986). The fact that measured diameters of SER profiles within the spines increased during recovery in all treatment groups does not necessarily negate the presence of some recovery from the effects of ethanol in spinous SER of our ethanolfed recovered rats. Dilatation of SER profiles in the spines during recovery was, in fact, lowest in the ethanol group when the percent of change in measured spinous SER diameters in nonrecovered and recovered rats in each of the three treatment groups were combined and compared (47% in chow-fed rats, 20%, in pair-fed rats, and 14% in ethanol-fed rats). An explanation for this smaller increase in SER dilatation within the spines of our ethanol-fed rats relates to two observations made on two separate groups of aged rats following similar long-term ethanol treatments. These observations include the lengthening of surviving dendritic terminal segments after deletion of dendritic terminals reported previously (Pentney & Quackenbush, 1990, 1991; Pentney, 1995) and the dilatation of SER in spines of intact PN dendrites reported here. These data suggest that two populations of spines were present on PN dendrites of recovered rats including new spines with normal SER formed on regenerated terminal segments and older spines with dilated SER found on internal and external branches that had not regressed with ethanol treatment. Mean measurements of SER profile diameter would increase during recovery because the older subpopulation of spines with swollen SER would be more frequent than new spines on regenerated terminals. In fact, dilatation of SER profiles appears to be a more widespread event than terminal segment deletion which has measurable effects on only ⵑ30% of the PN (Pentney, 1995). In the chow-fed rats, the dramatic increase in dilatation of the SER in spines during recovery may be a predictor of age-related spine loss since decreasing spine density has been reported in older rats (Pentney, 1986). The occurrence of the SER dilatation reported here justifies an investigation of the function of the SER in the PN. The fact that SER within the PN has been shown to respond to calcium deficits with severe morphological alterations (Garthwaite et al., 1992) suggests that the SER plays an important role in calcium metabolism. Calcium is essential to neuronal metabolism since it functions as a second messenger in many cellular processes including development, excitability, learning and memory (Koizumi et al., 1999). Continuously high levels of cytosolic calcium , however, are toxic to the neuron and, in the PN, an important function of the extensive SER network is to sequester and store intracellular calcium (Villa et al., 1991; Sacchetto et al., 1997). Calcium concentration within the SER is dependent not only on cytosolic calcium but also on membrane components such as receptors and calcium ATPase pumps (Villa et al., 1991; Sacchetto et al., 1997). Ethanol-related alterations in cytosolic calcium levels and the membrane components
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which control these levels could contribute to the dilatation of SER reported here as well as the deletion of PN dendritic terminals we have reported previously (Pentney & Quackenbush 1990, 1991; Pentney, 1995). A further examination of the correlation between these events will serve as the basis for future experiments. The results of this study demonstrate that long-term ethanol consumption caused dilatation of SER in Purkinje neuron dendritic arborizations in old rats. This dilatation was widespread in its distribution occurring within the dendritic shafts and spines throughout the PN dendritic arbor. The ethanol-related dilation of SER was also reversible following a recovery period within the dendritic shafts.
Acknowledgments We gratefully acknowledge the technical contributions of Ann Marie Hey and William Pentney . This work was supported by a grant from NIAAA/NIH (AA05592) and by Research Development/BRSG funds from the School of Medicine and Biomedical Sciences, University of Buffalo.
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