Long term alcohol consumption induces microtubular changes in the adult rat cerebellar cortex

Brain Research, 339 (1985) 195-199 Elsevier

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Long term alcohol consumption induces microtubular changes in the adult rat cerebellar cortex MANUEL M. PAULA-BARBOSA and MARIA AMleLIA TAVARES Department of Anatomy, Oporto School of Medicine, Porto (Portugal) (Accepted March 5th, 1985) Key words: microtubule - - dendrite - - axon - - alcohol - - cerebellum - - rat

The effects of prolonged alcohol consumption on the microtubules of Purkinje cell dendrites and granule cell axons were studied in adult rats fed alcohol for 1, 3, 6, 12 and 18 months and compared with respective age-matched controls. A significant consequential decrease in the number of dendritic microtubules in alcohol-fed rats was found when compared with the respective controls. Conversely, an increase in the number of these organelles was found in both ascending and parallel portion of the axons in the experimental animals. The possibility of a relationship between microtubular changes and previously reported cerebellar cortex alcohol-induced structural alterations is advanced. It is c o m m o n l y accepted that chronic and excessive consumption of alcohol induces striking structural and functional changes in the r o d e n t central nervous system 1A°,24,36. W e recently described m a r k e d degenerative changes in the cerebellar granule cell layer 30 and severe disruption of Purkinje cell dendritic trees of adult rats u n d e r those circumstances 31. Concomitantly, signs of axonal r e m o d e l i n g were described both in the granular33 and in the molecular layers 32 after 12 months of alcohol feeding. Microtubules are known to be a b u n d a n t in both axons and dendrites 21. T h e y are implicated in a variety of different cell functions as is the case of neuritic outgrowth, establishment and m a i n t e n a n c e of cell shape and neuroplasmic transportS,eL On the other hand, their vulnerability to certain drugs is well-established 8,26,27. Such is not the case of ethanol where conflicting results exist concerning its effects upon these organelles2,14,17,18. Taking into account that changes in Purkinje cell dendritic trees o b s e r v e d in h y p o t h y r o i d i s m 16 are closely linked with m i c r o t u b u l a r alterations9 and that parallel fibers show outgrowth capacities as described in e x p e r i m e n t a l 6 and pathological circumstances 20, we decided to study the organization of mi-

crotubules in those neuronal cell processes in o r d e r to ascertain whether there is any relationship between these organelles and the alcohol-induced cerebellar cortex structural changes referred to. The observations were carried out in animals used in previous studies21, 33. Briefly, 2-month-old rats were s e p a r a t e d in groups of 6. Half of the groups were given a 20% aqueous ethanol solution for periods of 1, 3, 6, 12 and 18 months; age-matched controls were pair-fed. A n i m a l s were perfused with a solution of 1% glut a r a l d e h y d e / l % f o r m a l d e h y d e in 0.12 M p h o s p h a t e buffer at p H 7.2. F r a g m e n t s were r e m o v e d from vermai lobules 4 - 6 (ref. 15) and processed according to the methods described by Palay and Chan-Palay 19. For the study of Purkinje cell dendrites and ascending portions of granule cell axons, semi-thick sections were cut from 3 blocks p e r animal, parallel to the surface of the folium. W h e n the most superficial cells of the Purkinje cell layer were reached, primary dendrites of Purkinje cells a p p e a r e d cross-sectioned in the surrounding molecular layer. Per animal, 6 primary dendrites (36 per group) and an average of 20 cross-sections of the ascending portions of granule cell axons per animal (120 p e r group) were

Correspondence: M. M. Paula-Barhosa, Department of Anatomy, Oporto School of Medicine, Al. Hern~mi Monteiro, Porto 420(/, Portugal. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

196 p h o t o g r a p h e d at r a n d o m and analysed at a final magnification of 60,000x. F o r the study of the parallel fiber portion of the axons, sections were cut perpendicular to the longer axis of the folia and the same n u m b e r of profiles were observed in the outer third of the molecular layer. The dendritic section areas were m e a s u r e d with a planimeter. The cross-sectioned areas of granule cells axons were measured with an a d e q u a t e acetate replica. The total n u m b e r of microtubules per profile

were counted and their number per unit of area calculated. The individual m o r p h o m e t r i c data was avezaged and means + S.D. calculated. Student's t-test was used for statistical analysis. Observations where P < 0.05 were considered significant. Microtubules in Purkinje cell dendrites of control rats a p p e a r e d regularly disposed through the entire cross-sectioned dendritic profile. A m o n g them a network of fine, filamentous material radiating from each microtubule was always present. Instead, after

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the ascending portions of granule cell axons from al-

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cohol-fed and control rats. O n the contrary, the parallel fiber portion of this axon often seemed to present a larger n u m b e r of microtubules after 6 months of alcohol consumption. The quantitative analysis of microtubules of the ascending portions of these axons

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showed a significant increase in their n u m b e r per unit surface in the 18-month alcohol-fed group (Fig. 3),

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(Fig. 3). The mean areas of cross-sectioned axons of controls and experimental groups showed no signifi-

Fig. 2. Effect of long-term alcohol consumption on the number of microtubules per ~m2 of cross-sectional area in Purkinje cell dendrites in groups of rats (n = 6) alcohol-fed for 1, 3, 6, 12 and 18 months. Columns represent the means and vertical lines S.D. Significance of differences: * P < 0.05, ** P < 0.02, • ** P < 0.005. 6 months of alcohol consumption, there were others

cant differences between them. These results indicate that microtubules from Purkinje cell dendrites and granule cell axons have a different, even opposite reaction to ethanol exposure. This dissimilarity is probably related to the different labilities shown by microtubules, in the various cell

interspersed with normal-looking dendritic profiles, in which microtubules were reduced and the filamentous material denser (Fig. 1). Furthermore, these

compartments, which leads to the thesis that these organelles may have different properties depending on their cell location 11,13. This hypothesis was recent-

organelles appear disarrayed with respect to the longer axis of the dendrite and the 'constant' distance existing between them in normal dendrites 4,25 was frequently replaced by areas in which they were

ly corroborated using antibodies against several microtubule-associated proteins (MAPs) which demonstrated that one of them, MAP2, known to stimulate microtubule assembly, was absent in axons, thus sug-

closely approximated. The quantitative study showed that after the first m o n t h of alcohol consumption there was a significant reduction in the n u m b e r of microtubules per unit of area in dendrites from alcohol-fed rats when compared with the respective

gesting the existence of differences between dendritic and axonal cytoskeletons 3,-~. The depletion of the microtubule cytoskeleton in Purkinje cell dendrites after long-term alcohol consumption could be the determinating factor in the

age-matched controls (Fig. 2). Qualitative differences were not detected between

genesis of their marked dendritic impoverishment observed in these circumstances. Although the im-

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Fig. 3. Effect of long-term alcohol consumption on the number of microtubules per,urn2of cross-sectional area in the ascending portion (AP) and in parallel fiber (PF) portion of granule cell axons in groups of rats (n = 6) alcohol-fed for 1,3, 6, 12 and 18 months. Columns represent the means and vertical lines SD. Significance of differences; * P < 0.05, ** P < 0.02, *** P < 0.005, **** P < 0.001.

198 portance of the in vivo cytoskeletal role assigned to the microtubule remains a matter of speculation8 it is tempting to make such an assumption since marked microtubular abnormalities were also found in chron-

interfere with neuroplasmic transport:.2:~ In addition to these dendritic alterations chronic alcohol consumption also induces granule cell toss ~,

ic hypothyroidism 9, where a severe hypoplasia of Purkinje cell dendrites is k n o w n to occur 16. Similar find-

Concomitantly with these degenerative processes, remodeling signs in the cerebellar cortex were indicated32, 33-34, conspicuously involving parallel fiber

ings were reported elsewhere by P u r p u r a et al. 23 and Bodik et al. 4, who described changes in dendrites of

boutons and leading to marked structural changes m the molecular layer32, 3a. Assuming that microtubules

cortical neurons in children with neurobehavioral de-

are implicated in the outgrowth of n e u r o n a l processesS,22,27,29 the reported increased n u m b e r of these or-

fects and could c o r r e l a t e t h e m with microtubular disarray. Furthermore, this attractive hypothesis is reinforced by data linking microtubules with neuroplasmic transport, both in axons 8,12,26 and in dendrites 7,26. In fact, these organelles are regarded as displaying a force-generating mechanism during the movements of cytoplasmic particlesS, 26. Microtubular disarray would, therefore, lead to a deficient distribution of cell constituants from their place of synthesis to distant places 8, contributing thus to the drastic degenerative alcohol effects observed in the Purkinje cell dendritic trees 31. This assumption is, moreover, supported by the presence of smooth endoplasmic reticulum inclusions found in Golgi cell dendrites after long-term alcohol consumption35, which might well

1 Abraham, W. C. and Hunter, B. E., An electrophysiological analysis of chronic ethanol neurotoxicity in the dentate gyrus: distribution of entorhinal afferents, Exp. Brain Res., 47 (1982) 61-68. 2 Berman, W. J., Joan-Gil, M. S., Jennett, R. B., Tuma, D., Sorrell, M. F. and Rubin, E., Ethanol, hepatocellular organelles, and microtubules. A morphometric study in vivo and in vitro, Lab. Invest., 48 (1983) 760-767. 3 Bernhardt, R. and Matus, A., Light and electron microscopic studies of the distribution of microtubule-associated protein 2 in rat brain: a difference between dendritic and axonal cytoskeletons, J. comp. Neurot., 226 (1984) 203-221. 4 Bodik, N., Stevens, J. K., Sasaki, S. and Purpura, D. P., Microtubular disarray in cortical dendrites and neurobehavioral failure. II. Computer reconstruction of perturbed microtubular arrays, Develop. Brain Res., 5 (1982) 299-309. 5 Burgoyne, R. D. and Cumming, R., Ontogeny of microtubule-associated protein 2 in rat cerebellum: differential expression of the doublet polypeptides, Neuroscience, 11 (1984) 157-167. 6 Chen, S. and Hillman, D. E., Plasticity of the parallel fiber - - Purkinje cell synapse by spine takeover and new synapse formation in the adult rat, Brain Research, 240 (1982) 21)5-220. 7 Droz, B., Koenig, H. L., Rambourg, A., Transport intracytoplasmatique de macromolecules et reticulum endoplas-

ganelles in granule cell axons after prolonged alcohol consumption can be related likewise to the abovem e n t i o n e d plastic changes. In short, we have shown that the degenerative and remodeling changes found in the adult rat cerebellar cortex after prolonged alcohol consumption may well be related to microtubutar alterations either in parallel or consequentially. Further studies are needed to elucidate the underlying mechanisms leading to such alterations. The authors are greatly indebted to Professor E. G. Gray for helpful discussion. This work was supported by grants from 1.N.I.C. (Lisbon) and Stiftung Volkswagenwerk 1-37424.

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