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Giant multivesicular bodies in the rat hippocampal pyramidal cells after chronic alcohol consumption
Neuroscienee Letters, 64 (1986) 345-349 Elsevier Scientific Publishers Ireland Ltd.
345
NSL 03809
GIANT M U L T I V E S I C U L A R B O D I E S IN T H E RAT H I P P O C A M P A L PYRAMIDAL C E L L S AFTER C H R O N I C A L C O H O L C O N S U M P T I O N
M. M. PAULA-BARBOSA*, M. M. BORGES, A. CADETE-LEITE and M. A. TAVARES
Department of Anatomy, Oporto School of Medicine, AI. Hernhni Monteiro, 4200 Porto (Portugal) (Received October 7th, 1985; Revised version received November 25th, 1985; Accepted December 4th, 1985)
Key words." multivesicular body hippocampus- r a t - alcohol
Multivesicular bodies (MVBs) with diameters up to 4.5 #m were observed in the hippocampal pyramidal cells of rats submitted to chronic alcohol consumption. A significant increase in the volumetric density (Vv) of these organelles was found in CAI pyramidal cells. Transitional forms of MVBs towards lysosomes were seen. A failure in MVB's enzymatic hydrolytic mechanisms, due to the prolonged alcohol aggression, could underlie its formation.
Past work has shown that chronic alcohol consumption in the rat induces marked degenerative changes in nerve cells [16], leading in some areas to unequivocal neuronal loss [16, 19]. The mechanisms which underlie these phenomena are not clearly understood, but we advanced that they could be related to changes in the activity of lysosomal enzymes [17], as it occurs in the liver [10]. Multivesicular bodies (MVBs) are commonly seen in neurons and assumed to belong to the cell lysosomal system [7, 11, 14]. Their participation in chromatolytic neuronal changes has been observed under experimental [7] and pathological [13] circumstances. Following our occasional finding of numerous MVBs of unexpectedly large dimensions in the hippocampus pyramidal cells of chronic alcohol-fed rats, we decided to study these organelles using qualitative and quantitative methods, to clarify the implications involved in the above-mentioned alcohol-induced neuronal degenerative changes. Eight-week-old, male, Sprague-Dawley rats, weighing 200 + 20 g, were separated in 10 different groups of 6 animals. Half of the groups were ethanol-fed for periods of 1, 3, 6, 12 and 18 months with a 20% aqueous ethanol solution [17]. The remainder were pair-fed and used as controls. The techniques described by Palay and ChanPalay [12] were used for processing the material. The hippocampal formation was dissected and coronal sections obtained. Ultrathin sections from Ammon's horn fields CA1 and CA3 were cut from 5 different blocks per animal, and from each section the first two pyramidal cells visualized were photographed at × 4000. At least *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
347 10 cells p e r a n i m a l a n d p e r field (CA1 a n d C A 3 ) were s t u d i e d at a final m a g n i f i c a t i o n o f x 12,000. T h e fraction o f cell c y t o p l a s m o c c u p i e d b y these organelles (Vv) was d e t e r m i n e d using the m e t h o d s d e s c r i b e d b y W e i b e l [20]. Q u a n t i t a t i v e results were c o m p a r e d using n o n - p a r a m e t r i c statistics ( M a n n - W h i t n e y U-test)~ o b s e r v a t i o n s were c o n s i d e r e d significant when P < 0.05. M V B s with d i a m e t e r s r a n g i n g b e t w e e n 0.2 a n d 0.5 # m were a b u n d a n t in p y r a m i d a l cells f r o m all g r o u p s o f animals, p r e s e n t i n g the s a m e characteristics as those described b y o t h e r a u t h o r s in different n e u r o n s [4, 11, 14]. W i t h the increasing time o f the e x p e r i m e n t , M V B s with d i a m e t e r s u p to 1/~m were c o m m o n l y f o u n d in a l c o h o l t r e a t e d a n i m a l s a n d a few in the p y r a m i d s f r o m controls. T h e presence o f large M V B s in c o n t r o l s agrees with the o b s e r v a t i o n s m a d e by C a s e y a n d F e l d m a n [4] w h o described identical structures in the aged rat m e d i a l nucleus o f the t r a p e z o i d b o d y . A f t e r 6 m o n t h s o f a l c o h o l feeding, progressively larger M V B s with d i a m e t e r s increasing with the time o f e x p e r i m e n t a n d reaching 4.5 /~m (Figs. 1-5) were freq u e n t l y o b s e r v e d in the apical pole o f b o t h C A 1 a n d C A 3 p y r a m i d a l cells o f a l c o h o l fed rats. As with the n o r m a l - s i z e d , the g i a n t M V B s were in close r e l a t i o n s h i p with the G o l g i c o m p l e x [5] (Fig. 3) a n d s u r r o u n d e d b y n u m e r o u s c o a t e d vesicles (Figs. 3 a n d 5). T u b u l a r processes p r o j e c t i n g f r o m their surface [11, 14] were often o b s e r v e d (Fig. 5). The fuzzy c o a t involving M V B s was m o s t o f the time e x t r e m e l y developed, m a k i n g difficult the visualization o f their limiting m e m b r a n e (Figs. 2-5). F l a t elevations at their surface (Figs. 3-5) a n d d o m e - s h a p e d figures (Figs. 3-5) were m o r e num e r o u s t h a n in n o r m a l - s i z e d MVBs. T h e i r m a t r i x was always o f the light type [11, 14], frequently with a m o r p h o u s m a t e r i a l (Fig. 3). Vesicles were n u m e r o u s with different sizes a n d shapes (Figs. 2, 3 a n d 5). O n a few occasions, M V B s with dense m a t e r i a l similar to the l y s o s o m a l m a t r i x , suggesting t r a n s i t i o n a l forms to l y s o s o m e s [!, 9], were seen (Fig. 5). G i a n t M V B s were frequently o b s e r v e d close to n o r m a l - s i z e d (Fig. 3), b u t we never detected m o r e t h a n one g i a n t M V B p e r neuron. The results o f the m o r p h o m e t r i c analysis are represented in Fig. 6. A significant
Fig. 1. Pyramidal cells from the hippocampus CAI field. In their apical poles two profiles presumably belonging to MVBs (arrows) are seen with maximum diameters of 3.8 kLmand 2/~m. Note their close relalionship with numerous lipofuscin granules. 18-month alcohol-fed rat. Toluidine blue. x 1040. Fig. 2. MVB with a diameter of 3.5 ,um in the apical pole of a CA3 pyramidal cell with numerous pleomorphic vesicles. 18-month alcohol-fed rat. x 9000. Fig. 3. Normal-sized (asterisk) and giant MVBs in a CA1 pyramidal cell. The latter presents numerous dome-shaped figures (arrows) and flat elevations (arrowheads) and is covered by a conspicuous fuzzy coat. Amorphous material among vesicles (double arrowheads) is seen. Note in its proximity a Golgi apparatus (G) and coated vesicles. 18-month alcohol-fed rat. x 30,000. Fig. 4. Higher magnification of MVBs shown in Fig. 3. A dome-shaped figure (arrow), a flat elevation (arrowhead) and the fuzzy coat are clearly seen. 18-month alcohol-fed rat. x 60,000. Fig. 5. MVB in a CA1 pyramidal cell with a tubular evagination (arrow). A lysosome (arrowhead) with a membrane and vesicles similar to those of MVB is seen. 18-month alcohol-fed rat. x 48,000.
348
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Fig. 6. Graphic representation of the volumetric density (Vv) of multivesicular bodies of CAI-CA3 pyramidal cells. Columns represent means and vertical bars 1 S.D. *P<0.05, **P<0.002.
increase in the fraction of cell cytoplasm (Vv) occupied by MVBs in CA1 pyramidal cells was observed after 3 months of alcohol consumption. This regional difference was rather unexpected, as giant MVBs were abundant in both CAI and CA3 fields, being observed in up to 2% of neurons in 12- and 18-month alcohol-fed groups. Moreover, in spite of the different location and connectivity that CA1 and CA3 pyramidal cells have within the hippocampal synaptic circuitry, neurocytological changes were not observed between them, which makes it difficult to advance any solid explanation for the aforementioned quantitative discrepancies. MVBs were rarely the specific aim of past studies, and, thus, in the existing extensive literature [8] concerning these organelles, their origin, function and fate [5, 8] remain an unsolved problem. There is, however, a growing body of evidence that they can display multiple roles, such as in the hydrolysis of macromolecules [1], the synthesis of lipids [18] and cell membrane transfer [6]. The first of these roles is by far the most explored, particularly with the aid of tracers [8]. Different macromolecules were shown to be transferred to MVBs and then hydrolysed by means of their enzymes. This capacity displayed by MVBs together with the morphological evidence of transitional forms of these organelles towards lysosomes observed in this study, supports the hypothesis that both belong to the same functional compartment of the cell [1, 7, 11]. Considering the interrelationship between lysosomes and lipofuscin granules [3, 15], the aforementioned hypothesis is strongly supported by the recent finding of large amounts of prematurely formed pigment in the cerebellar cortex of chronic alcohol-fed rats [17]. To our knowledge, the largest MVBs observed in previous studies were 2 #m in diameter. These were located in cells of the vaginal epithelium of the rat [9]. MVBs twice that size were commonly found in this study. An explanation for their presence in the hippocampus of alcohol-fed rats is difficult to find. However, the existence in its limiting membrane of numerous dome-shaped figures indicating a marked membrane activity might well be the result of the continuous alcohol aggression, which could lead to a failure of MVBs enzymatic lytic mechanisms and, thus, to an accumulation of non-hydrolysed substances. As cells from the hippocampal formation are particularly sensitive to different experimental conditions [19] and to aging [2], this
349
view is in agreement with the numerous observations of MVBs abnormalities related to changes in cell physiology. The a u t h o r s are i n d e b t e d to P r o f e s s o r E.G. G r a y for helpful criticisms. This w o r k w a s s u p p o r t e d b y I . N . I . C . a n d ' S t i f t u n g V o l k s w a g e n w e r k ' (1-61408). I Abrahams, S.J. and Holtzman, E., Secretion and endocytosis in insulin-stimulated rat adrenal medulla cells, J. Cell Biol., 56 (1973) 54(~558. 2 Brizzee, K.R. and Ordy, J.M., Age pigments, cell loss and hippocampal function, Mech. Ageing Develop., 9 (1979) 143-162. 3 Brunk, V. and Ericsson, J.L.E., Electron microscopical studies on rat brain neurons. Localization of acid phosphatase and mode of formation of lipofuscin bodies, J. Ultrastruct. Res., 38 (1972) 1--15. 4 Casey, M.A. and Feldman, M.L., Aging in the rat medial nucleus of the trapezoid body. II. Electron microscopy, J. Comp. Neurol., 232 (1985) 401J,13. 5 Friend, D.S. and Farquhar, M.G., Functions of coated vesicles during protein absorption in the rat vas deferens, J. Cell Biol., 35 (1967) 357-376. 6 Herzog, V. and Miller, F., Membrane retrieval in epithelial ceils of isolated thyroid follicles, Eur. J. Cell Biol., 19 (1979) 203 215. 7 Holtzman, E., Novikoff, A.B. and Villaverde, H., Lysosomes and GERL in normal and chromatolytic neurons of the rat ganglion nodosum, J. Cell Biol., 33 (1967) 419~,35. 8 Mata, L.R., Corpos multivesiculares da c61ula animal. Perspectiva morfo-funcional. In A.P. Carvalho, V.M.C. Madeira and E.M.V. Pires (Eds.), Ci~ncia Biol6gica, lnstituto de Zoologia da Universidade de Coimbra, Coimbra, 1984, pp. 1 39. 9 Merker, H.J., l~lber das vorkommen multivesicul/irer einschlussk6rper (multivesicular bodies) im vaginalepithel der Ratte, Z. Zellforsch., 68 (1965) 618-630. 10 Mczey, E., Potter, J.J., Ruby, J.S., Brandes, D., Romero, J., Tsunenobu, T. and Halsted, C.H., Effect of ethanol feeding on hepatic lysosomes in the monkey, Lab. Invest., 43 (1980) 88 93. I I Novikoff, A.B., Lysosomes in nerve cells. In H. Hyd~n (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 255 319. 12 Palay, S.L. and Chan Palay, V., Cerebellar Cortex: Cytology and Organization, Springer, Berlin, 1974. 13 Paula-Barbosa, M.M., Mota-Cardoso, R., Faria, R. and Cruz, C., Multivesicular bodies in cortical dendrites of two patients with Alzheimer's disease, J. Neurol. Sci., 36 (1978) 259 264. 14 Peters, A., Palay, S.L. and Webster, H.F., The Fine Structure of the Nervous System: The Neurons and Supporting Cells, W.B. Saunders Company, Philadelphia, 1976, pp. 38~40. 15 Sekhon, S.S. and Maxwell, D.S., Ultrastructural changes in neurons of the spinal anterior horn of ageing mice with particular reference to the accumulation of lipofuscin pigment, J. Neurocytol.. 3 (1974) 59 72. 16 Tavares, M.A. and Paula-Barbosa, M.M., Alcohol-induced granule cell loss in the cerebellar cortex of the adult rat, Exp. Neurol., 78 (1982) 574-582. 17 Tavares, M.A., Paula-Barbosa, M.M., Barroca, H. and Volk, B., Lipofuscin granules in cerebellar intcrneurons after long-term alcohol consumption in the adult rat, Anat. Histol. Embryol., 171 (1985) 61 69. 18 Wake, K., Development of vitamin A-rich lipid droplets in multivesicular bodies of rat liver stellate cells, J. Cell Biol., 63 (1974) 683 691. 19 Walker, D.W., Barnes, D.E., Riley, J.N., Hunter, B.E. and Kubanis, P., Neuronal loss in hippocampus induced by prolonged ethanol consumption in rats, Science, 209 (1980) 711-713. 20 Wcibel, E.R., Stereological techniques for electron microscopic morphometry. In M.A. Hayat (Ed.), Principles and Techniques of Electron Microscopy, Van Nostrand Reinhold Company, New York, 1973, pp. 239 296.
345
NSL 03809
GIANT M U L T I V E S I C U L A R B O D I E S IN T H E RAT H I P P O C A M P A L PYRAMIDAL C E L L S AFTER C H R O N I C A L C O H O L C O N S U M P T I O N
M. M. PAULA-BARBOSA*, M. M. BORGES, A. CADETE-LEITE and M. A. TAVARES
Department of Anatomy, Oporto School of Medicine, AI. Hernhni Monteiro, 4200 Porto (Portugal) (Received October 7th, 1985; Revised version received November 25th, 1985; Accepted December 4th, 1985)
Key words." multivesicular body hippocampus- r a t - alcohol
Multivesicular bodies (MVBs) with diameters up to 4.5 #m were observed in the hippocampal pyramidal cells of rats submitted to chronic alcohol consumption. A significant increase in the volumetric density (Vv) of these organelles was found in CAI pyramidal cells. Transitional forms of MVBs towards lysosomes were seen. A failure in MVB's enzymatic hydrolytic mechanisms, due to the prolonged alcohol aggression, could underlie its formation.
Past work has shown that chronic alcohol consumption in the rat induces marked degenerative changes in nerve cells [16], leading in some areas to unequivocal neuronal loss [16, 19]. The mechanisms which underlie these phenomena are not clearly understood, but we advanced that they could be related to changes in the activity of lysosomal enzymes [17], as it occurs in the liver [10]. Multivesicular bodies (MVBs) are commonly seen in neurons and assumed to belong to the cell lysosomal system [7, 11, 14]. Their participation in chromatolytic neuronal changes has been observed under experimental [7] and pathological [13] circumstances. Following our occasional finding of numerous MVBs of unexpectedly large dimensions in the hippocampus pyramidal cells of chronic alcohol-fed rats, we decided to study these organelles using qualitative and quantitative methods, to clarify the implications involved in the above-mentioned alcohol-induced neuronal degenerative changes. Eight-week-old, male, Sprague-Dawley rats, weighing 200 + 20 g, were separated in 10 different groups of 6 animals. Half of the groups were ethanol-fed for periods of 1, 3, 6, 12 and 18 months with a 20% aqueous ethanol solution [17]. The remainder were pair-fed and used as controls. The techniques described by Palay and ChanPalay [12] were used for processing the material. The hippocampal formation was dissected and coronal sections obtained. Ultrathin sections from Ammon's horn fields CA1 and CA3 were cut from 5 different blocks per animal, and from each section the first two pyramidal cells visualized were photographed at × 4000. At least *Author for correspondence. 0304-3940/86/$ 03.50 © 1986 Elsevier Scientific Publishers Ireland Ltd.
347 10 cells p e r a n i m a l a n d p e r field (CA1 a n d C A 3 ) were s t u d i e d at a final m a g n i f i c a t i o n o f x 12,000. T h e fraction o f cell c y t o p l a s m o c c u p i e d b y these organelles (Vv) was d e t e r m i n e d using the m e t h o d s d e s c r i b e d b y W e i b e l [20]. Q u a n t i t a t i v e results were c o m p a r e d using n o n - p a r a m e t r i c statistics ( M a n n - W h i t n e y U-test)~ o b s e r v a t i o n s were c o n s i d e r e d significant when P < 0.05. M V B s with d i a m e t e r s r a n g i n g b e t w e e n 0.2 a n d 0.5 # m were a b u n d a n t in p y r a m i d a l cells f r o m all g r o u p s o f animals, p r e s e n t i n g the s a m e characteristics as those described b y o t h e r a u t h o r s in different n e u r o n s [4, 11, 14]. W i t h the increasing time o f the e x p e r i m e n t , M V B s with d i a m e t e r s u p to 1/~m were c o m m o n l y f o u n d in a l c o h o l t r e a t e d a n i m a l s a n d a few in the p y r a m i d s f r o m controls. T h e presence o f large M V B s in c o n t r o l s agrees with the o b s e r v a t i o n s m a d e by C a s e y a n d F e l d m a n [4] w h o described identical structures in the aged rat m e d i a l nucleus o f the t r a p e z o i d b o d y . A f t e r 6 m o n t h s o f a l c o h o l feeding, progressively larger M V B s with d i a m e t e r s increasing with the time o f e x p e r i m e n t a n d reaching 4.5 /~m (Figs. 1-5) were freq u e n t l y o b s e r v e d in the apical pole o f b o t h C A 1 a n d C A 3 p y r a m i d a l cells o f a l c o h o l fed rats. As with the n o r m a l - s i z e d , the g i a n t M V B s were in close r e l a t i o n s h i p with the G o l g i c o m p l e x [5] (Fig. 3) a n d s u r r o u n d e d b y n u m e r o u s c o a t e d vesicles (Figs. 3 a n d 5). T u b u l a r processes p r o j e c t i n g f r o m their surface [11, 14] were often o b s e r v e d (Fig. 5). The fuzzy c o a t involving M V B s was m o s t o f the time e x t r e m e l y developed, m a k i n g difficult the visualization o f their limiting m e m b r a n e (Figs. 2-5). F l a t elevations at their surface (Figs. 3-5) a n d d o m e - s h a p e d figures (Figs. 3-5) were m o r e num e r o u s t h a n in n o r m a l - s i z e d MVBs. T h e i r m a t r i x was always o f the light type [11, 14], frequently with a m o r p h o u s m a t e r i a l (Fig. 3). Vesicles were n u m e r o u s with different sizes a n d shapes (Figs. 2, 3 a n d 5). O n a few occasions, M V B s with dense m a t e r i a l similar to the l y s o s o m a l m a t r i x , suggesting t r a n s i t i o n a l forms to l y s o s o m e s [!, 9], were seen (Fig. 5). G i a n t M V B s were frequently o b s e r v e d close to n o r m a l - s i z e d (Fig. 3), b u t we never detected m o r e t h a n one g i a n t M V B p e r neuron. The results o f the m o r p h o m e t r i c analysis are represented in Fig. 6. A significant
Fig. 1. Pyramidal cells from the hippocampus CAI field. In their apical poles two profiles presumably belonging to MVBs (arrows) are seen with maximum diameters of 3.8 kLmand 2/~m. Note their close relalionship with numerous lipofuscin granules. 18-month alcohol-fed rat. Toluidine blue. x 1040. Fig. 2. MVB with a diameter of 3.5 ,um in the apical pole of a CA3 pyramidal cell with numerous pleomorphic vesicles. 18-month alcohol-fed rat. x 9000. Fig. 3. Normal-sized (asterisk) and giant MVBs in a CA1 pyramidal cell. The latter presents numerous dome-shaped figures (arrows) and flat elevations (arrowheads) and is covered by a conspicuous fuzzy coat. Amorphous material among vesicles (double arrowheads) is seen. Note in its proximity a Golgi apparatus (G) and coated vesicles. 18-month alcohol-fed rat. x 30,000. Fig. 4. Higher magnification of MVBs shown in Fig. 3. A dome-shaped figure (arrow), a flat elevation (arrowhead) and the fuzzy coat are clearly seen. 18-month alcohol-fed rat. x 60,000. Fig. 5. MVB in a CA1 pyramidal cell with a tubular evagination (arrow). A lysosome (arrowhead) with a membrane and vesicles similar to those of MVB is seen. 18-month alcohol-fed rat. x 48,000.
348
1'0 I|
1M
o.sl
~]co.'r.o,
3M
mALCOHOL
6M
*
12M
B ''
o o.6 ~'= i i
~
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1 I
. . . . . cA~
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eA~
cA3
18M
i i
, ''
:
:
t
I
I
i
eA~
cA3
'
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Fig. 6. Graphic representation of the volumetric density (Vv) of multivesicular bodies of CAI-CA3 pyramidal cells. Columns represent means and vertical bars 1 S.D. *P<0.05, **P<0.002.
increase in the fraction of cell cytoplasm (Vv) occupied by MVBs in CA1 pyramidal cells was observed after 3 months of alcohol consumption. This regional difference was rather unexpected, as giant MVBs were abundant in both CAI and CA3 fields, being observed in up to 2% of neurons in 12- and 18-month alcohol-fed groups. Moreover, in spite of the different location and connectivity that CA1 and CA3 pyramidal cells have within the hippocampal synaptic circuitry, neurocytological changes were not observed between them, which makes it difficult to advance any solid explanation for the aforementioned quantitative discrepancies. MVBs were rarely the specific aim of past studies, and, thus, in the existing extensive literature [8] concerning these organelles, their origin, function and fate [5, 8] remain an unsolved problem. There is, however, a growing body of evidence that they can display multiple roles, such as in the hydrolysis of macromolecules [1], the synthesis of lipids [18] and cell membrane transfer [6]. The first of these roles is by far the most explored, particularly with the aid of tracers [8]. Different macromolecules were shown to be transferred to MVBs and then hydrolysed by means of their enzymes. This capacity displayed by MVBs together with the morphological evidence of transitional forms of these organelles towards lysosomes observed in this study, supports the hypothesis that both belong to the same functional compartment of the cell [1, 7, 11]. Considering the interrelationship between lysosomes and lipofuscin granules [3, 15], the aforementioned hypothesis is strongly supported by the recent finding of large amounts of prematurely formed pigment in the cerebellar cortex of chronic alcohol-fed rats [17]. To our knowledge, the largest MVBs observed in previous studies were 2 #m in diameter. These were located in cells of the vaginal epithelium of the rat [9]. MVBs twice that size were commonly found in this study. An explanation for their presence in the hippocampus of alcohol-fed rats is difficult to find. However, the existence in its limiting membrane of numerous dome-shaped figures indicating a marked membrane activity might well be the result of the continuous alcohol aggression, which could lead to a failure of MVBs enzymatic lytic mechanisms and, thus, to an accumulation of non-hydrolysed substances. As cells from the hippocampal formation are particularly sensitive to different experimental conditions [19] and to aging [2], this
349
view is in agreement with the numerous observations of MVBs abnormalities related to changes in cell physiology. The a u t h o r s are i n d e b t e d to P r o f e s s o r E.G. G r a y for helpful criticisms. This w o r k w a s s u p p o r t e d b y I . N . I . C . a n d ' S t i f t u n g V o l k s w a g e n w e r k ' (1-61408). I Abrahams, S.J. and Holtzman, E., Secretion and endocytosis in insulin-stimulated rat adrenal medulla cells, J. Cell Biol., 56 (1973) 54(~558. 2 Brizzee, K.R. and Ordy, J.M., Age pigments, cell loss and hippocampal function, Mech. Ageing Develop., 9 (1979) 143-162. 3 Brunk, V. and Ericsson, J.L.E., Electron microscopical studies on rat brain neurons. Localization of acid phosphatase and mode of formation of lipofuscin bodies, J. Ultrastruct. Res., 38 (1972) 1--15. 4 Casey, M.A. and Feldman, M.L., Aging in the rat medial nucleus of the trapezoid body. II. Electron microscopy, J. Comp. Neurol., 232 (1985) 401J,13. 5 Friend, D.S. and Farquhar, M.G., Functions of coated vesicles during protein absorption in the rat vas deferens, J. Cell Biol., 35 (1967) 357-376. 6 Herzog, V. and Miller, F., Membrane retrieval in epithelial ceils of isolated thyroid follicles, Eur. J. Cell Biol., 19 (1979) 203 215. 7 Holtzman, E., Novikoff, A.B. and Villaverde, H., Lysosomes and GERL in normal and chromatolytic neurons of the rat ganglion nodosum, J. Cell Biol., 33 (1967) 419~,35. 8 Mata, L.R., Corpos multivesiculares da c61ula animal. Perspectiva morfo-funcional. In A.P. Carvalho, V.M.C. Madeira and E.M.V. Pires (Eds.), Ci~ncia Biol6gica, lnstituto de Zoologia da Universidade de Coimbra, Coimbra, 1984, pp. 1 39. 9 Merker, H.J., l~lber das vorkommen multivesicul/irer einschlussk6rper (multivesicular bodies) im vaginalepithel der Ratte, Z. Zellforsch., 68 (1965) 618-630. 10 Mczey, E., Potter, J.J., Ruby, J.S., Brandes, D., Romero, J., Tsunenobu, T. and Halsted, C.H., Effect of ethanol feeding on hepatic lysosomes in the monkey, Lab. Invest., 43 (1980) 88 93. I I Novikoff, A.B., Lysosomes in nerve cells. In H. Hyd~n (Ed.), The Neuron, Elsevier, Amsterdam, 1967, pp. 255 319. 12 Palay, S.L. and Chan Palay, V., Cerebellar Cortex: Cytology and Organization, Springer, Berlin, 1974. 13 Paula-Barbosa, M.M., Mota-Cardoso, R., Faria, R. and Cruz, C., Multivesicular bodies in cortical dendrites of two patients with Alzheimer's disease, J. Neurol. Sci., 36 (1978) 259 264. 14 Peters, A., Palay, S.L. and Webster, H.F., The Fine Structure of the Nervous System: The Neurons and Supporting Cells, W.B. Saunders Company, Philadelphia, 1976, pp. 38~40. 15 Sekhon, S.S. and Maxwell, D.S., Ultrastructural changes in neurons of the spinal anterior horn of ageing mice with particular reference to the accumulation of lipofuscin pigment, J. Neurocytol.. 3 (1974) 59 72. 16 Tavares, M.A. and Paula-Barbosa, M.M., Alcohol-induced granule cell loss in the cerebellar cortex of the adult rat, Exp. Neurol., 78 (1982) 574-582. 17 Tavares, M.A., Paula-Barbosa, M.M., Barroca, H. and Volk, B., Lipofuscin granules in cerebellar intcrneurons after long-term alcohol consumption in the adult rat, Anat. Histol. Embryol., 171 (1985) 61 69. 18 Wake, K., Development of vitamin A-rich lipid droplets in multivesicular bodies of rat liver stellate cells, J. Cell Biol., 63 (1974) 683 691. 19 Walker, D.W., Barnes, D.E., Riley, J.N., Hunter, B.E. and Kubanis, P., Neuronal loss in hippocampus induced by prolonged ethanol consumption in rats, Science, 209 (1980) 711-713. 20 Wcibel, E.R., Stereological techniques for electron microscopic morphometry. In M.A. Hayat (Ed.), Principles and Techniques of Electron Microscopy, Van Nostrand Reinhold Company, New York, 1973, pp. 239 296.