Ethanol-induced dendritic alterations in hippocampal granule cells

Brain Research, 477 (1989)373-377 Elsevier

373

BRE 23294

Ethanobinduced dendritic alterations in hippocampa! granule cells D. Durand 3, J.A. Saint-Cyr 2, N.

G u r e v i c h I and P.L. C a f i e n I

tAddiction Research Foundation, Playfair Neuroscience Unit, 2Dept. of Anatomy and Psychology, University of Toronto, Toronto (Canada) and SThe Applied Neural Control Laboratory, Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH (U.S.A.) (Accepted 20 September 1988)

Key words: Ethanol~ Chronic intake; Neuron; Morphology

The effects of chronic ethanol intake were studied on the morphology of rat hippocampal granule cells. Sprague-Dawley rats w e r e exposed~to ethanol in a liquid diet for 5 months followed by a 3 week withdrawal period. A control group was fed similar amounts of the s a m e diet but with ethanol replaced by maltose-dextrins. Intracellular recordings were performed using the hippocampal slice preparation and the granule cells from animals of both groups were injected with HRP. The ethanol treatment produced a significant increase in the average length of the dendrites of granule cells compared to the control group. Chronic ethanol intake produced a decrease in the number of dendrites in the proximal region of the tree (80-180 pro) but also a significant increase in the number of dendrites in the distal portion (260-340/~m). The decrease in the number of proximal dendrites suggests that et'nai~olcou!d be affecting a population of neurons with afferent inputs in the proximal region of the tree or that ethanol could interfere with the normal maturation p r o c e s s e s of the granule cells. The increase in the number of dendrites in the distal region of the tree suggests, however, an accelerated growth or sprouting of dendrites in the molecular layer. Golgi staining techniques have revealed a significant effect of chronic ethano| intake on dendritic spines :a aad reduced dendritic branching in hippocampal CA1 pyramidal and granule cells in the hippocampus 12"15. Fifteen to 25% of granule cells in the hippocampus and Purkinje cells in the cerebellum are lost after chronic ethanol intake 19'2°. The Golgi stain does not always fill the whole cell, and therefore accurate studies of dendritic lengths and branching dendrites are difficult. Animal studies of the effects of chronic ethanol intake have revealed contradictory results such as dendritic growth 12 and dendritic damage 15 in CA1 pyramidal cells. It was decided to use horseradish peroxidase (HRP), an enzyme injected intracellularly, which has been reported to completely fill the dendrites, somata and axons of the neurons injected. The H R P injections were made subsequent to intracellular recordings of the electrophysiology associated with ethanolinduced brain damage 6's which showed that chronic etaanol intake can markedly affect inhibitory poten-

tials and membrane electrotonic parameters. Here, we report the results of the morphological analysis performed in these same neurons. Twenty-two adult Sprague-Dawley rats (200 g) individually housed, were given ad lihitum access to a liquid diet for a period of 5 months. The animals were randomly divided into two groups.. An ethanol group was fed the Lieber-Decarli liquid diet 4 provided in Richter tubes containing 35% of its calories as ethanol. A pair-fed control group was fed the same diet with ethanol replaced by an equivalent caloric. amount of maltose-dextrins. The ettt~mol group received the ethanol diet for 20 weeks and was then switched to the control diet for the remaining 3 weeks. The ethanol intake started at 15 g/kg/day and quickly stabilized at 10 g/kg/day. At the time of sacrifice, the animals were anesthetized with ether, decapitated and the hippocampus was rapidly dissected. Four hundred-/~m slices were then cut for the standard in vitro preparation 7,s. The techniques for the intracellular recording and

Correspondence: D. Durand, Applied Neural Control Laboratory, Department of Biomedical En~,ineering. Case Western Reserve University, Cleveland, OH 44106, U.S.A. 0006-8993/89/$03.50© 1989Elsevier Science Publishers B.V. (Biomedical Div'~ion)

374 the following equation:

the H R P injection and processing have been already published as well as the equations for the calculation and measurement of the branching power, and the somatic surface area. The branching ratio is defined as the ratio of the diameter of the parent and daughter branches. The branching power N is defined by

where dl is a parent branch and d2. 3 a r e the daughter branches. The length of the dendritic segments was

A HlpgOcamDa

I

IleOUre

120. , .

B 10 a ÷+÷

CONTROL

000 ~THANOL %d 0.

10.0-

t. £]ZSTANCE (um)

Fig. 1. Analysisof dendritic branches. A: sh~ftedlinear analysisof dendritic branching. The soma of each cell analyzed is positionea within the granule cell layer (GL) as measured in the tissue shoe and the numberof dendrites crossingequally spaced lines (30/zm)parallel to the cell body layer is recorded for each cell. The cell density was also measured in the ventral half of the granule cell layer (VGL) and in the dorsal half of the layer (DGL). Note also that cells located at the bottom part of the layer (dorsal) have on average a longer dendritic tree than cells located on the top of the layer (ventral). Scale bar -= 50/~m. B: number of dendrites at fixed distances from the top of the granule cell layer. The number of intersections between dendrites and lines is averaged for all the cells and plotted. Note the significantdecrease in the number of dendrites proximally and the increase distally for neurons in the ethanol grour when compared to neurons in the control group.

375 calculated in the 3-dimensional space and entered on the p r o j e c t e d image. The total dendritic length (TL) and the average dendritic length ( A L ) (average distance between the terminal points and the soma) were estimated. The dendritic trees were analyzed using a shifted linear analysis9"12. The number of intersections between the dendrites and a set of lines perpendicular to the main axis of the tree was counted at various distances from the soma with a resolution of 3 0 # m (Fig. 1). Following the microscopic measurements, the slices were rehydrated using increasing concentration of phosphate buffer solution. The slices were then introduced into large slabs o f cortical tissue and the blocks frozen at - 6 0 °C. Forty/~m sections were then cut and directly mounted on gel subbed slides and a Nissl staining was done. T h e widths of both the layers were measured in 3 - 5 sections per slice in the middle third of the dorsal blade of the hippocampal slice. The cell layer was then divided in two and the cell density measured in the same sections for the dorsal half and the ventral half. This analysis was done for 3 - 5 sections over a distance of 200/~m per slice in the middle third of the dorsal blade of the hippocampus. The results of the morphometric analyses for the two treatment groups are tabulated in Table I. The

total dendritic length was increased in the ethanol group but not significantly. However, the cells from the ethanol-fed animals had significantly longer dendritic trees (1-tailed t-test, P < 0.025). No difference was observed for the soma surface area and the branching power n and ratio R. The results o f the shifted linear analysis show a significant decrease in the number from 80 to 180/~m and an increase in the number of branches from 260 to 340 jura (Fig. 1B) from neurons in the ethanol group when compared to neurons from animals in the control group. The increase in the dendritic length and the number of dendritic branches of the ethanol-fed rats could not be explained by a difference of these animals' body weight; 4.3 + 0.3 g (mean + S.D.) for control animals and 433 + 41.3 g for ethanol-fed animals measured before sacrifice. Both groups gained weight at a similar rate with no significant difference during the 5-month liquid diet. Similarly, there were no differences between the two groups in the weight of the contralaterai hippocampus 66.8 + 5.19 mg (n = 10) for the control group versus 65.6 + 9.12 mg (n = 8) for the ethanol group. It has been reported that neurons situated at the t o p of the layer have shorter dendritic trees than neurons at the bottom of the layer since their dendrites all extend to the hippocampal fissure 9 (Fig. 1). More-

TABLE I Morphological measurements in granule cells comparing ethanol-fed rats and their pair-fed contro!

The mean (M) and standard deviation (S.D.) of the parameters listed are tabulated for both control- and ethanol-fed animals. The statistical analysis was done using a one-tailed t-test (N.S. = not significant). The number of samples n refers to the number of cells for the first three parameters. For the branching ratio and power, n is the number of branch points randomly selected on the neurons of each group. For the cell density measurements, n is the number of sections analyzed (3-5 per slice). Control n

Total dendritic length ~m) Average dendritic length (,um) Somatic surface area (urn-') B, anching power (n) Branching ratio (R) Cell density in the d,,. ~,alhalf(no, of ozll~ x 1000/gin2) Ceil density in the ventra| half (no. of cells x 1000/~lm2) Cell density across the whole layer (no. of cells x 1000//~m2)

Ethanol Mean

Mean

S.D.

815.00 39.00 39.00 0.18 0.36

11 11 20 32 32

N.S. <0.025 N.S. N.S. N.S.

14.07

2.89

68

<0.030

94

16.77

3.32

68

N.S.

94

15.42

3.31

68

N.S.

S.D.

n

763.00 47.00 54.00 0.20 0.49

12 12 19 17 17

2988.00 329.00 495.00 1.56 1.32

15.07

3.42

94

15,90

4.10

15.49

3.31

2,444.00 287.00 470.00 1.57 1.35

376 over, the branching pattern of these neurons differs and this effect could explain the difference in the dendritic tree between the two groups. Therefore, the neuronal density in the ventral half versus the dorsal half of the cell layer was measured in both groups (Fig. 1). The results (Table I) show that there was no statistical difference between groups for the neuronal density across the whole layer. However, the cell density in the dorsal half of the layer was significantly lower in the ethanol group as measured by the number of cells times 1000 p e r g m 2 (mean + S.D.: 15.07 + 3.42) compared to the control group (14.07 + 2.89). These results suggest that some neurons are indeed lost at the top of the layer but that the loss is small (6%) compared to the increase in the number of dendrites (160% at 300 ram). The decreased dendritic branching reported above has also been observed in the CA1 region of the hippocampus t2` However, the increase in the dendritic length of ethanol-fed animals compared to the control group was unexpected since one would expect a 'dying-back' phenomenon as previously reported for the aging process 16. This increase could not be explained by an increased weight of the ethanol-fed animals or enlarged hippocampi in the ethanol group or by a biased sampling of the longer neurons as shown by the cell density measurements. A similar increase in dendritic length was aiso detected in a study of aging in humans ~. Cortical cells from aged subjects had longer dendritic trees than the younger subjects. From these results it was postulated that the dendrites of the layer II pyramidal neurons in the human parahippocampal gyrus were growing during normal aging. Other studies have shown dendritic growth in the central ne~'ous system of adult and aged rats t°'~7"~s. In the case of chronic ethanol intake, a similar pattern of decreased number of dendrites proximally and increased number of dendrites distally was also detected in pyram~al cells';. The mechanisms proposed for the proximal decrease in the number of

dendrites were that chronic ethanol intake could damage the specific afferents in the proximal region leading to transneuronal degeneration II or chronic ethanol could delay the normal growth process of proximal dendrites of CA1 pyramidal cellss. The mechanism for the rarprising increase in the length and the number of dendrites can be attributed to a period of accelerated growth most likely during the withdrawal phase following the intake of ethanol 12. Sprouting has been shown to occur following deafferentation 14. Cerebral atrophy in abstinent alcoholics has been shown to be partially reversible 2'3 and could he explained in part by a growth of the dendrites during the withdrawal phase. The increase in dendritic length of neurons from ethanol-fed rats was also accompanied by an increase in the electrotonic length (L) of these neurons s. The analysis of the electrotonic parameters of these cells also showed an increase in membrane specific resistance s. This last parameter is essential foi neuronal integratk~,~, since an increase in the membrane resistance can:~:~crea~e attenuation of voltages along dendrites, inc:ease the membrane time constant and thereby significantly alter the neuronal processing capability. This study confirms the fact that chronic intake of ethanol can cause significant alterations in the organization of dendritic trees in the hippocampus. The morphological alte_ra,.ions may be correlated with the physiological dendritic properties such as long-term potentiation 7 and electrotonic membrane properties s. These results support the hypothesis that following prolonged alcohol consumption there is regeneration of dendrites compensating for the damaging effects of chronic ethanol intake.

1 Buel, S.J. and Coleman, P.D., Dendritic growth in the aged human brain and failure of growth in senile dementia, Science, 206 (1979~852-854. 2 Carlen, P.L., Wilkinson, A., Wortzman, G. and Rankin, J.G., Reversible cerebral atrophy in recently abstinent alcoholics measur
3 Carlen, P.L., Wilkinson, A., Wortzman, G. and Holgate, R.C., Partially reversible cerebral atrophy and functional improvement in abstinent alcoholics, Can. J. Neurol. Sci., 11 (1984) 441--446. 4 Decarli, L.M. and Lieber, C.S., Fatty liver in the rat after prolonged intake of ethanol with a nutritionally adequate new liquid diet, J. Nutr., 91 (1967) 331-336.

This research was supported by the MRC of Canada and by the NIAAA. The autho~ would like to thank Sharon Sasaki for her assistance in the histological analysis of the samples.

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