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Journal of the Anatomical Society of India

Effect of Ethanol on Dendrites Of Hippocampal Neurons

Author(s): Mitra N.K. & Mukherjee A.

Vol. 50, No. 1 (2001-01 - 2001-06)

Department of Anatomy, M.G.M. Medical College, Kamothe, Navi Mumbai-INDIA

For Reprints, request the first author.

Abstract

Many clinical and experimental studies have established impairment of short term memory as a salient characteristicfeature of cognitive dysfunction in chronic alcoholics. Hippocampus has been reported to be involved with formation and retention of recentmemory. Previous studies (Mitra et al 1999) have found reduction in density of normal looking neurons per square mm. of section in CA-1,CA-2 and CA-3 areas of stratum pyramidalis of hippocampus in albino mice fed with 10% ethanol ad libitum. With a view to correlate thesechanges in dendritic arborization of hippocampal neurons, adult albino mice (n = 10) were fed with 10% ethanol ad libitum for 4 months.Control group was fed with water in place of ethanol. Brains were fixed with Golgi-Cox solution for 3 months and Golgi stained CA-1hippocampal neurons were traced using camera lucida. Reduction in dendritic arborization of proximal order dendrites and attenuation ofdistal order dendrites were observed in CA-1 hippocampal pyramidal neurons in ethanol fed mice compared to control group.

Key words: Alcohol Ethyl, Hippocampus, Golgi-Cox, Morphology, Mice.

Introduction:

Chronic consumption of alcohol is associated with disturbances of mnemonic functions and behavioural deficit. Korsakoffs & Serbski (1892) first described impairment of short term memory in chronic alcoholism. Milner (1959) in a clinical study found that chronic alcoholic patients and patients with hippocampal damage are similar performers in short term memory tasks, Rebecca Rausch (1993) counted volumetric cell densities of hippocampal region in resected temporal lobe of patients suffering from intractable epilepsy and tested verbal and non-verbal memory pre-operative and post-operatively. He found that degree of hippocampal neuronal loss was significantly correlated with learning & recall score of unrelated word pairs. The aim of the present study was to find out whether chronic alcohol intake affects the morphology of dendritic arborization of hippocampal pyramidal neurons or not in adult mice to extrapolate the basis of deficit in short term memory of chronic alcholism.

Materials and Methods:

Twenty adult albino mice weighing 25-30 gms each, divided into 2 groups constituted the material for the study. Group E (10) received 10% ethanol v/ v in water ad libitum for 4 months. Group C (10) as control was fed plain water ad libitum during this period. Both the groups received standard mouse feed ad libitum during this period. At the end of 4 months, the animals were anaesthetized with ether and intra-cardiac perfusion of normal saline followed by 10% formal saline was done.

The brains of both the groups were dissected out, blotted and weight was taken. The right cerebral hemispheres of both the groups were dissected out and immersed in Golgi-Cox solution for 3 months. After 3 months of fixation, the brains were dehydrated, cleared and embedded in paraffin. 35 micron coronal sections were done at the level of hippocampus. The sections were blackened by placing in 10% ammonia for an hour. Then following quick dehydration in graded alcohol, clearing was done in cedarwood oil and sections were mounted in thick canada balsam. The left half of the hemispheres after fixation with 10% formal saline and processing were embedded in paraffin.8 Micron coronal sections at the level of hippocampus were cut and stained with Kluver- Barrera to find out qualitative histological changes following ethanol intake. The Golgi-stained sections were screened for adequate impregnation of cornu-ammonis pyramidal neurons. Due to the C-shaped bend of cornu ammonis layer of pyramidal neurons and diffuse arrangement of CA-1 neurons, only CA-1 neuronal dendrites could be traced satisfactorily under camera lucida. CA-2 and CA-3 neuronal dendrites could not be traced properly as they were densely packed. Only adequately impregnated CA-1 pyramidal neurons were selected for study. The first six neurons encountered in each animal when moving from medial to lateral in the sections were selected for quantitative analysis. Camera Lucida drawings of dendritic arborization were made at a magnification of 400x. According to method of Sholl 1953, concentric circles were superimposed over the drawings of dendritic tree with increasing radius equal to geometric equivalent of 20 micron at a magnification of 400x so that cell body was placed in the centre of the innermost circle (Fig.1). The number of dendrites intersecting each circle were counted and compared group wise.

Observations and Results:

The weight of whole brain including cerebral hemisphere, cerebellum and brain stem in two groups of mice are shown in Table 1. The weights were lower in ethanol fed group E compared to

TABLE 1 BRAIN WEIGHTS OF TWO GROUPS OF MICE

  Group C Group E
Range 0.40 - 0.48 gms. 0.36- -.46 gms
Mean 0.44 gms. 0.41 gms.
S. D. ± ±0.025 ± 0.027
GROUP C = CONTROL
GROUP E = ETHANOL FED

control group C. CA-1 CA-2 CA-3 neurons of stratum pyramidalis of cornu-ammonis of hippocampus in control group of adult mice are shown in Fig. 2. Several non-pyramidal neurons were observed in dentate gyrus, stratum oriens and stratum radiatum-lacunosum but they were not included in this study due to less prominent dendritic network. In control group of animals, the CA-1 pyramidal cells showed (Fig. 2 &3) a single main apical dendrite with oblique segments branching from it. At the base multiple primary basilar dendrites extended radially. The proximal portions of the dendrites (arrow in Fig.3) were less densely covered with spines than the distal portions. CA-2 neurons had the largest cell body which were oblong (Fig.2) and had 2 main basilar dendrites emanating from the two ends of the base which subsequently got branched. CA-2 dendrites did not show prominent spines. CA-3 pyramidal cells were larger than CA-1 cells (Fig.4). The middle and basal part of these cells had rounded appearance compared to the CA-1 cells. Apical dendrites branched closer to the cell body in these cells and spines were larger and prominent compared to those in CA-1 cells (arrow in Fig.4). Axons of the cells were seen as thick black bands proximal to cell body and were not stained distally towards the edge of the sections. Fig. 5 shows dendritic arborization of distal part of the apical stem dendrites of control group of CA-1 neuron. In some of the ethanol fed animals in group E, cell bodies were smaller compared to that in control group of animals (Fig. 7). But some CA-1 pyramidal cells (as shown in Fig. 6) showed cell bodies of size similar to that of control groups of animals. Dendritic branching of CA-1 neurons were found to be attenuated in all the group E mice compared to that in group C (Fig. 6, Fig. 7 Compared to Fig. 3, Fig. 5). Dendrites emanating particularly from basal part of the cells were found stunted in appearance (Fig.7). Dendritic spines were also apparently smaller in size and seem to be reduced in number compared to that in control group C. Fig. 8 & Fig. 9 show the qualitative change in CA-1 neurons following ethanol intake. The control group of CA-1 cells showed prominent nissl granules, nucleolus and well defined nuclear membrane whereas the experimental ethanol fed group of the CA-1 cells showed lack of nissl granules, pale nucleolus, discountinuity of nuclear membrance (x in Fig. 9) Neuropil also showed vacuolation and luxol fast blue stained myelinated fibres were darker, coarser and fragmented.

In quantitative study (Table 2), the count of the dendritic intersections with concentric circles in both the groups of animals were highest (7 to 8 in group C & 3 to 5 in group E) in innermost concentric circles. In the outermost concentric circle, dendritic intersections were ranging between 1 to 2 in control group whereas no intersections could be counted in ethanol fed group E as distal order dendrites were found to be absent in the mice of ethanol fed group. The dendritic intersections of CA-1 pyramidal neurons were reduced significantly in ethanol fed group E (p<0.001) compared to that in control group C in concentric circles of 20, 40 and 60 microns. The dendritic intersections could not be counted in 80 and 100 micron concentric circles in ethanol fed group E animals as the dendritic tree was found to be attenuated in distal order dendrites following intake of ethanol.

TABLE 2 MEAN + (S. D.). COUNT OF DENDRITIC INTER SECTIONS OF CA-1 NEURONS WITH CONCENTRIC CIRCLES IN CAMERA LUCIDA DRAWINGS

  20m 40m 60m 80m 100m
GROUP C 7.43 ± 0.38 6.83 ± 0.36 5.2 ± 0.15 3.54 ± 0.16 1.26 ± 0.86
GROUP E 4.85 + 0.79* 3.27 + 1.9* 1.16 + 0.56* - -
*represent statistical significance p < 0.001, comparisons were made with group C
GROUP C = CONTROL, GROUP E = ETHANOL FED

Discussion:

Jha & Nag, 1994 in a clinical and neurological evaluation of 62 chronic alcoholics consuming indigenous alcohol found significant impairment of cognitive functions especially in calculation, immediate recall and attention.

Mitra et al, 1999 found significant reduction (p<0.001) in count of density of normal looking neurons per sq. mm section in CA-1, CA-2, CA-3 areas of hippocampus in mice fed with 10% ethanol for 6 months. Freund & Walker 1971 established evidence of impaired maze learning and avoidance of learning process in mice by neurotoxic effect of alcohol. The present study showed reduction in dendritic arborization in proximal order dendrites and attenuation of distal order dendrites of CA-1 pyramidal neurons in hippocampus of albino mice fed with 10% ethanol ad libitum for 4 months. There is also evidence of qualitative changes in the form of loss of nissl granules in CA-1 neurons following the effect of ethanol. Zhou et al (1991) found reduction of serotonergic fibres in hippocampal neurons in selectively bred alcohol preferring rats. Hence it can be concluded that neurotoxic effects of alcohol selectively damages cornu ammonis pyramidal neurons of hippocampus which may be the probable cause of deficit in learning process and short term memory in chronic alcohol abuse.

References :

  1. Freund, G., Walker, D. W (1971) : Impairment of avoidance learning by prolonged ethanol consumption in mice. Journal of Pharmacology & Experimental Therapeutics, 292 174 - 284.
  2. Jha, S. and Nag, D. (1984) : Some observations on clinical, cognitive and neurophysiological changes in subjects consuming indigenous alcohol. Indian Journal of Physiology & Pharmacology, 38(4): 277-280.
  3. Korsakoff, S. S., Serbski, W. (1892) : Ein fall von poly neurifischer psychose nit antopsie. Archive fure Psychiatrie und Nerven Krankhiten, 23 : 112-134.
  4. Milner, B. (1959): The memory deficit in bilateral hippocampal lesions. Psychiatric. Research Report. 11 : 43-52.
  5. Mitra, NK., Ghosh, S. K. and Angalagan, J. (1999) : Effect of alcohol on hippocampal neurons-A histomorphometric study. Biomedicine, 19 (2) : 155-162
  6. Rebecca Rausch (1993) : Hippocampal neuronal loss and memory scores before and after temporal lobe surgery for epilepsy Archives of Neurology, 50 : 812-817.
  7. Sholl, D. A (1953) : Dendritic organization in the neurons of the visual and motor cortices of the cat. Journal of Anatomy 87 : 387-406.
  8. Zhou, F. C., Bledsoe, S., Lumeng. L., Li, T. K. (1991) : Immunostained serotonergic fibres are decreased in selected brain regions of alcohol preferring rats. Alcohol, 8(6) : 425-431.

J. Anat. Soc. India 50(1) 28-30 (2001)

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Diagram showing the projection of concentric circles around the camera lucida drawing of CA-1 hippocampal neurons.

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Fig. 2. Photomicrograph of hippocampal area in control group of mice showing CA-1, CA-2 & CA-3 neurons (Golgi-cox-x100).

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Fig. 3. Photomicropgraph of CA-1 neuron in control group of mice. Arrow points towards dendritic spine (Golgi-Cox-x400).

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Fig 4: Photomicrograph of CA-3 neuron in control group of mice. Arrow points towards prominent bulbous spine (Golgi-Cox 400).

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Fig 5: Photomicrograph of a part of distal portion of branching of apical dendrite of CA-1 neuron in control group of mice (Golgi-Cox—x400).

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Fig 6: Photomicrograph of CA-1 neurons in ethanol fed group of mice. Compared to Control group, the branching of apical dendrites are less (Golgi-Cox—x400).

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Fig. 7. Photomicrograph of CA-1 neurons in ethanol fed group of mice. Cell bodies are smaller, dendritic Fig. 8. Photomicrograph of CA-1 neurons in control group branchings are attenuated (Golgi-Cox-x400). of mice. Nissl granules are prominent as a rim around nuclear membrane (Kluver-Barrera&-X1000) Fig 8: Photomicrograph of CA-1 neurons in control group of mice. Nissl granules are prominent as a rim around nuclear membrane Kluver-Barrera—X1000)

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Fig. 9. Photomicrograph of CA-1 neurons in ethanol fed group of mice. Nissl granules are absent with pale nucleoli and darker, coarser, fragmented myelinated fibres (Kluver-Barrera-X1000)

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