Brain Research 879 2000 55–64 www.elsevier.com locate bres
Research report
Short-term ethanol exposure alters calbindin D28k and glial fibrillary acidic protein immunoreactivity in hippocampus of mice
a , a
a b
b
Irawan Satriotomo , Takanori Miki , Masahiro Itoh , Kiyoshi Ameno , Iwao Ijiri ,
a
Yoshiki Takeuchi
a
Department of Anatomy , Faculty of Medicine, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Ikenobe, Kagawa 761-0793, Japan
b
Department of Forensic Medicine , Faculty of Medicine, Kagawa Medical University, 1750-1, Miki-cho, Kita-gun, Ikenobe, Kagawa 761-0793 Japan
Accepted 18 July 2000
Abstract
The effects of a short-term ethanol treatment on hippocampus have been studied in mice exhibiting intoxication signs. The alterations of neurons and astrocytes as well as quantitative changes of calbindin D28k-immunoreactivity and glial fibrillary acidic protein-
immunoreactivity GFAP-IR in selected regions of the dorsal hippocampus were examined using anti-calbindin and anti-GFAP monoclonal anti-body mAb, respectively. The administration of 6 v v ethanol during first week led to the neuronal death and
decrease of the total number of calbindin-IR neurons in the examined brain regions. Moreover, the calbindin positive neurons were shown to have diminished processes following short-term ethanol exposure. These neuronal changes were associated with the increase of the
GFAP-IR astrocytes. Hypertrophy of cell bodies and cytoplasmic processes of reactive astrocytes were also seen. In addition, dense, thick and highly-stained GFAP-IR cells with long processes in granular cell layer appeared entering into molecular layer of dentate gyrus. In
agreement with the discrepancy percentage of neuronal cell loss and increase of reactive astrocytes detected by calbindin and GFAP-IR using image quantitative analysis, the regional differences in the vulnerability to the neurotoxic effects following short-term ethanol
exposure were found: CA3.CA2.CA1.DG. These findings also illustrate the importance of correlation between calbindin and GFAP-IR when determining the morphological alteration of neuron and astroglial following short-term ethanol treatment. The disruption
of calbindin and GFAP could affect neuronal-astroglial interaction, resulting in disturbance of behaviors dependent on hippocampus.
2000 Elsevier Science B.V. All rights reserved.
Theme : Neural basis of behaviour
Topic : Drugs of abuse: alcohol, barbiturates, and benzodiazepines
Keywords : Hippocampus; Short-term ethanol exposure; Intoxication signs; Glial fibrillary acidic protein GFAP; Calbindin D28k
1. Introduction resulted in a wide variety of behavioral tasks and processes
[7]. Recently it has been recognized that the method used Ethanol exposure has been shown to result in alteration
for ethanol exposure may be important, both in the in morphology and function of the central nervous system
induction of dependence and degree of damage inflicted on CNS. Behaviorial studies on the effects of ethanol
the CNS. In this context, it has been demonstrated that treatment suggest that the hippocampus, which has been
high peak blood ethanol concentrations maintained for particularly identified as one of the targets for neurotoxic
relatively short periods of time may be critical in disturb- effects, is more sensitive than other regions and plays a
ing brain functions not only in developing brain [28,29] prominent role in memory and learning processes [9].
but also mature brain studies [5,35]. Damage to this structure by acute ethanol treatment
Previous studies suggested that glutamate excitotoxicity- like processes and related changes in calcium levels were
implied in the toxic effects of alcohol [25]. Such a
Corresponding author. Tel.: 181-87-891-2087; fax: 181-87-891-
mechanism is also possible in hippocampus, since gluta-
2088. E-mail address
: irawankms.ac.jp I. Satriotomo.
mate is the main excitatory neurotransmitter in the hip-
0006-8993 00 – see front matter
2000 Elsevier Science B.V. All rights reserved. P I I : S 0 0 0 6 - 8 9 9 3 0 0 0 2 7 2 9 - 3
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pocampus and the importance of Ca for the functioning
diet except that sucrose was substituted isocalorically for and survival of hippocampal cells has been well estab-
ethanol. After 4–5 days, the mice which exhibited stage 2 lished. Calcium binding proteins have been implicated as
or 3 of intoxication signs according to Freund’s classifica- important regulators of neuronal degeneration in pathologi-
tion [10] were selected for morphological observation. The cal process [1]. Calbindin-D28k, which is a member of the
intoxication signs consisted of four stages, such as hyper- superfamily of calcium binding proteins is implicated in
reactivity and tremor stage 1; episode of rapid beating the regulation of intracellular calcium and as a marker of a
motion of the tail, slow, broad-based gait and rapid neuronal subpopulation [30]. In the adult mammalian
backward movements retropulsion; stereotype or repeti- brain, calbindin was thought to be present only in neurons,
tive movement stage 2; generalized tonic–clonic convul- which is believed to serve a neuroprotective role by its
sion stage 3; and death during convulsion stage 4. The calcium-buffering abilities [23].
approval of the Kagawa Medical University Animal Com- Astrocytes were originally thought to play only suppor-
mittee was obtained for this study. tive roles in the brain. Recently, there have been many
The animals were deeply anaesthetized with sodium reports concerning the functions of astrocytes including
pentobarbital 40 mg kg i.p.. To measure the blood neurotransmitter uptake [15], synthesis and secretion of
alcohol concentration BAC when the animals showed the neurotrophic factors [11]. Analysis targets of astrocytic
intoxication signs, blood was taken from the axilla-vessels reaction led the hypothesis that astrocytes are primary
of the mice and analyzed by the gas chromatograph targets of chemical injury and mediates the phenotypic
Shimadzu GC-8A, Tokyo, Japan. Four mice of the expression of chemical injury in the CNS [2]. Glial
control group and 6 mice of the experimental group were fibrillary acidic protein GFAP, which is the major protein
then perfused transcardially with a fixative of 10 for- of the intermediate filaments of astroglial cells, is the most
malin neutral buffer solution pH 7.4, Wako Co. Ltd., commonly used method to examine the distribution of
Osaka, Japan for preparation of paraffin embedded sec- astrocytes and the hypertrophy of astrocytes in response to
tions. Serial 7 mm-thick sections were cut coronally and neural degeneration or injury [21].
stained with cresyl-violet. The remaining animals of each Our previous report about the effects of short-term
group were perfused with 0.02 M phosphate buffered ethanol exposure on the suprachiasmatic nucleus SCN of
saline PBS, pH 7.4 followed by a fixative of 4 the hypothalamus of mice demonstrated that there was no
paraformaldehyde in PBS. The brains were removed and change on the calbindin D28k-IR with the increase on the
cryoprotected by immersion in 30 w v sucrose at 48C GFAP-IR [33]. The question arise that whether similar
until the brain sank. Serial 40 mm-thick frozen sections immunoreactivity changes are seen or not in the hippocam-
were cut coronally and processed for immunohistochemis- pus. Therefore, the aim of the present study was to
try. Free floating sections were immunostained for calbin- investigate the alteration calbindin D28k-IR neurons and
din D28k or GFAP using monoclonal antibody. To stain GFAP-IR astrocytes in the hippocampus of mice exhibiting
for either GFAP or calbindin D28k, the sections were intoxication signs in response to short-term ethanol treat-
incubated in the following solutions and between each ment. To understand which are particularly vulnerable or
steps the sections were rinse with PBS: 10 normal goat invulnerable to short-term ethanol exposure, the possible
serum in PBS with 0.2 Triton X-100 for 30 min; anti- interaction between short-term sensitivity to ethanol and
GFAP rabbit antibody 1:2000 dilution, DAKO, Glostrup, differences in the subfields of hippocampus were also
Denmark or anti-calbindin D28k mouse monoclonal anti- examined.
body 1:500 dilution, Sigma Chemical Company, St. Louis, MO overnight; biotinylated anti rabbit IgG or
biotinylated anti mouse IgG for 1 h; avidin–biotin complex
2. Materials and methods Vectastain, Elite ABC Kit, Vector Lab. Burlingame USA
