Neuroscience Letters, 120 (1990) 271-274 271 Elsevier Scientific Publishers Ireland Ltd.

NSL 07374

Acute administration of ethanol suppresses pentylenetetrazole-induced c-fos expression in rat brain

Fei Le, Peter Wilce 1, Ian Cassady 2, David Hume a and Brian Shanley 1

1Alcohol Research Unit, Department of Biochemistry and 2Centre for Molecular Biology and Biotechnology, University of Queensland, Queensland (Australia)

(Received 1 March 1990; Revised version received 13 August 1990; Accepted 27 August 1990)

Key words: Alcohol; c-fos; Proto-oncogene; Pentylenetetrazole; Northern blot

The effect of acute ethanol administration on pentylenetetrazole-induced c-fos expression in rat brain was studied. Pentylenetetrazole induced the rapid and transient expression ofc-fos mRNA in rat brain. Maximal induction at a dose of 30 mg/kg was detected within 30 rain and persisted for 60 min. Thereafter c-fos gene expression decreased to control levels by 180 rain. No increase in c-fos mRNA was evident at doses of pentylene- tetrazole ,<,< 20 mg/kg, whereas maximal elevation was seen at 30 or 40 mg/kg. This action was inhibited by acute ethanol treatment (blood alcohol level/> 100 mg/dl). Acute ethanol treatment alone had no effect on c-fos gene expression.

The involvement of the inhibitory neurotransmitter, ~,-aminobutyric acid (GABA), in the actions of ethanol on the central nervous system has been demonstrated by a number of workers [7]. For instance, it is known that sedation, sleep and anaesthesia produced by relatively high doses of ethanol in rodents (2.0-5.0 g/kg b.wt.) are enhanced by GABA agonists and counteracted by GABA antagonists [2, 4, 10]. Furthermore, ethanol has been reported to potentiate the inhibition of cortical neurons by GABA in cats [16] and to inhibit the firing of pars reticulata neurons in the rat through a GABA- ergic mechanism [13].

The mode of action of ethanol on the GABA-benzo- diazepine receptor complex may be multifacetted. Etha- nol has been found to potentiate GABA-mediated 36Cl-

fluxes in cultured spinal cord neurons [12] and in synap- toneurosomes [21]. Liljequist et al. [8] and Hillmann et al. [6] have suggested that ethanol alters the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to the picrotoxin site on the GABA-benzodiazepine receptor complex, both in vitro and in vivo. De Vries et al. [3] and Hillmann et al. [5] have shown a disruption of the inter- action between the receptor subunits after chronic etha- nol treatment.

There is substantial evidence that the convulsant ac- tion of pentylenetetrazole (PTZ) is mediated via the GABA-benzodiazepine receptor complex. PTZ has been

Correspondence: F. Le, Alcohol Research Unit, Department of Bio- chemistry, University of Queensland, Queensland 4072, Australia.

shown to interact competitively with the binding of both [3H]diazepam [19] and [35S]TBPS to the GABA-benzo- diazepine receptor complex [18]. Moreover, Squires et al. [20] have shown for several tetrazole derivatives that there is a good correlation between relative affinity for the [3sS]TBPS binding site and convulsant potency.

Recently, PTZ has been reported to induce expression of the proto-oncogene c-fos in mouse brain [14]. The induction of c-fos by PTZ was found to be completely abolished by the administration of diazepam [14], sug- gesting an action via the GABA-benzodiazepine recep- tor. In view of the known interaction of ethanol with the GABA-benzodiazepine receptor complex, we investi- gated the effect of acute sub-hypnotic doses of ethanol on PTZ-induced expression of c-fos.

A complete c-fos cDNA probe was kindly provided by Prof. J. Pettigrew (Centre for Vision, Touch and Hear- ing, University of Queensland, Australia) and fl-actin cDNA probe was provided by Dr. J.C. Wallace, Univer- sity of Adelaide, Australia. Probes were labeled with [32p]dCTP (370 MBq/ml) by the random priming meth- od (Pharmacia-Oligolabelling Kit or Amersham Multi- prime DNA labelling Kit) and purified by Sephadex G-50 column chromatography.

Male Wistar rats (300-400 g b.wt.) were injected intra- peritoneally with varying doses of PTZ or ethanol in saline. Control rats received an equivalent volume of saline. Where both drugs were administered, ethanol was injected 10 min before PTZ treatment. The animals were killed by decapitation 60 min thereafter. The effect of

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272

0 15 30 60 90 180 min

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8 0 0

6 0 0

N

~ 4 0 0

0 U

2 2 0 O

- - ~ - act in 0 o lo

0 15 3 0 60 go 180

Penty lenete t rozo le t ime course ( ra in)

Fig. I. A: time course of pentylenetetrazole induction of c-fos expression. Animals were injected with pentylenetetrazole (30 mg/kg b.wt.) and killed by decapitation at the times indicated. Total brain RNA was prepared from groups each containing two or three animals, separated by agarose gel electrophoresis and transferred by Northern blotting as described in the text. Filters were probed with rat c-fos cDNA and chicken ~-actin cDNA

probes. B: determination of the relative band intensities in A was performed by image analysis as described in the text.

ethanol alone was examined by injection either 10 min

or 1 h before sacrifice. Blood alcohol levels were deter-

mined using the method o f Lundquist [9]. Whole brains

were rapidly removed and immediately frozen in liquid

nitrogen. For each observation brains from 2 or 3 ani-

rnals were pooled and used for the preparation o f total

R N A by the method o f Brooker et al. [1]. After ethanol

precipitation, R N A was dissolved in 1 x TE buffer (10

m M Tris-HC1, 0.1 m M E D T A , pH 8.0) and stored at

- 2 0 ° C . Quanti tat ion and purity were assessed by

0 10 20 30 40 mg kg -~

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o

o

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-- ~ - ac t in

8 0 0 .

600 -

4 0 0

2 0 0

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0 1Q 20 30 40

Penty lene te t razo le dose- response (mg / kg )

Fig. 2. A: dose response curve for pentylenetetrazole induction ofc-fos expression. Animals were injected with pentylenetetrazole at the doses shown. They were killed 60 min after treatment and total brain RNA was prepared from groups each containing two or three animals, treated and analysed

as described in Fig. 1. B: determination of the relative band intensities in A was performed by image analysis as described in the text.

273

measurement of the absorbance at 260 and 280 nm. Northern blot analysis was performed using Hybond-N nylon membranes (Amersham, Australia) according to the protocol supplied by the manufacturer. Total RNA (approximately 50 pg) was separated by electrophoresis in 1% (w/v) agarose-formaldehyde gels and transferred onto Hybond-N membranes. Membranes were prehybri- dized for 2-4 h at 42°C in hybridization buffer (5 x SSC, 50% (v/v) deionized formamide, 5 x Denhardt's, 50 pg/ ml denatured herring sperm DNA) and then hybridized with 32p-labeled DNA probes for 16-24 h at 42°C in hybridization buffer. After washing in 0.5 x SSC con- taining 0.1% (w/v) sodium dodecylsulfate (SDS) at 62°C, membranes were exposed to Fuji X-ray film for 4-24 h. Band intensities were measured by the AVID image ana- lysis system (Biomedical Computing Systems, Austra- lia.).

Figs. 1 and 2 represent the results of a single experi- ment and Figs. 3 and 4 are representive of 3 separate experiments. Increased c-fos expression was detected within 15 min of PTZ injection and remained elevated for 90 min, before returning to the pretreatment value by 180 min (Fig. 1). This time course is very similar to that previously reported in mice [14]. The dose-response curve is shown in Fig. 2. There was no increase in c-fos mRNA at a dose of PTZ ~< 20 mg/kg b.wt. However, a sharp elevation of c-fos mRNA level was seen at 30 mg/

kg b.wt. No further increase in c-fos gene expression was observed at higher doses. Doses of PTZ less than 20 m[~/ kg b.wt. failed to induce convulsions. However, at doses of 30 mg/kg b.wt. or above, convulsions occurred within 2-6 min after the injection of PTZ. It would appear that induction of c-fos mRNA is a consequence of the con- vulsant effect of PTZ, as has been observed previously in mice [14]. Alterations in the level of fl-actin mRNA were not produced by PTZ administration (Figs. 1 and 2).

Northern blot analysis indicated that low doses of eth- anol (blood alcohol level ~< 50 mg/dl) had no significant effect on PTZ-induced synthesis of brain c-fos mRNA (Fig. 3). Higher doses of ethanol (blood alcohol level ~>100 mg/dl) obviously decreased the level of c-fos mRNA induced by PTZ. Ethanol alone, at either low or high doses over a short or longer period, did not induce c-fos gene expression (Fig. 4).

In neurons, c-fos expression can be induced by phar- macological, electrical, surgical and physiological stimuli [15]. The PTZ-induced seizure model has proved useful in elucidating the biochemical events following c-fos expression. In Fos-specific areas, e.g. the hippocampus, a brief period of stimulation results in the induction of c-fos expression which is rapidly followed by the accu- mulation of Fos and Fos-related proteins. These pro- teins participate in AP-1 DNA binding and presumably

1 2 3 4 5 6

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B lood e t h a n o l level ( m g / d l )

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Fig. 3. A: effect of ethanol on pentylenetetrazole-induced c-fos expression. Animals were injected with pentylenetetrazole alone (30 mg/kg b.wt., lane 1), or after ethanol administration to give the blood alcohol concentrations shown in B. Ethanol was injected 10 minutes before administration of pentylenetetrazole (30 mg/kg). Animals were killed 60 min after pentylenetetrazole treatment and total brain RNA was prepared from groups each containing two or three animals, treated and analysed as described in Fig. 1. B: determination of the relative band intensities in A was performed

by image analysis as described in the text.

274

1 2 3 4 5 6

- - C - fos

~-octin

Fig. 4. Effect of ethanol on c-fos expression. Animals were injected with saline (lane 1), pentylenetetrazole (30 mg/kg b.wt., lane 2), 0.5 ml of ethanol (16% w/v) (lanes 3, 4) or 5 ml of ethanol (16% w/v) (lanes 5, 6). Animals were killed 10 min (lanes 3, 5) or 60 min (lanes 1, 2, 4, 6) after treatment. Total brain RNA was prepared from groups each

containing two or three animals and analysed as described in Fig. 1.

have effects on express ion o f o t h e r genes, e.g. p r ep ro -

enkepha l in [15]. The molecu la r mechan i sm o f increased

c-fos expression, af ter seizures induced by PTZ, is

t hough t to resul t f rom selective b lockade o f the ch lor ide

channel th rough in te rac t ion with the G A B A - b e n z o d i a -

zepine recep tor complex [17]. Induc t ion o f c-fos expres-

sion by P T Z is comple te ly abo l i shed by d i azepam [14]

which acts as an an t i convu l san t th rough agonis t ic inter-

ac t ion with the G A B A - b e n z o d i a z e p i n e receptor com-

plex [11]. As ind ica ted above m a n y o f the acute p h a r m a -

cological effects o f e thano l can be re la ted to its effects on

G A B A e r g i c neuro t ransmiss ion [7]. Therefore , it is

r easonab le to speculate tha t the G A B A - b e n z o d i a z e p i n e

receptor complex m a y be specifically involved in the in-

te rac t ion o f e thano l and P T Z on c-fos expression, as

opposed to a nonspecif ic an t i convu l san t ac t ion o f e tha-

nol.

The results o f the present s tudy suggest a n u m b e r o f

avenues for fur ther invest igat ion. In view o f the sensiti-

vity o f the P T Z mode l to inh ib i t ion at relat ively low

b lood e thano l concen t ra t ions , fur ther s tudies using tech-

niques such as in .situ hybr id i za t ion o r immunoh i s to -

chemis t ry cou ld yield interest ing in fo rma t ion a b o u t re-

g ional sensi t ivi ty o f the b ra in to the effects o f e thanol .

Fos is involved in a n u m b e r o f b ra in phenomena , includ-

ing gene expression, p las t ic i ty and deve lopmen t [15]. I f

e thanol has more general effects on the induc t ion o f c-fos by phys io logica l st imuli , this could well expla in m a n y o f

its diverse ac t ions on bra in deve lopmen t and funct ion.

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