Molecular Brain Research, 15 (1992) 327-331
327
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00
BRES 70485
M u s c i m o l - i n d u c e d reduction of G A B A A receptor a l-subunit m R N A in primary cultured cerebral cortical neurons Masaaki Hirouchi, Seitaro Ohkuma and Kinya Kuriyama Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto (Japan) (Accepted 19 May 1992)
Key words: Muscimol; GABA A receptor ai-subunit; mRNA; Neuron; Primary culture; RNA blot analysis; Ribonuclease protection assay
The expression of mRNA for GABA A receptor ai-subunit in mouse cerebral cortical neurons in primary culture was examined using RNA blot analysis and ribonuclease protection assay following the treatment of neurons with muscimol, a selective agonist of GABA A receptor. The level of mRNA for GABA A receptor al-SUbunit showed a decrease in comparison with that in non-treated cells, whereas no changes in the level of fl-actin mRNA were noted under the same experimental conditions. This muscimol-induced reduction in GABAA receptor cq-subunit mRNA was counteracted by the simultaneous exposure of neurons to both bicuculline, an antagonist of GABA A receptor, and muscimol. The expression of mRNA for GABA A receptor al-subunit also showed a decline by the treatment of cells with flunitrazepam alone, an agonist of benzodiazepine receptor, and this change was also abolished by the simultaneous exposure of cells to flunitrazepam and Ro15-1788, an antagonist for central benzodiazepine receptor. These results suggest that the continuous stimulation of cerebral GABA A receptor complex may induce the reduced expression of mRNA for the receptor complex.
INTRODUCTION G A B A ( y - a m i n o b u t y r i c acid) A r e c e p t o r is a heterooligomeric p r o t e i n complex consisting of several h o m o l o g o u s m e m b r a n e - s p a n n i n g s u b u n i t s 18. Recently, c D N A c l o n i n g has revealed that G A B A A r e c e p t o r consists of several s u b u n i t s d e n o t e d as a , /3, y, 6 a n d p5,20,23,24. I n addition, the h e t e r o g e n e i t y in each s u b u n i t has i n d i c a t e d the p r e s e n c e of h o m o l o g o u s s u b u n i t s such as 0~1-0~13'14'21'26 a n d a l t e r n a t i v e splicing s u b u n i t s such as /34, y~,tl. T h e s e G A B A g r e c e p t o r s u b u n i t s c o n s e r v e d four t r a n s m e m b r a n e d o m a i n s similar to those in o t h e r i o n o t r o p i c receptors t. T h e r e f o r e , it has b e e n c o n c l u d e d that C l - ion c h a n n e l of G A B A A receptor b e l o n g s to a family of l i g a n d - g a t e d ion c h a n nels 25. W e previously r e p o r t e d the results of c D N A c l o n i n g o n G A B A A r e c e p t o r t~t-subunit from the h u m a n b r a i n 8. T h e expression of m R N A for G A B A A r e c e p t o r a~s u b u n i t , e x a m i n e d by R N A blot h y b r i d i z a t i o n using this c D N A as a probe, has i n d i c a t e d that t h e r e are two m a j o r m R N A species in b o t h rat a n d m o u s e brains. I n
addition, we have d e m o n s t r a t e d that the expression of m R N A for G A B A A r e c e p t o r al-SUbunit rapidly increases d u r i n g the d e v e l o p m e n t of p r i m a r y c u l t u r e d n e u r o n s 9. Since it is i m p o r t a n t to clarify w h e t h e r or not the expression of m R N A for G A B A A receptor is m o d u lated by a c o n t i n u o u s s t i m u l a t i o n of the receptor, the level of m R N A for G A B A A r e c e p t o r a t - s u b u n i t has b e e n e x a m i n e d following the t r e a t m e n t of p r i m a r y c u l t u r e d cerebral cortical n e u r o n s with muscimol, a selective G A B A A r e c e p t o r agonist. MATERIALS AND METHODS
Materials
[Ol-32P]dCTP and [a-32P]UTP were obtained from New England Nuclear (Boston, MA, USA). FASTTrack for mRNA isolation was purchased from Invitrogen (San Diego, USA). Ambion Inc. (TX, USA) was a source of ribonuclease protection assay kit (RPA II). Other drugs used were of analytical grade and commercially available. Neuronal cultures Primary culture of mouse cerebral cortical neurons was carried out as described previously9'10'17. The neopaUium was isolated from
Correspondence: K. Kuriyama, Department of Pharmacology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan. Fax: (81) 75-241-0824.
328 15-days-old fetus of ddy strain mouse under ether anesthesia, trypsinized and filtered through a nylon mesh. Isolated cells were inoculated on a poly-L-lysine-coated dish containing modified Eagle's minimum essential medium (MEM) supplemented with 15% fetal calf serum and cultured for 3 days. After the treatment with 10/xM cytosine arabinoside in MEM containing 10% horse serum for 24 h, the culture medium was exchanged to MEM with 10% horse serum. Thereafter, the culture medium was exchanged to fresh one (MEM with 10% horse serum) every 4 days. Preparation of mRNA mRNA was obtained from neurons cultured for 10 days in all experiments. After washing neuronal cells with phosphate-buffered saline, neurons were scraped off from a culture dish with a rubber policeman. Poly(A) + RNAs were isolated from these cells using FASTTrack. RNA blot analysis RNA blot hybridization was performed as previously described 8. Denatured poly(A) + RNA (5 /xg) was electrophoresed on 1.5% agarose gel containing formaldehyde, and blotted onto a nitrocellulose filter. The filter was hybridized with 50 mM sodium phosphate buffer (pH 7.0), 5 × standard saline citrate (SSC), 50 p~g/ml salmon sperm DNA, 1 × Denhardt's solution, 30% formamide, 10% dextran sulfate and 3zp-labeled probe at 42°C. After the hybridization, the filter was washed with 2× SSC and 0.1% sodium lauryl sulfate at 50°C. The A5 eDNA fragment of GABA A receptor oq-subunit from the human brain was labeled with [a-32p]dCTP by the random-primed DNA labeling method and the denatured labeled DNA was used as a probe s. Ribonuclease protection assay The detection of mRNA for GABA A receptor at-subunit was carried out by the ribonuclease protection method according to the instruction manual for RPA II. Poly(A) ÷ RNA (0.8 /zg) obtained from neuronal cells was used for each assay. The A5 eDNA fragment (420 base pairs (bp), Hirouchi et al. s) was ligated within the EcoRI site of pBluescriptlI KS(+). Following the digestion of EcoT 221, the A5 eDNA fragment (257 bp) was transcribed to its anti-sense RNA with [a-32p]UTP by the catalytic actions of T3 RNA polymerase. The 32p-labeled anti-sense RNA for /3-actin (250 bp) was used as a control. These 32p-labeled anti-sense RNAs were incubated with each sample RNA at 42°C. The hybridized RNA was then digested with RNase A and RNase T1. The protected RNA was electrophoresed on 50% urea/6% polyacrylamide gel. The detection of RNA was performed using Image analyzer (BAS 2000, Fujifilm).
A
C
tomb
7.4
m
5.3
--
2 . 8
--
1.9
---
1.6
•
Fig. 1. RNA blot analysis on GABA A receptor cq-subunit mRNA in primary cultured neurons following treatment with muscimol. A: control. B: 1 day treatment prior to the isolation. C: 4 days treatment prior to the isolation. RNA molecular weight markers (kb) are indicated on the left hand side.
In a d d i t i o n , t h e t i m e c o u r s e o f c h a n g e s in t h e expression of mRNA
for G A B A A r e c e p t o r
at-subunit
f o l l o w i n g t h e t r e a t m e n t w i t h m u s c i m o l was e x a m i n e d using a solution hybridization
procedure
s u c h as ri-
b o n u c l e a s e p r o t e c t i o n assay. T h e level o f m R N A
for
G A B A A r e c e p t o r a z - s u b u n i t in cells t r e a t e d w i t h m u s c i m o l for 4 h o u r s s h o w e d a s i g n i f i c a n t r e d u c t i o n , alt h o u g h little c h a n g e in t h e e x p r e s s i o n was r e c o g n i z e d following the treatment reduction of mRNA
for 1 o r 2 h (Fig. 2). T h i s
was also f o u n d a f t e r t h e e x p o s u r e
o f m u s c i m o l for 72 h. T h e l e v e l o f m R N A
for G A B A A
r e c e p t o r a ] - s u b u n i t in t h e cells i n c u b a t e d for 24 h w i t h muscimol
RESULTS
B
was
about
70%
of the
level d e t e c t e d
in
n o n - t r e a t e d cells. In c o n t r a s t , t h e e x p r e s s i o n o f m R N A
Effect o f m u s c i m o l treatment on G A B A A receptor a 1subunit m R N A M o u s e c e r e b r a l c o r t i c a l n e u r o n s in p r i m a r y c u l t u r e w e r e c u l t u r e d for 10 days. T h e G A B A A r e c e p t o r a ] subunit mRNA
in n e u r o n a l cells, e x p o s e d to 10 / z M
.•
~
~
I
...a._
100
m u s c i m o l for 1 o r 4 days p r i o r to t h e i s o l a t i o n o f e a c h p o l y ( A ) + R N A , was a n a l y z e d by R N A b l o t h y b r i d i z a -
50
tion. Fig. 1 s h o w s t h e a u t o r a d i o g r a p h y o f R N A
b l o t hy-
b r i d i z a t i o n for G A B A A r e c e p t o r
cq-subunit mRNA.
Although the expression of mRNA
for GABA A recep-
t o r a ~ - s u b u n i t in p r i m a r y c u l t u r e d n e u r o n s s i g n i f i c a n t l y r e d u c e d f o l l o w i n g t h e t r e a t m e n t w i t h m u s c i m o l for 1 day, t h e m R N A
in t h e cells t r e a t e d w i t h m u s c i m o l for
4 days e x h i b i t e d s i m i l a r e x t e n t o f t h e e x p r e s s i o n c o m p a r e d w i t h t h a t in c o n t r o l .
as
0 I 2 4 24 48 72 Incubation T i u ( h r s )
96
Fig. 2. Time course of changes in the level of GABA A receptor a~-subunit mRNA in primary cultured neurons following treatment with muscimol. Values are mean ± S.E.M. obtained from 5 to 6 separate determinations. * P < 0.05, * * P < 0.0l, compared with the value obtained at 0 time (Scheffe's test).
329 GABAAReceptor c(,-Subonit mRNA(| of Control l 50 100
•
1-1-
I0o
t
1
_.t_
Control
~ ¢
,.o,.o, ~
so
I
[ 0
1
2
4
24 48 72 96
Incubation Timelhrs) Fig. 3. Time course of changes in the level of fl-actin m R N A in primary cultured neurons following treatment with muscimol. Values are m e a n +S.E.M. olrtained from three separate determinations.
in the cells exposed to muscimol for 96 h was similar to that in non-treated cells as shown in Fig. 1. The expression of mRNA for/3-actin was simultaneously studied in primary cultured neurons exposed to 10/zM muscimol. The expression of mRNA for fl-actin had no significant change following the exposure to muscimol (Fig. 3). The muscimol-induced reduction in the expression of mRNA for GABA A receptor t~-subunit was also dose-dependent (Fig. 4).
Effect of bicuculline on muscimol-induced reduction in the expression of GABA~ receptor t~l-SUbunit mRNA Whether the reduction of GABA A receptor ~-subunit mRNA induced by muscimol is mediated via GABA A receptor has been investigated by simultaneous exposure of cells to both muscimol and bicuculline, a GABA A receptor antagonist. The simultaneous treatment of cells with bicuculline and muscimol for 1 day abolished the muscimol-induced reduction of mRNA for GABA A receptor al-subunit, although bicuculline alone showed no effect on the expression of GABA g receptor al-SUbunit mRNA (Fig. 5).
!
~
i
|
i-
luso i moI + nicoeuliino
i I
i
,]"
Bicuculline
t I
I
Fig. 5. Effect of bicuculline on muscimol-induced reduction in G A B A A receptor a r s u b u n i t m R N A in primary cultured neurons. The concentration of each drug used was 10 ~ M . Values are m e a n + S.E.M. obtained from three separate determinatiohs. * P < 0.05 (Bonferroni's test).
Effect of benzodiazepine derivatives on the expression of GABA 4 receptor al-SUbunit mRNA Flunitrazepam, one of benzodiazepine derivatives, is an agonist for benzodiazepine receptor coupled with GABA A receptor. Following the treatment of cells with flunitrazepam for 1 day, the expression of mRNA for GABA A receptor a~-subunit significantly decreased(Fig. 6). Although Ro15-1788, an antagonist of central benzodiazepine receptor, had no effect on the expression of GABA A receptor al-SUbunit mRNA, this drug antagonized the reduction of mRNA induced by flunitrazepam alone (Fig. 6). On the other hand, fiunitrazepam had no significant accentuating effect on the muscimol-induced suppression of the mRNA expression for GABA A receptor al-SUbunit. Furthermore, Ro15-1788 did not exhibit significant antagonistic effect on the suppression of the mRNA expression observed in the presence of both muscimol and flunitrazepam. These results suggest that flunitrazepam may have little accentuating effect on the muscimol-induced suppression of the mRNA expression under experimental conditions employed in this study.
,
}loo
100
o,-~
~
so
i'
o~,
~ r, 5o
C
..I
i
0
m
.
1 10 Husclmol (/Jill
i
100
Fig. 4. Effect of muscimol on the level of G A B A A receptor Otl-SUbunit m R N A in primary cultured neurons, aPercent of the value obtained in the absence of muscimol.
F
F~R
H
N+F N+F+R R
Fig. 6. Effect of benzodiazepine derivatives on the level of G A B A A receptor cq-subunit m R N A in primary cultured neurons. C, control; M, muscimol; F, flunitrazepam; R, Ro15-1788. The concentration of each drug used was 10/zM. Values are m e a n + S . E . M , obtained from 6 to 14 separate determinations. * P < 0.05, compared with control (Aspin-Welch's test).
330 DISCUSSION
In this study, it was found that the exposure of primary cultured neurons to muscimol, a GABA A receptor agonist, induced reduction of the mRNA expression of GABA A receptor al-SUbunit. This effect of muscimol was dose-dependent and antagonized by bicuculline, a G A B A A receptor antagonist. Furthermore, the treatment of neuronal cells with flunitrazepam decreased the expression of m R N A for GABA A receptor ax-subunit. This effect of flunitrazepam was inhibited by the simultaneous treatment of Ro15-1788, an antagonist of central type of benzodiazepine receptor. It has been well established that benzodiazepines increase the G A B A A receptor binding, since G A B A A receptor is functionally coupled with benzodiazepine receptor TM. Therefore, it is reasonable to assume that the activation of benzodiazepine receptor may induce the reduction of m R N A for GABA A receptor at-subunit via G A B A A receptor. However, the reduction of m R N A expression following the treatment with both muscimol and flunitrazepam was not additive. Although the exact mechanisms underlying this lack of the accentuating effect of flunitrazepam on muscimol-induced suppression of the m R N A expression are unclear at present, it seems likely that muscimol and endogenous G A B A in neuronal cells already fully activate GABA A receptor. This notion has been supported by the fact that the treatment with flunitrazepam alone is effective. Considering these results, it is concluded that the muscimol-induced reduction of G A B A A receptor a~-subunit mRNA is due to the activation of GABA A receptor. In fact, we have previously reported that the mouse cerebral cortical neurons in primary culture used in this study predominantly consist of GABAergic neurons and exhibited the development of G A B A A receptor coupled with benzodiazepine receptor ~2. Chronic exposure of cultured cells to G A B A A receptor agonist induced the down-regulation of G A B A A / b e n z o d i a z e p i n e receptor complex 22. Furthermore, the decrease in the expression of GABA A receptor subunit mRNA in the rat brain following continuous administration of diazepam in vivo has been reported 7. These reports coincided well with the present results that the continuous activation of GABA A receptor induced the decreased expression of GABA A receptor al-subunit mRNA. Therefore, it is likely that the down-regulation of GABA A receptor caused by continuous stimulation with agonist is attributed to the reduced expression of GABA A receptor subunit mRNA, although its exact mechanism is not clear at present.
The treatment with antagonists such as bicuculline or Ro15-1788 was not effective on the expression of mRNA for GABA A receptor al-subunit, suggesting that the expression of m R N A for GABA A receptor a~-subunit may be regulated by positive signal transduction. On the other hand, the structure of gene promoter for chicken GABA A receptor al-SUbunit has been reported 2. However, the overall structure of GABA A receptor a l-SUbunit gene as well as its regulatory mechanism of the transcription is unknown. In the case of /3z-adrenergic receptor, Malbon and his coworkers reported that the down-regulation of /32-adrenergic receptor m R N A was accompanied by the decline of steady-state level of/32-adrenergic receptor m R N A 6. In contrast, the agonist-induced decline of a~-adrenergic receptor m R N A was resulted from the changes in transcription rate for the a~-adrenergic receptor gene 16. Furthermore, the transcriptional regulation by cAMP response element as a promotor of both receptors is also derived by signal transduction via the activation of receptor 6'~6. These data assume to support a hypothesis that the muscimol-induced reduction of the expression of G A B A A receptor al-SUbunit m R N A observed in this study results from the transcriptional regulation by signal transduction via the activation of G A B A A receptor. Nevertheless, it remains a possibility that the m R N A level is regulated by post-transcriptional mechanism. In addition, it has been reported that the expression of GABA A receptor subunit m R N A is regulated by the activation of N-methyl-D-aspartate-sensitive glutamate receptor, suggesting that the expression of GABA A receptor subunit m R N A may be also regulated by unknown mechanisms functionally related to NMDA receptor ~5. The muscimol-induced reduction of G A B A A receptor al-SUbunit mRNA disappeared in the treatment with muscimol for 96 h. However, the molecular mechanism underlying this phenomenon is not clear at present. To clarify these points, studies on the regulatory mechanism of transcription for G A B A A receptor a~subunit gene as well as those of other subunits of GABA a receptor complex remain to be conducted. Acknowledgement. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1 Barnard, E.A., Darlison, M.G. and Seeburg, P., Molecular biology of the GABA A receptor: the receptor/channel superfamily, Trends Neurosci.., 10 (1987) 502-509. 2 Bateson, A.N., Ultsch, A., Harvey, J,, Sutherland, M.L., Lasham, A., Glencorse, T.A., Barnard, E.A. and Darlison, M.G., The chicken GABA A receptor: cDNA cloning and structural analysis
331 of the at-subunit gene promoter. In A.M.G. Stella, J.D. Vellis and J.R. Perezpolo (Eds.), Regulation of Gene Expression in the Nervous System, Wiley-Liss, New York, 1990, pp. 241-252. 3 Bateson, A.N., Lasham, A. and Darlison, M.G., y-Aminobutyric acid A receptor heterogeneity is increased by alternative splicing of a novel /3-subunit gene transcript, J. Neurochem., 56 (1991) 1437-1440. 4 Collins, S., Bouvier, M., Bolanowski, M.A., Caron, M.G. and Lefkowitz, R.J., cAMP stimulates transcription of the fl2-adrenergic receptor gene in response to short-term agonist exposure, Proc. Natl. Acad. ScL USA, 86 (1989) 4853-4857. 5 Cutting, G.R., Lu, L., O'Hara, B.F., Kasch, L.M., MontroseRafizadeh, C., Donovan, D.M., Shimada, S., Antonarakis, S.E., Guggino, W.B., Uhl, G.R and Kazazian, Jr. H.H., Cloning of the ~/-aminobutyric acid (GABA) Pt cDNA: a GABA receptor subunit highly expressed in the retina, Proc. Natl. Acad. ScL USA, 88 (1991) 2673-2677. 6 Hadcock, J.R. and Malbon, C.C., Down-regulation of fl-adrenergic receptors: agonist-induced reduction in receptor mRNA levels, Proc. Natl. Acad. Sci. USA, 85 (1988) 5021-5025. 7 Heninger, C., Saito, N., Tallman, J.F., Garrett, K.M., Vitek, M.P., Duman, R.S. and Gallager, D.W., Effects of continuous diazepam administration on GABA A subunit mRNA in rat brain, J. Mol. Neurosci., 2 (1990) 101-107. 8 Hirouchi, M., Kuwano, R., Katagiri, T., Takahashi, Y. and Kuriyama, K., Nucleotide and deduced amino acid sequences of the GABA A receptor a-subunit from human brain, Neurochem. Int., 15 (1989) 33-38. 9 Hirouchi, M., Ohkuma, S. and Kuriyama, K., Expression of mRNA for GABA A receptor at-subunit in mouse brain, Neurochem. Int., 19 (1991) 375-382. 10 Hirouchi, M., Ohkuma, S. and Kuriyama, K., Expression of mRNA for Gsa and Gi2a in primary cultured mouse cerebral cortical neurons, .Neuroreport, 2 (1991) 766-768. 11 Kofuji, P., Wang, J.B., Hoss, S.J., Huganir, R.L. and Butt, D.R., Generation of two forms of the 3,-aminobutyric acid A receptor 3,2-subunit in mice by alternative splicing, J. Neurochem., 56 (1991) 713-715. 12 Kuriyama, K., Tomono, S., Kishi, M., Mukainaka, T. and Ohkuma, S., Development of ~,-aminobutyric acid(GABA)ergic neurons in cerebral cortical neurons in primary culture, Brain Res., 416 (1987) 7-21. 13 Levitan, E.S., Schofield, P.R., Burt, D.R., Rhee, L.M., Wisden, W., Kohler, M., Fujita, N., Rodriguez, H.F., Stephenson, A., Darlison, M.G., Barnard, E.A. and Seeburg, P.H., Structural and functional basis for GABA A receptor heterogeneity, Nature, 335 (1988) 76-79.
14 Luddens, H., Pritchett, D.B. , Kohler, M., Killisch, I., Keinanen, K., Monyer, H., Sprengel, R. and Seeburg, P.H., Cerebellar GABA A receptor selective for a behavioural alcohol antagonist, Nature, 346 (1990) 648-651. 15 Memo, M., Bovolin, P., Costa, E. and Grayson, D.R., Regulation of 7-aminobutyric acid A receptor subunit expression by activation of N-methyl-o-aspartate-sensitive glutamate receptors, Mol. Pharmacol., 39 (1991) 599-603. 16 Morris, G.M., Hadcock, J.R. and Malbon, C.C., Cross-regulation between G-protein-coupled receptors, J. Biol. Chem., 266 (1991) 2233-2238. 17 Ohkuma, S., Tomono, S., Tanaka, Y., Kuriyama, K. and Mukainaka, T., Development of taurine biosynthesizing system in cerebral cortical neurons in primary culture, Int. J. Dev. Neurosci., 4 (1986) 383-395. 18 Olsen, R.W., GABA-benzodiazepine barbiturate receptor interactions, J. Neurochem., 37 (1981) 1-13. 19 Olsen, R.W. and Tobin, A.J., Molecular biology of GABA A receptors, FASEB J., 4 (1990) 1469-1480. 20 Pritchett, D.B., Sontheimer, H., Shivers, B.D., Ymer, S., Kettenmann, H., Schofield, P.R. and Seeburg, P.H., Importance of a novel GABA A receptor subunit for benzodiazepine pharmacology, Nature, 338 (1989) 582-585. 21 Pritchett, D.B. and Seeburg, P.H., y-Aminobutyric acid A receptor as-subunit creates novel type II benzodiazepine receptor pharmacology, J. Neurochem., 54 (1990) 1802-1804. 22 Roca, D.J., Rozenberg, I., Farrant, M. and Farb, D.H., Chronic agonist exposure induces down-regulation and allosteric uncoupling of the ~,-aminobutyric acid/benzodiazepine receptor complex, Mol. Pharmacol., 37 (1990) 37-43. 23 Schofield, P.R., Darlison, M.G., Fujita, N., Burt, D.R., Stephenson, F.A., Rodriguez, H., Rhee, L.M., Ramachandran, J., Reale, V., Glencorse, T.A., Seeburg, P.H. and Barnard, E.A., Sequence and functional expression of the GABA A receptor shows a ligand-gated receptor super family, Nature, 328 (1987) 221-227. 24 Shivers, B.D., Killisch, I., Sprengel, R., Sontheimer, H., Kohler, M., Schofield, P.R. and Seeburg, P.H., Two novel GABA A receptor subunits exist in distinct neuronal subpopulations, Neuron, 3 (1989) 327-337. 25 Stevens, C.F., Channel families in the brain, Nature, 328 (1987) 198-199. 26 Wisden, W., Herb, A., Wieland, H., Keinanen, K., Luddens, H. and Seeburg, P.H., Cloning, pharmacological characteristics and expression pattern of the rat GABA A receptor a 4 subunit, FEBS Lett., 289 (1991) 227-230.
327
© 1992 Elsevier Science Publishers B.V. All rights reserved 0169-328x/92/$05.00
BRES 70485
M u s c i m o l - i n d u c e d reduction of G A B A A receptor a l-subunit m R N A in primary cultured cerebral cortical neurons Masaaki Hirouchi, Seitaro Ohkuma and Kinya Kuriyama Department of Pharmacology, Kyoto Prefectural University of Medicine, Kyoto (Japan) (Accepted 19 May 1992)
Key words: Muscimol; GABA A receptor ai-subunit; mRNA; Neuron; Primary culture; RNA blot analysis; Ribonuclease protection assay
The expression of mRNA for GABA A receptor ai-subunit in mouse cerebral cortical neurons in primary culture was examined using RNA blot analysis and ribonuclease protection assay following the treatment of neurons with muscimol, a selective agonist of GABA A receptor. The level of mRNA for GABA A receptor al-SUbunit showed a decrease in comparison with that in non-treated cells, whereas no changes in the level of fl-actin mRNA were noted under the same experimental conditions. This muscimol-induced reduction in GABAA receptor cq-subunit mRNA was counteracted by the simultaneous exposure of neurons to both bicuculline, an antagonist of GABA A receptor, and muscimol. The expression of mRNA for GABA A receptor al-subunit also showed a decline by the treatment of cells with flunitrazepam alone, an agonist of benzodiazepine receptor, and this change was also abolished by the simultaneous exposure of cells to flunitrazepam and Ro15-1788, an antagonist for central benzodiazepine receptor. These results suggest that the continuous stimulation of cerebral GABA A receptor complex may induce the reduced expression of mRNA for the receptor complex.
INTRODUCTION G A B A ( y - a m i n o b u t y r i c acid) A r e c e p t o r is a heterooligomeric p r o t e i n complex consisting of several h o m o l o g o u s m e m b r a n e - s p a n n i n g s u b u n i t s 18. Recently, c D N A c l o n i n g has revealed that G A B A A r e c e p t o r consists of several s u b u n i t s d e n o t e d as a , /3, y, 6 a n d p5,20,23,24. I n addition, the h e t e r o g e n e i t y in each s u b u n i t has i n d i c a t e d the p r e s e n c e of h o m o l o g o u s s u b u n i t s such as 0~1-0~13'14'21'26 a n d a l t e r n a t i v e splicing s u b u n i t s such as /34, y~,tl. T h e s e G A B A g r e c e p t o r s u b u n i t s c o n s e r v e d four t r a n s m e m b r a n e d o m a i n s similar to those in o t h e r i o n o t r o p i c receptors t. T h e r e f o r e , it has b e e n c o n c l u d e d that C l - ion c h a n n e l of G A B A A receptor b e l o n g s to a family of l i g a n d - g a t e d ion c h a n nels 25. W e previously r e p o r t e d the results of c D N A c l o n i n g o n G A B A A r e c e p t o r t~t-subunit from the h u m a n b r a i n 8. T h e expression of m R N A for G A B A A r e c e p t o r a~s u b u n i t , e x a m i n e d by R N A blot h y b r i d i z a t i o n using this c D N A as a probe, has i n d i c a t e d that t h e r e are two m a j o r m R N A species in b o t h rat a n d m o u s e brains. I n
addition, we have d e m o n s t r a t e d that the expression of m R N A for G A B A A r e c e p t o r al-SUbunit rapidly increases d u r i n g the d e v e l o p m e n t of p r i m a r y c u l t u r e d n e u r o n s 9. Since it is i m p o r t a n t to clarify w h e t h e r or not the expression of m R N A for G A B A A receptor is m o d u lated by a c o n t i n u o u s s t i m u l a t i o n of the receptor, the level of m R N A for G A B A A r e c e p t o r a t - s u b u n i t has b e e n e x a m i n e d following the t r e a t m e n t of p r i m a r y c u l t u r e d cerebral cortical n e u r o n s with muscimol, a selective G A B A A r e c e p t o r agonist. MATERIALS AND METHODS
Materials
[Ol-32P]dCTP and [a-32P]UTP were obtained from New England Nuclear (Boston, MA, USA). FASTTrack for mRNA isolation was purchased from Invitrogen (San Diego, USA). Ambion Inc. (TX, USA) was a source of ribonuclease protection assay kit (RPA II). Other drugs used were of analytical grade and commercially available. Neuronal cultures Primary culture of mouse cerebral cortical neurons was carried out as described previously9'10'17. The neopaUium was isolated from
Correspondence: K. Kuriyama, Department of Pharmacology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamikyo-ku, Kyoto 602, Japan. Fax: (81) 75-241-0824.
328 15-days-old fetus of ddy strain mouse under ether anesthesia, trypsinized and filtered through a nylon mesh. Isolated cells were inoculated on a poly-L-lysine-coated dish containing modified Eagle's minimum essential medium (MEM) supplemented with 15% fetal calf serum and cultured for 3 days. After the treatment with 10/xM cytosine arabinoside in MEM containing 10% horse serum for 24 h, the culture medium was exchanged to MEM with 10% horse serum. Thereafter, the culture medium was exchanged to fresh one (MEM with 10% horse serum) every 4 days. Preparation of mRNA mRNA was obtained from neurons cultured for 10 days in all experiments. After washing neuronal cells with phosphate-buffered saline, neurons were scraped off from a culture dish with a rubber policeman. Poly(A) + RNAs were isolated from these cells using FASTTrack. RNA blot analysis RNA blot hybridization was performed as previously described 8. Denatured poly(A) + RNA (5 /xg) was electrophoresed on 1.5% agarose gel containing formaldehyde, and blotted onto a nitrocellulose filter. The filter was hybridized with 50 mM sodium phosphate buffer (pH 7.0), 5 × standard saline citrate (SSC), 50 p~g/ml salmon sperm DNA, 1 × Denhardt's solution, 30% formamide, 10% dextran sulfate and 3zp-labeled probe at 42°C. After the hybridization, the filter was washed with 2× SSC and 0.1% sodium lauryl sulfate at 50°C. The A5 eDNA fragment of GABA A receptor oq-subunit from the human brain was labeled with [a-32p]dCTP by the random-primed DNA labeling method and the denatured labeled DNA was used as a probe s. Ribonuclease protection assay The detection of mRNA for GABA A receptor at-subunit was carried out by the ribonuclease protection method according to the instruction manual for RPA II. Poly(A) ÷ RNA (0.8 /zg) obtained from neuronal cells was used for each assay. The A5 eDNA fragment (420 base pairs (bp), Hirouchi et al. s) was ligated within the EcoRI site of pBluescriptlI KS(+). Following the digestion of EcoT 221, the A5 eDNA fragment (257 bp) was transcribed to its anti-sense RNA with [a-32p]UTP by the catalytic actions of T3 RNA polymerase. The 32p-labeled anti-sense RNA for /3-actin (250 bp) was used as a control. These 32p-labeled anti-sense RNAs were incubated with each sample RNA at 42°C. The hybridized RNA was then digested with RNase A and RNase T1. The protected RNA was electrophoresed on 50% urea/6% polyacrylamide gel. The detection of RNA was performed using Image analyzer (BAS 2000, Fujifilm).
A
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Fig. 1. RNA blot analysis on GABA A receptor cq-subunit mRNA in primary cultured neurons following treatment with muscimol. A: control. B: 1 day treatment prior to the isolation. C: 4 days treatment prior to the isolation. RNA molecular weight markers (kb) are indicated on the left hand side.
In a d d i t i o n , t h e t i m e c o u r s e o f c h a n g e s in t h e expression of mRNA
for G A B A A r e c e p t o r
at-subunit
f o l l o w i n g t h e t r e a t m e n t w i t h m u s c i m o l was e x a m i n e d using a solution hybridization
procedure
s u c h as ri-
b o n u c l e a s e p r o t e c t i o n assay. T h e level o f m R N A
for
G A B A A r e c e p t o r a z - s u b u n i t in cells t r e a t e d w i t h m u s c i m o l for 4 h o u r s s h o w e d a s i g n i f i c a n t r e d u c t i o n , alt h o u g h little c h a n g e in t h e e x p r e s s i o n was r e c o g n i z e d following the treatment reduction of mRNA
for 1 o r 2 h (Fig. 2). T h i s
was also f o u n d a f t e r t h e e x p o s u r e
o f m u s c i m o l for 72 h. T h e l e v e l o f m R N A
for G A B A A
r e c e p t o r a ] - s u b u n i t in t h e cells i n c u b a t e d for 24 h w i t h muscimol
RESULTS
B
was
about
70%
of the
level d e t e c t e d
in
n o n - t r e a t e d cells. In c o n t r a s t , t h e e x p r e s s i o n o f m R N A
Effect o f m u s c i m o l treatment on G A B A A receptor a 1subunit m R N A M o u s e c e r e b r a l c o r t i c a l n e u r o n s in p r i m a r y c u l t u r e w e r e c u l t u r e d for 10 days. T h e G A B A A r e c e p t o r a ] subunit mRNA
in n e u r o n a l cells, e x p o s e d to 10 / z M
.•
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I
...a._
100
m u s c i m o l for 1 o r 4 days p r i o r to t h e i s o l a t i o n o f e a c h p o l y ( A ) + R N A , was a n a l y z e d by R N A b l o t h y b r i d i z a -
50
tion. Fig. 1 s h o w s t h e a u t o r a d i o g r a p h y o f R N A
b l o t hy-
b r i d i z a t i o n for G A B A A r e c e p t o r
cq-subunit mRNA.
Although the expression of mRNA
for GABA A recep-
t o r a ~ - s u b u n i t in p r i m a r y c u l t u r e d n e u r o n s s i g n i f i c a n t l y r e d u c e d f o l l o w i n g t h e t r e a t m e n t w i t h m u s c i m o l for 1 day, t h e m R N A
in t h e cells t r e a t e d w i t h m u s c i m o l for
4 days e x h i b i t e d s i m i l a r e x t e n t o f t h e e x p r e s s i o n c o m p a r e d w i t h t h a t in c o n t r o l .
as
0 I 2 4 24 48 72 Incubation T i u ( h r s )
96
Fig. 2. Time course of changes in the level of GABA A receptor a~-subunit mRNA in primary cultured neurons following treatment with muscimol. Values are mean ± S.E.M. obtained from 5 to 6 separate determinations. * P < 0.05, * * P < 0.0l, compared with the value obtained at 0 time (Scheffe's test).
329 GABAAReceptor c(,-Subonit mRNA(| of Control l 50 100
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Incubation Timelhrs) Fig. 3. Time course of changes in the level of fl-actin m R N A in primary cultured neurons following treatment with muscimol. Values are m e a n +S.E.M. olrtained from three separate determinations.
in the cells exposed to muscimol for 96 h was similar to that in non-treated cells as shown in Fig. 1. The expression of mRNA for/3-actin was simultaneously studied in primary cultured neurons exposed to 10/zM muscimol. The expression of mRNA for fl-actin had no significant change following the exposure to muscimol (Fig. 3). The muscimol-induced reduction in the expression of mRNA for GABA A receptor t~-subunit was also dose-dependent (Fig. 4).
Effect of bicuculline on muscimol-induced reduction in the expression of GABA~ receptor t~l-SUbunit mRNA Whether the reduction of GABA A receptor ~-subunit mRNA induced by muscimol is mediated via GABA A receptor has been investigated by simultaneous exposure of cells to both muscimol and bicuculline, a GABA A receptor antagonist. The simultaneous treatment of cells with bicuculline and muscimol for 1 day abolished the muscimol-induced reduction of mRNA for GABA A receptor al-subunit, although bicuculline alone showed no effect on the expression of GABA g receptor al-SUbunit mRNA (Fig. 5).
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Fig. 5. Effect of bicuculline on muscimol-induced reduction in G A B A A receptor a r s u b u n i t m R N A in primary cultured neurons. The concentration of each drug used was 10 ~ M . Values are m e a n + S.E.M. obtained from three separate determinatiohs. * P < 0.05 (Bonferroni's test).
Effect of benzodiazepine derivatives on the expression of GABA 4 receptor al-SUbunit mRNA Flunitrazepam, one of benzodiazepine derivatives, is an agonist for benzodiazepine receptor coupled with GABA A receptor. Following the treatment of cells with flunitrazepam for 1 day, the expression of mRNA for GABA A receptor a~-subunit significantly decreased(Fig. 6). Although Ro15-1788, an antagonist of central benzodiazepine receptor, had no effect on the expression of GABA A receptor al-SUbunit mRNA, this drug antagonized the reduction of mRNA induced by flunitrazepam alone (Fig. 6). On the other hand, fiunitrazepam had no significant accentuating effect on the muscimol-induced suppression of the mRNA expression for GABA A receptor al-SUbunit. Furthermore, Ro15-1788 did not exhibit significant antagonistic effect on the suppression of the mRNA expression observed in the presence of both muscimol and flunitrazepam. These results suggest that flunitrazepam may have little accentuating effect on the muscimol-induced suppression of the mRNA expression under experimental conditions employed in this study.
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Fig. 4. Effect of muscimol on the level of G A B A A receptor Otl-SUbunit m R N A in primary cultured neurons, aPercent of the value obtained in the absence of muscimol.
F
F~R
H
N+F N+F+R R
Fig. 6. Effect of benzodiazepine derivatives on the level of G A B A A receptor cq-subunit m R N A in primary cultured neurons. C, control; M, muscimol; F, flunitrazepam; R, Ro15-1788. The concentration of each drug used was 10/zM. Values are m e a n + S . E . M , obtained from 6 to 14 separate determinations. * P < 0.05, compared with control (Aspin-Welch's test).
330 DISCUSSION
In this study, it was found that the exposure of primary cultured neurons to muscimol, a GABA A receptor agonist, induced reduction of the mRNA expression of GABA A receptor al-SUbunit. This effect of muscimol was dose-dependent and antagonized by bicuculline, a G A B A A receptor antagonist. Furthermore, the treatment of neuronal cells with flunitrazepam decreased the expression of m R N A for GABA A receptor ax-subunit. This effect of flunitrazepam was inhibited by the simultaneous treatment of Ro15-1788, an antagonist of central type of benzodiazepine receptor. It has been well established that benzodiazepines increase the G A B A A receptor binding, since G A B A A receptor is functionally coupled with benzodiazepine receptor TM. Therefore, it is reasonable to assume that the activation of benzodiazepine receptor may induce the reduction of m R N A for GABA A receptor at-subunit via G A B A A receptor. However, the reduction of m R N A expression following the treatment with both muscimol and flunitrazepam was not additive. Although the exact mechanisms underlying this lack of the accentuating effect of flunitrazepam on muscimol-induced suppression of the m R N A expression are unclear at present, it seems likely that muscimol and endogenous G A B A in neuronal cells already fully activate GABA A receptor. This notion has been supported by the fact that the treatment with flunitrazepam alone is effective. Considering these results, it is concluded that the muscimol-induced reduction of G A B A A receptor a~-subunit mRNA is due to the activation of GABA A receptor. In fact, we have previously reported that the mouse cerebral cortical neurons in primary culture used in this study predominantly consist of GABAergic neurons and exhibited the development of G A B A A receptor coupled with benzodiazepine receptor ~2. Chronic exposure of cultured cells to G A B A A receptor agonist induced the down-regulation of G A B A A / b e n z o d i a z e p i n e receptor complex 22. Furthermore, the decrease in the expression of GABA A receptor subunit mRNA in the rat brain following continuous administration of diazepam in vivo has been reported 7. These reports coincided well with the present results that the continuous activation of GABA A receptor induced the decreased expression of GABA A receptor al-subunit mRNA. Therefore, it is likely that the down-regulation of GABA A receptor caused by continuous stimulation with agonist is attributed to the reduced expression of GABA A receptor subunit mRNA, although its exact mechanism is not clear at present.
The treatment with antagonists such as bicuculline or Ro15-1788 was not effective on the expression of mRNA for GABA A receptor al-subunit, suggesting that the expression of m R N A for GABA A receptor a~-subunit may be regulated by positive signal transduction. On the other hand, the structure of gene promoter for chicken GABA A receptor al-SUbunit has been reported 2. However, the overall structure of GABA A receptor a l-SUbunit gene as well as its regulatory mechanism of the transcription is unknown. In the case of /3z-adrenergic receptor, Malbon and his coworkers reported that the down-regulation of /32-adrenergic receptor m R N A was accompanied by the decline of steady-state level of/32-adrenergic receptor m R N A 6. In contrast, the agonist-induced decline of a~-adrenergic receptor m R N A was resulted from the changes in transcription rate for the a~-adrenergic receptor gene 16. Furthermore, the transcriptional regulation by cAMP response element as a promotor of both receptors is also derived by signal transduction via the activation of receptor 6'~6. These data assume to support a hypothesis that the muscimol-induced reduction of the expression of G A B A A receptor al-SUbunit m R N A observed in this study results from the transcriptional regulation by signal transduction via the activation of G A B A A receptor. Nevertheless, it remains a possibility that the m R N A level is regulated by post-transcriptional mechanism. In addition, it has been reported that the expression of GABA A receptor subunit m R N A is regulated by the activation of N-methyl-D-aspartate-sensitive glutamate receptor, suggesting that the expression of GABA A receptor subunit m R N A may be also regulated by unknown mechanisms functionally related to NMDA receptor ~5. The muscimol-induced reduction of G A B A A receptor al-SUbunit mRNA disappeared in the treatment with muscimol for 96 h. However, the molecular mechanism underlying this phenomenon is not clear at present. To clarify these points, studies on the regulatory mechanism of transcription for G A B A A receptor a~subunit gene as well as those of other subunits of GABA a receptor complex remain to be conducted. Acknowledgement. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture, Japan.
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