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GABAergic regulation of striatal opioid gene expression
Molecular Brain Research, 10 (1991) 49-54 t~) 1991 Elsevier Science Publishers B.V. (Biomedical Division) 0169-328X/91/$03.50 ADONIS 0169328X9170280L
49
BRESM 70280
GABAergic regulation of striatal opioid gene expression Susanne Reimer and Volker H611t Physiologisches Institut der Universitiit Miinchen, Munich (F.R. G.)
(Accepted 13 November 1990) Key words: Proenkephalin; Prodynorphin; In situ hybridization; Striatum; y-Aminobutyric acid; y-Aminobutyric acid-transaminase inhibitor
Peptides derived from prodynorphin and preproenkephalin are located in GABAergic striatal projection neurons. We have used nucleic acid hybridization techniques to investigate the role of GABA in the regulation of striatal opioid peptide gene expression. Rats were treated with the GABA-transaminase inhibitors aminooxy acetic acid, ethanolamine O-sulphate and y-vinyl-GABA for one week. The GABA levels in the striatum were significantly elevated after each treatment. The GABA-transaminase-inhibitors decreased the striatal levels of the opioid peptides met-enkephalin and dynorphin(1-8) and concomitantly decreased the concentrations of the mRNAs coding for proenkephalin and prodynorphin. These findings indicate that GABA exerts an inhibitory influence on prodynorphin and proenkephalin gene expression in the striatum. The mechanisms underlying these inhibitions are discussed. INTRODUCTION G A B A , one of the main inhibitory neurotransmitters in the central nervous system, is known to have several n e u r o p e p t i d e r g i c cotransmitters in striatal efferents. G A B A and M e t - e n k e p h a l i n ( M E ) are coexistent in striatopallidal neurons projecting to the external segment of the pallidum 19. G A B A and d y n o r p h i n ( 1 - 8 ) ( D Y N ) coexist in striatopallidal neurons projecting to the internal pallidal segment 1°. This implies that opioids might play an i m p o r t a n t role in e x t r a p y r a m i d a l functions and dysfunctions. I n d e e d , the levels of a n u m b e r of peptides have been found to be changed in neurologic and psychiatric diseases. M E levels are decreased in substantia nigra and striatum of Parkinsonian patients 1"25. Huntington's chorea is characterized by a loss of striatal p r o j e c t i o n neurones 22,24, especially those containing meto r leu-enkephalin and substance P. A d v a n c e d stages show a nearly c o m p l e t e loss of striatal p r o j e c t i o n neurones 24. T h e r e are several reports in the literature showing that G A B A e r g i c drugs exert an inhibitory influence on striatal M E p e p t i d e levels. Thus, stimulation of G A B A receptors in vitro resulted in an inhibited release of M E from striatal slices 2°. Treatment of rats with benzodiazepines, which act via a G A B A e r g i c mechanism, resulted in d e c r e a s e d striatal M E levels examined in vivo 7. Similarly, application of the G A B A - t r a n s a m i n a s e inhibitor aminooxyacetic acid ( A O A A ) also r e d u c e d striatal M E 8. Sivam and H o n g found decreased M E levels and
increased p r o e n k e p h a l i n ( P E N K ) m R N A - l e v e l s in the striatum of rats following r e p e a t e d administration of A O A A 26. T h e r e is no information available on the influence of G A B A on p e p t i d e s derived from p r o d y n o r p h i n ( P D Y N ) . The aim of this study was therefore, to investigate the influence of the classical n e u r o t r a n s m i t t e r G A B A on striatal D Y N levels and on P D Y N biosynthesis after injections of various G A B A - t r a n s a m i n a s e inhibitors. Peptide levels were d e t e r m i n e d by R I A , P E N K and P D Y N m R N A levels by in situ hybridization and blot techniques. MATERIALS AND METHODS Surgical procedures Male Sprague-Dawley rats weighing 200-250 g (Charles-River Wiga, Sulzfeld) were treated intraperitoneally with GABA-transaminase (GABA-T) inhibitors. Concentrations of 40, 60 and 80 mg/kg AOAA (Sigma Chemicals), 100 mg/kg ethanolamine Osulphate (EOS, Sigma Chemicals), 300 mg/kg EOS and 800 mg/kg y-vinyI-GABA (GVG, Merrel Dow, Strassbourg) were used. The application of the substances was once daily over a period of one week. Control animals received 0.9% saline injections. The rats were kept under controlled dark/light conditions and had free access to food and water. Twenty-four h after the last injection the animals were killed, the brains removed and immediately frozen on dry ice. For RNA extraction, the striatum was dissected out of the brain and weighed before freezing. The tissues were stored at -70 °C until used. Determination of GABA levels GABA concentrations were determined using an enzymaticmicrofluorometric assay as described by Mansky et al. 16. This assay is based on the enzymatic conversion of GABA to succinate by
Correspondence: S. Reimer, Physiologisches Institut der Universit/it Miinchen, Pettenkoferstrasse 12, D-8000 Munich 2, F.R.G.
50 GABA transaminase and succinic semialdehyde dehydrogenase. The conversion of the substrate is accompanied by an equimolar reduction of NADP. The NADPH 2 formed by the reaction can be measured fluorometrically. Tissue pieces were homogenized by brief sonication in 150/~1 of 0.1 N HCI and a 10 pl aliquot was taken for protein determination 15. The rest was centrifuged at 10,000 g for 10 rain and the supernatant used for the GABA determination.
In situ hybridization The method of in situ hybridization and the preparation of the probes was performed as described previously17'1s. Briefly, 10 #m cryostat sections were mounted onto polylysine-coated slides, air-dried and fixed for 15 min in 4% paraformaldehyde. The slides were kept at 4 °C under 70% ethanol until use. Following treatment with 0.2 N HCI (10 min), 0.1/~g/ml proteinase K (12 min), 0.25% acetic anhydride (3 min) and 4% paraformaldehyde, the sections were prehybridized for I h at 37°C with 5× Denhardts (1× Denhardts: 0.02% polyvinylpyrrolidone; 0.02% bovine serum albumine; 0.02% Ficoll), 0.5 mg/ml denatured salmon sperm DNA, 5x SSC (Ix SSC: 0.15 M NaC1; 0.015 M trisodium citrate; pH 7.0), 50 mM sodium phosphate buffer pH 7.0 and 50% deionized formamide. The slides were then dehydrated in ethanol and hybridized overnight with the radiolabelled probe together with 10 mg/ml yeast tRNA into the prehybridization buffer at 50 °C. When using the PDYN cDNA probe, the buffer additionally contained 6% dextran sulphate. For determination of PENK mRNA, a SrnaI-SacI restriction fragment representing approximately 1000 bases of rat PENK mRNA sequence was used 29. The fragment was nick-translated with a-[32p]dATP. Probe specific activity was 2 × 108 cpm/#g. For determination of PDYN mRNA a 100 mer cDNA oligonucleotide was synthesized which corresponded to nucleotides 488-587 of rat PDYN mRNA 6. The oligonucleotide was radiolabeiled using a specific 15 mer antisense primer and Klenow enzyme using a-[32p]dATP as described previously17. Probe specific activity was 1 x 108 cpm//ag. Probe concentrations used were 12,000 and 10,000 cpm/gl for the PENK cDNA and PDYN cDNA probes, respectively. Following hybridization, sections were washed under conditions of increasing stringency (2 x SSC, 1 × SSC and 0.1 x SSC for PENK cDNA probe; 6x SSC, 3x SSC and 1× SSC for PDYN cDNA probe) for a total time of 2 h at 50 °C, dehydrated in ethanol and apposed to Kodak X-AR films for 3-5 days. Densitometry was performed using the Java (Jandel) analysis program. Northern blot analysis The Northern blot analysis was performed as described previously 13. Briefly total RNA was extracted from the tissues using the LiCi-method2. Aliquots of 5 #g RNA were denatured with glyoxal, run on a 1.2% agarose electrophoresis gel and transferred to nylon sheets (Nytran, Schleicher and Schiill). Equal loading of the RNA samples was checked by ethidium bromide staining of the gels after transfer followed by densitometric scanning of negative photographs. Filters were baked for two h at 80 °C, prehybridized, and hybridized overnight with either PENK or PDYN cRNA probes at 60 °C. The hybridization buffer used was the same as that used for in situ hybridization with the exception that it contained 0.1% sodium dodecylsulphate. The probes were obtained by subcloning the cDNA sequences into the Bluescribe vector (Stratagene, San Diego, CA), which allows to prepare single-stranded sense or antisense RNA transcripts using T3 or T7 RNA-Polymerase. After hybridization, the filters were washed, dried and exposed to X-ray films at -70 °C. Autoradiograms were scanned using a LKB ultroscan II laser densitometer. Peptide RIAs For determination of peptide levels the tissues were processed for RIA as described previously ~z. The tissues were homogenized in 1 M acetic acid and centrifuged. The supernatants were lyophilized,
redissolved in RIA buffer and measured for immunoreactive peptides using antisera against ME (a gift from Dr. K.H. Voigt, Marburg, F.R.G.) and DYN (a gift from Dr. E. Weber, Portland, U.S.A.). The characteristics of the antisera and their crossreactivities have been described previously3,27. RESULTS In an initial series of e x p e r i m e n t s , the effect of v a r i o u s GABA-T
inhibitors
(AOAA,
EOS
and
GVG)
on
G A B A levels in the rat s t r i a t u m w e r e e x a m i n e d . A p p l i c a t i o n of 80 m g / k g A O A A ,
a d o s e which was
used in a p r e v i o u s study 26, r e s u l t e d in 100% m o r t a l i t y within the first two days. 5 0 % m o r t a l i t y o c c u r r e d to application of 60 m g / k g A O A A . In a d d i t i o n , it i n d u c e d convulsions similar to stage 4 - 5 kindling seizures 23. T h e r e f o r e the d o s e o f A O A A E v e n at this d o s e , A O A A
was r e d u c e d to 40 mg/kg.
c a u s e d s e v e r e s y m p t o m s of
ataxia, k a t a t o n i a , s e d a t i o n and l a c r i m a t i o n . E O S (100 mg/kg; 300 mg/kg) and G V G (800 mg/kg) also i n d u c e d ataxia, k a t a t o n i a , s e d a t i o n a n d l a c r i m a t i o n , but the s y m p t o m s w e r e less s e v e r e . N o n e o f t h e animals t r e a t e d with a p p l i e d died.
GVG
or
EOS
at
either
concentration
Influence on striatal G A B A levels T h e e f f e c t i v e n e s s of t h e v a r i o u s G A B A - T inhibitors in increasing G A B A levels in s t r i a t u m was d e t e r m i n e d . Fig. 1 shows the alterations in striatal G A B A - l e v e l s m e a s u r e d in n m o l G A B A / m g p r o t e i n a f t e r daily i.p. application of 40 m g / k g A O A A , 300 m g / k g E O S o r 800 m g / k g G V G for
T
.=_ oa 10 -4-T E cn •t.D ,z
5
E C
0
GVG EOS AOAA Fig. 1. Striatal GABA concentrations following application of 800 mg/kg GVG, 100 mg/kg EOS and 40 mg/kg AOAA. Open bar represents control C, shaded bars represent values of rats treated with the GABA-transaminase inhibitors. All values are the means _+ standard deviation of four measurements and are significant (*P < 0.05) according to ANOVA followed by Scheffe's test.
51 TABLE I
TABLE II
Effect olEOS administration on ME and D YN peptide levels in the rat striatum
Quantitative analysis of in-situ hybridization autoradiographs from striatal sections of animals treated with 40 mg/kg A OAA, 300 mg/kg EOS or 800 mg/kg GVG for 7 days
Rats were injected daily (300 mg/kg EOS) for one week. Peptides were measured by RIA. The values are means + S.E.M. of eight animals each.
Dose EOS (mg/kg)
Met-enkephalin (pmol/mg)
Dynorphin (1-8) (pmol/mg)
C 100 300
22.85 + 3.37 19.34 + 4.14" 17.50 + 2.94*
46.40 + 4.61 42.90 + 6.06 40.10 + 5.65*
* Significance (P < 0.05) when compared to control group (C) using ANOVA followed by Scheffe's test.
The sections were hybridized with the PENK or PDYN cDNA probe respectively. The results are the means + S.E.M. of 4 experiments.
C AOAA EOS GVG
PENK mRNA level (% of control)
PD YN mRNA level (% of control)
100 + 5.7 74 + 10.2" 62 + 8.6* 49 + 4.4*
100 + 6.8 n.d. 53 + 4.2* 54 + 3.9*
* Significant differences (P < 0.05) when compared to control group (C) using Student's two-tailed t-test, n.d., not determined.
7 days. A O A A , E O S and G V G produced a 2.5- to 4-fold increase in striatal G A B A levels.
Effect o f E O S on striatal opioid peptide levels The effect of 100 mg/kg and 300 mg/kg E O S on striatal M E and D Y N peptide levels was measured. Treatment of rats with 300 mg/kg E O S caused a significant decrease in the level of both M E and D Y N in the striatum (23% and 14%, respectively). A t 100 mg/kg E O S resulted in a significant decrease in striatal M E of 15% but did not alter D Y N levels (see Table I).
Effect on striatal P E N K and P D Y N m R N A levels In situ hybridization showed strong hybridization signals for P E N K m R N A (Fig. 2A) and P D Y N m R N A (Fig. 2C) in the striatum, which were greatest in the ventromedial area. Following t r e a t m e n t with 300 mg/kg EOS, the striatal P E N K (Fig. 2B) and P D Y N (Fig. 2D) m R N A signals were reduced. Similar autoradiographs were observed following t r e a t m e n t with A O A A and G V G (data not shown). Quantitative densitometry of data from four in situ hybridization experiments revealed
A
B
Fig. 2. Autoradiographs of in situ hybridizations of striatai brain slices following treatment with the GABA-transaminase inhibitor EOS. A and C are slices from control animals hybridized with PENK (A) and PDYN (C) cDNA respectively. B and D are slices from animals treated with 300 mg/kg EOS daily for one week hybridized with PENK (B) and PDYN (D) cDNA, respectively.
52 75%) in the P E N K and P D Y N m R N A hybridization signal following treatment with 300 mg/kg EOS and 800 mg/kg G V G (Table III). DISCUSSION
2.2 ~
/ C
I
I EOS
\ GVG
I
I ii
1.45 Kb
Fig. 3. Autoradiogram of a Northern blot of striatal tissue of EOS and GVG-treated animals. The blots were hybridized with cRNAs for PENK mRNA (1.45 kb) and PDYN mRNA (2.2 kb). The autoradiograms are two typical examples from 6 experiments in each group. a significant decrease (between 28% and 50%) in both R N A species (see Table II). Representative R N A blots showed single bands corresponding to m R N A species with a size of 1.45 kb when hybridized with the P E N K c D N A probe and of 2.2 kb when hybridized with the P D Y N c D N A probe (Fig. 3). These are the expected sizes for the m R N A species 6'29. Quantitative densitometry of R N A gels derived from 6 experiments showed a marked decrease (between 50 and
TABLE III Quantitative analysis of RNA blots from striatal tissues of rats treated with EOS (300 mg/kg) and GVG (800mg/kg)
The blots were hybridized with the PENK cRNA and PDYN cRNA probe respectively. The values represent mean _+ S.E.M. of 6 animals.
C EOS GVG
PENKmRNA level (% of control)
PD YNmRNA level (% of control)
100 + 5.6 40 + 4.3* 25 + 2.8*
100 + 6.9 35 + 2.7* 50 + 4.6*
* Significant values (P < 0.01) when compared to control group (C) using ANOVA followed by Scheffe's test.
Our results show for the first time that G A B A exerts an inhibitory influence on P D Y N gene expression in the striatum. Thus, elevation of G A B A levels by the chronic administration of G A B A - T inhibitors in rats decreased striatal concentrations of P D Y N m R N A and DYN. In addition, we also found reduced ME levels in the striatum after the G A B A - T inhibitor treatment. This finding is in agreement with earlier reports which showed that the activation of the G A B A e r g i c transmission decreased striatal ME levels in v i v o 7'26 and in vitro 1~. However, our findings that chronic treatment with G A B A - T inhibitors decreases the P E N K m R N A levels is in disagreement with the results of Sivam and Hong 26, who found a significant elevation in striatal P E N K m R N A levels and a decrease in the ME levels in rat chronically treated with A O A A . These authors suggested that activation of G A B A e r g i c transmission enhanced striatal release of M E to a greater degree than it augmented P E N K biosynthesis, which resulted in a decrease in the level of the peptide. This interpretation, however, appears to be unlikely, since G A B A e r g i c drugs have been shown to decrease release of ME from the striatum in vitro 2° and in vivo 4'5. In their experiments Sivam and H o n g 26 used a dose of A O A A (80 mg/kg) which was higher than that used in our experiments (40 mg/kg). We found that A O A A applied at even 40 mg/kg produced marked behavioural alterations such as seizures and convulsions. These behavioural symptoms were much less frequently seen after E O S and G V G , in line with the observation that A O A A is a less specific G A B A - T inhibitor than EOS and G V G 21. Similarly, our observation that A O A A increases striatal P E N K m R N A levels to a similar degree as G V G (4-fold), but is less efficient in reducing striatal G A B A levels, indicates that mechanisms unrelated to the activity of A O A A on G A B A - T , possibly counteracting the effect of G A B A , might be involved. It is not unreasonable to assume that the convulsive effect of A O A A might be responsible for its stimulatory effect on striatal P E N K m R N A levels. In fact, increased striatal P E N K m R N A levels following treatment leading to kindling stages 2 and 5 have been reported 14. On the other hand, it is possible that there is a strain difference in drug susceptibility, since Sivam and Hong z6 used Fischer 344 rats whereas Sprague-Dawley rats were used in our experiments. P D Y N m R N A levels in the striatum were also reported to be increased after convulsive treatment 28. Our
53 results showing that both opioid p e p t i d e m R N A s are d e c r e a s e d provide evidence for an inhibitory G A B A e r g i c control on two p e p t i d e systems ( P E N K and P D Y N ) in the rat striatum. T h e r e are several possible mechanisms underlying this effect. G A B A e r g i c neurones projecting from the striatum to the external segment of the pallidum possess M E as co-transmitter 19. Striatofugal neurones projecting to the internal segment of the pallidum and to the substantia nigra contain G A B A - and P D Y N - d e r i v e d p e p t i d e s m. Inhibition of G A B A - T augments G A B A transmission in these p r o j e c t i o n neurones, which might lead to a r e d u c e d neuronal activity via feed-back inhibition, resulting in a r e d u c e d biosynthesis of opioid peptides. This m a y explain the decreased levels of P E N K and P D Y N m R N A in the striatum which contains the cell bodies of the p r o j e c t i o n neurones. F u r t h e r m o r e , G A B A and M E are k n o w n to exist in intrinsic neurones of the striatum, and activation of G A B A e r g i c neurones in striatal slices in vitro has been shown to inhibit M E release 2°.
questions on the basis of p e p t i d e levels, since the concentration of a p e p t i d e reflects the balance b e t w e e n release and biosynthesis. T h e r e f o r e , the release of opioid peptides has to be d e t e r m i n e d in the striatum and the various terminal fields of the striatal p r o j e c t i o n neurones in vivo using microdialysis techniques. This m e t h o d is presently established in o u r laboratory. G A B A - m i m e t i c drugs are used in the clinical treatm e n t o f H u n t i n g t o n ' s chorea (for review see Enna9). The levels of P E N K derived p e p t i d e s in the striatum of H u n t i n g t o n ' s chorea patients are u n c h a n g e d 1°. It remains to be d e t e r m i n e d w h e t h e r or n o t a t r e a t m e n t of these patients with G A B A e r g i c drugs m a y also cause a reduction in opioidergic activity in the striatum and if so, w h e t h e r this decrease is involved in the t h e r a p e u t i c effect of the drugs. In s u m m a r y , the d a t a p r e s e n t e d provide evidence for an inhibitory action of G A B A on striatal P D Y N and P E N K biosynthesis.
Increased G A B A e r g i c activity reduces striatal M E and D Y N p e p t i d e levels to a lesser degree than biosynthesis. This might indicate that the a u g m e n t e d G A B A e r g i c transmission also reduces the striatal release of the opioid peptides. It is also possible that G A B A differentially affects opioid p e p t i d e biosynthesis and/or release in striatal intrinsic neurones as c o m p a r e d to striatofugal p r o j e c t i o n neurones. It is difficult to answer these
Acknowledgements. We are very grateful to Dr. S. Sabol for the gift of the rat proenkephalin clone. We wish to thank Drs. W. Wuttke and H. Jarry, G6ttingen, F.R.G., for the determination of the GABA levels. We are very grateful to Merrel Dow Inc., Strassbourg, France for the gift of gammavinyl GABA and to Ms. Gabi Horn for excellent technical assistance. The animal experiments were performed in the year 1987 at the Max-Planck-Institute for Psychiatry, Martinsried, at the department of Dr. A. Herz. The work was supported by Deutsche Forschungsgemeinschaft (Schwerpunkt: Neuropeptide).
ABBREVIATIONS
GABA-T GVG ME PDYN PENK
AOAA DYN EOS GABA
aminooxyacetic acid dynorphin(1-8) ethanolamine O-sulphate y-aminobutyric acid
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49
BRESM 70280
GABAergic regulation of striatal opioid gene expression Susanne Reimer and Volker H611t Physiologisches Institut der Universitiit Miinchen, Munich (F.R. G.)
(Accepted 13 November 1990) Key words: Proenkephalin; Prodynorphin; In situ hybridization; Striatum; y-Aminobutyric acid; y-Aminobutyric acid-transaminase inhibitor
Peptides derived from prodynorphin and preproenkephalin are located in GABAergic striatal projection neurons. We have used nucleic acid hybridization techniques to investigate the role of GABA in the regulation of striatal opioid peptide gene expression. Rats were treated with the GABA-transaminase inhibitors aminooxy acetic acid, ethanolamine O-sulphate and y-vinyl-GABA for one week. The GABA levels in the striatum were significantly elevated after each treatment. The GABA-transaminase-inhibitors decreased the striatal levels of the opioid peptides met-enkephalin and dynorphin(1-8) and concomitantly decreased the concentrations of the mRNAs coding for proenkephalin and prodynorphin. These findings indicate that GABA exerts an inhibitory influence on prodynorphin and proenkephalin gene expression in the striatum. The mechanisms underlying these inhibitions are discussed. INTRODUCTION G A B A , one of the main inhibitory neurotransmitters in the central nervous system, is known to have several n e u r o p e p t i d e r g i c cotransmitters in striatal efferents. G A B A and M e t - e n k e p h a l i n ( M E ) are coexistent in striatopallidal neurons projecting to the external segment of the pallidum 19. G A B A and d y n o r p h i n ( 1 - 8 ) ( D Y N ) coexist in striatopallidal neurons projecting to the internal pallidal segment 1°. This implies that opioids might play an i m p o r t a n t role in e x t r a p y r a m i d a l functions and dysfunctions. I n d e e d , the levels of a n u m b e r of peptides have been found to be changed in neurologic and psychiatric diseases. M E levels are decreased in substantia nigra and striatum of Parkinsonian patients 1"25. Huntington's chorea is characterized by a loss of striatal p r o j e c t i o n neurones 22,24, especially those containing meto r leu-enkephalin and substance P. A d v a n c e d stages show a nearly c o m p l e t e loss of striatal p r o j e c t i o n neurones 24. T h e r e are several reports in the literature showing that G A B A e r g i c drugs exert an inhibitory influence on striatal M E p e p t i d e levels. Thus, stimulation of G A B A receptors in vitro resulted in an inhibited release of M E from striatal slices 2°. Treatment of rats with benzodiazepines, which act via a G A B A e r g i c mechanism, resulted in d e c r e a s e d striatal M E levels examined in vivo 7. Similarly, application of the G A B A - t r a n s a m i n a s e inhibitor aminooxyacetic acid ( A O A A ) also r e d u c e d striatal M E 8. Sivam and H o n g found decreased M E levels and
increased p r o e n k e p h a l i n ( P E N K ) m R N A - l e v e l s in the striatum of rats following r e p e a t e d administration of A O A A 26. T h e r e is no information available on the influence of G A B A on p e p t i d e s derived from p r o d y n o r p h i n ( P D Y N ) . The aim of this study was therefore, to investigate the influence of the classical n e u r o t r a n s m i t t e r G A B A on striatal D Y N levels and on P D Y N biosynthesis after injections of various G A B A - t r a n s a m i n a s e inhibitors. Peptide levels were d e t e r m i n e d by R I A , P E N K and P D Y N m R N A levels by in situ hybridization and blot techniques. MATERIALS AND METHODS Surgical procedures Male Sprague-Dawley rats weighing 200-250 g (Charles-River Wiga, Sulzfeld) were treated intraperitoneally with GABA-transaminase (GABA-T) inhibitors. Concentrations of 40, 60 and 80 mg/kg AOAA (Sigma Chemicals), 100 mg/kg ethanolamine Osulphate (EOS, Sigma Chemicals), 300 mg/kg EOS and 800 mg/kg y-vinyI-GABA (GVG, Merrel Dow, Strassbourg) were used. The application of the substances was once daily over a period of one week. Control animals received 0.9% saline injections. The rats were kept under controlled dark/light conditions and had free access to food and water. Twenty-four h after the last injection the animals were killed, the brains removed and immediately frozen on dry ice. For RNA extraction, the striatum was dissected out of the brain and weighed before freezing. The tissues were stored at -70 °C until used. Determination of GABA levels GABA concentrations were determined using an enzymaticmicrofluorometric assay as described by Mansky et al. 16. This assay is based on the enzymatic conversion of GABA to succinate by
Correspondence: S. Reimer, Physiologisches Institut der Universit/it Miinchen, Pettenkoferstrasse 12, D-8000 Munich 2, F.R.G.
50 GABA transaminase and succinic semialdehyde dehydrogenase. The conversion of the substrate is accompanied by an equimolar reduction of NADP. The NADPH 2 formed by the reaction can be measured fluorometrically. Tissue pieces were homogenized by brief sonication in 150/~1 of 0.1 N HCI and a 10 pl aliquot was taken for protein determination 15. The rest was centrifuged at 10,000 g for 10 rain and the supernatant used for the GABA determination.
In situ hybridization The method of in situ hybridization and the preparation of the probes was performed as described previously17'1s. Briefly, 10 #m cryostat sections were mounted onto polylysine-coated slides, air-dried and fixed for 15 min in 4% paraformaldehyde. The slides were kept at 4 °C under 70% ethanol until use. Following treatment with 0.2 N HCI (10 min), 0.1/~g/ml proteinase K (12 min), 0.25% acetic anhydride (3 min) and 4% paraformaldehyde, the sections were prehybridized for I h at 37°C with 5× Denhardts (1× Denhardts: 0.02% polyvinylpyrrolidone; 0.02% bovine serum albumine; 0.02% Ficoll), 0.5 mg/ml denatured salmon sperm DNA, 5x SSC (Ix SSC: 0.15 M NaC1; 0.015 M trisodium citrate; pH 7.0), 50 mM sodium phosphate buffer pH 7.0 and 50% deionized formamide. The slides were then dehydrated in ethanol and hybridized overnight with the radiolabelled probe together with 10 mg/ml yeast tRNA into the prehybridization buffer at 50 °C. When using the PDYN cDNA probe, the buffer additionally contained 6% dextran sulphate. For determination of PENK mRNA, a SrnaI-SacI restriction fragment representing approximately 1000 bases of rat PENK mRNA sequence was used 29. The fragment was nick-translated with a-[32p]dATP. Probe specific activity was 2 × 108 cpm/#g. For determination of PDYN mRNA a 100 mer cDNA oligonucleotide was synthesized which corresponded to nucleotides 488-587 of rat PDYN mRNA 6. The oligonucleotide was radiolabeiled using a specific 15 mer antisense primer and Klenow enzyme using a-[32p]dATP as described previously17. Probe specific activity was 1 x 108 cpm//ag. Probe concentrations used were 12,000 and 10,000 cpm/gl for the PENK cDNA and PDYN cDNA probes, respectively. Following hybridization, sections were washed under conditions of increasing stringency (2 x SSC, 1 × SSC and 0.1 x SSC for PENK cDNA probe; 6x SSC, 3x SSC and 1× SSC for PDYN cDNA probe) for a total time of 2 h at 50 °C, dehydrated in ethanol and apposed to Kodak X-AR films for 3-5 days. Densitometry was performed using the Java (Jandel) analysis program. Northern blot analysis The Northern blot analysis was performed as described previously 13. Briefly total RNA was extracted from the tissues using the LiCi-method2. Aliquots of 5 #g RNA were denatured with glyoxal, run on a 1.2% agarose electrophoresis gel and transferred to nylon sheets (Nytran, Schleicher and Schiill). Equal loading of the RNA samples was checked by ethidium bromide staining of the gels after transfer followed by densitometric scanning of negative photographs. Filters were baked for two h at 80 °C, prehybridized, and hybridized overnight with either PENK or PDYN cRNA probes at 60 °C. The hybridization buffer used was the same as that used for in situ hybridization with the exception that it contained 0.1% sodium dodecylsulphate. The probes were obtained by subcloning the cDNA sequences into the Bluescribe vector (Stratagene, San Diego, CA), which allows to prepare single-stranded sense or antisense RNA transcripts using T3 or T7 RNA-Polymerase. After hybridization, the filters were washed, dried and exposed to X-ray films at -70 °C. Autoradiograms were scanned using a LKB ultroscan II laser densitometer. Peptide RIAs For determination of peptide levels the tissues were processed for RIA as described previously ~z. The tissues were homogenized in 1 M acetic acid and centrifuged. The supernatants were lyophilized,
redissolved in RIA buffer and measured for immunoreactive peptides using antisera against ME (a gift from Dr. K.H. Voigt, Marburg, F.R.G.) and DYN (a gift from Dr. E. Weber, Portland, U.S.A.). The characteristics of the antisera and their crossreactivities have been described previously3,27. RESULTS In an initial series of e x p e r i m e n t s , the effect of v a r i o u s GABA-T
inhibitors
(AOAA,
EOS
and
GVG)
on
G A B A levels in the rat s t r i a t u m w e r e e x a m i n e d . A p p l i c a t i o n of 80 m g / k g A O A A ,
a d o s e which was
used in a p r e v i o u s study 26, r e s u l t e d in 100% m o r t a l i t y within the first two days. 5 0 % m o r t a l i t y o c c u r r e d to application of 60 m g / k g A O A A . In a d d i t i o n , it i n d u c e d convulsions similar to stage 4 - 5 kindling seizures 23. T h e r e f o r e the d o s e o f A O A A E v e n at this d o s e , A O A A
was r e d u c e d to 40 mg/kg.
c a u s e d s e v e r e s y m p t o m s of
ataxia, k a t a t o n i a , s e d a t i o n and l a c r i m a t i o n . E O S (100 mg/kg; 300 mg/kg) and G V G (800 mg/kg) also i n d u c e d ataxia, k a t a t o n i a , s e d a t i o n a n d l a c r i m a t i o n , but the s y m p t o m s w e r e less s e v e r e . N o n e o f t h e animals t r e a t e d with a p p l i e d died.
GVG
or
EOS
at
either
concentration
Influence on striatal G A B A levels T h e e f f e c t i v e n e s s of t h e v a r i o u s G A B A - T inhibitors in increasing G A B A levels in s t r i a t u m was d e t e r m i n e d . Fig. 1 shows the alterations in striatal G A B A - l e v e l s m e a s u r e d in n m o l G A B A / m g p r o t e i n a f t e r daily i.p. application of 40 m g / k g A O A A , 300 m g / k g E O S o r 800 m g / k g G V G for
T
.=_ oa 10 -4-T E cn •t.D ,z
5
E C
0
GVG EOS AOAA Fig. 1. Striatal GABA concentrations following application of 800 mg/kg GVG, 100 mg/kg EOS and 40 mg/kg AOAA. Open bar represents control C, shaded bars represent values of rats treated with the GABA-transaminase inhibitors. All values are the means _+ standard deviation of four measurements and are significant (*P < 0.05) according to ANOVA followed by Scheffe's test.
51 TABLE I
TABLE II
Effect olEOS administration on ME and D YN peptide levels in the rat striatum
Quantitative analysis of in-situ hybridization autoradiographs from striatal sections of animals treated with 40 mg/kg A OAA, 300 mg/kg EOS or 800 mg/kg GVG for 7 days
Rats were injected daily (300 mg/kg EOS) for one week. Peptides were measured by RIA. The values are means + S.E.M. of eight animals each.
Dose EOS (mg/kg)
Met-enkephalin (pmol/mg)
Dynorphin (1-8) (pmol/mg)
C 100 300
22.85 + 3.37 19.34 + 4.14" 17.50 + 2.94*
46.40 + 4.61 42.90 + 6.06 40.10 + 5.65*
* Significance (P < 0.05) when compared to control group (C) using ANOVA followed by Scheffe's test.
The sections were hybridized with the PENK or PDYN cDNA probe respectively. The results are the means + S.E.M. of 4 experiments.
C AOAA EOS GVG
PENK mRNA level (% of control)
PD YN mRNA level (% of control)
100 + 5.7 74 + 10.2" 62 + 8.6* 49 + 4.4*
100 + 6.8 n.d. 53 + 4.2* 54 + 3.9*
* Significant differences (P < 0.05) when compared to control group (C) using Student's two-tailed t-test, n.d., not determined.
7 days. A O A A , E O S and G V G produced a 2.5- to 4-fold increase in striatal G A B A levels.
Effect o f E O S on striatal opioid peptide levels The effect of 100 mg/kg and 300 mg/kg E O S on striatal M E and D Y N peptide levels was measured. Treatment of rats with 300 mg/kg E O S caused a significant decrease in the level of both M E and D Y N in the striatum (23% and 14%, respectively). A t 100 mg/kg E O S resulted in a significant decrease in striatal M E of 15% but did not alter D Y N levels (see Table I).
Effect on striatal P E N K and P D Y N m R N A levels In situ hybridization showed strong hybridization signals for P E N K m R N A (Fig. 2A) and P D Y N m R N A (Fig. 2C) in the striatum, which were greatest in the ventromedial area. Following t r e a t m e n t with 300 mg/kg EOS, the striatal P E N K (Fig. 2B) and P D Y N (Fig. 2D) m R N A signals were reduced. Similar autoradiographs were observed following t r e a t m e n t with A O A A and G V G (data not shown). Quantitative densitometry of data from four in situ hybridization experiments revealed
A
B
Fig. 2. Autoradiographs of in situ hybridizations of striatai brain slices following treatment with the GABA-transaminase inhibitor EOS. A and C are slices from control animals hybridized with PENK (A) and PDYN (C) cDNA respectively. B and D are slices from animals treated with 300 mg/kg EOS daily for one week hybridized with PENK (B) and PDYN (D) cDNA, respectively.
52 75%) in the P E N K and P D Y N m R N A hybridization signal following treatment with 300 mg/kg EOS and 800 mg/kg G V G (Table III). DISCUSSION
2.2 ~
/ C
I
I EOS
\ GVG
I
I ii
1.45 Kb
Fig. 3. Autoradiogram of a Northern blot of striatal tissue of EOS and GVG-treated animals. The blots were hybridized with cRNAs for PENK mRNA (1.45 kb) and PDYN mRNA (2.2 kb). The autoradiograms are two typical examples from 6 experiments in each group. a significant decrease (between 28% and 50%) in both R N A species (see Table II). Representative R N A blots showed single bands corresponding to m R N A species with a size of 1.45 kb when hybridized with the P E N K c D N A probe and of 2.2 kb when hybridized with the P D Y N c D N A probe (Fig. 3). These are the expected sizes for the m R N A species 6'29. Quantitative densitometry of R N A gels derived from 6 experiments showed a marked decrease (between 50 and
TABLE III Quantitative analysis of RNA blots from striatal tissues of rats treated with EOS (300 mg/kg) and GVG (800mg/kg)
The blots were hybridized with the PENK cRNA and PDYN cRNA probe respectively. The values represent mean _+ S.E.M. of 6 animals.
C EOS GVG
PENKmRNA level (% of control)
PD YNmRNA level (% of control)
100 + 5.6 40 + 4.3* 25 + 2.8*
100 + 6.9 35 + 2.7* 50 + 4.6*
* Significant values (P < 0.01) when compared to control group (C) using ANOVA followed by Scheffe's test.
Our results show for the first time that G A B A exerts an inhibitory influence on P D Y N gene expression in the striatum. Thus, elevation of G A B A levels by the chronic administration of G A B A - T inhibitors in rats decreased striatal concentrations of P D Y N m R N A and DYN. In addition, we also found reduced ME levels in the striatum after the G A B A - T inhibitor treatment. This finding is in agreement with earlier reports which showed that the activation of the G A B A e r g i c transmission decreased striatal ME levels in v i v o 7'26 and in vitro 1~. However, our findings that chronic treatment with G A B A - T inhibitors decreases the P E N K m R N A levels is in disagreement with the results of Sivam and Hong 26, who found a significant elevation in striatal P E N K m R N A levels and a decrease in the ME levels in rat chronically treated with A O A A . These authors suggested that activation of G A B A e r g i c transmission enhanced striatal release of M E to a greater degree than it augmented P E N K biosynthesis, which resulted in a decrease in the level of the peptide. This interpretation, however, appears to be unlikely, since G A B A e r g i c drugs have been shown to decrease release of ME from the striatum in vitro 2° and in vivo 4'5. In their experiments Sivam and H o n g 26 used a dose of A O A A (80 mg/kg) which was higher than that used in our experiments (40 mg/kg). We found that A O A A applied at even 40 mg/kg produced marked behavioural alterations such as seizures and convulsions. These behavioural symptoms were much less frequently seen after E O S and G V G , in line with the observation that A O A A is a less specific G A B A - T inhibitor than EOS and G V G 21. Similarly, our observation that A O A A increases striatal P E N K m R N A levels to a similar degree as G V G (4-fold), but is less efficient in reducing striatal G A B A levels, indicates that mechanisms unrelated to the activity of A O A A on G A B A - T , possibly counteracting the effect of G A B A , might be involved. It is not unreasonable to assume that the convulsive effect of A O A A might be responsible for its stimulatory effect on striatal P E N K m R N A levels. In fact, increased striatal P E N K m R N A levels following treatment leading to kindling stages 2 and 5 have been reported 14. On the other hand, it is possible that there is a strain difference in drug susceptibility, since Sivam and Hong z6 used Fischer 344 rats whereas Sprague-Dawley rats were used in our experiments. P D Y N m R N A levels in the striatum were also reported to be increased after convulsive treatment 28. Our
53 results showing that both opioid p e p t i d e m R N A s are d e c r e a s e d provide evidence for an inhibitory G A B A e r g i c control on two p e p t i d e systems ( P E N K and P D Y N ) in the rat striatum. T h e r e are several possible mechanisms underlying this effect. G A B A e r g i c neurones projecting from the striatum to the external segment of the pallidum possess M E as co-transmitter 19. Striatofugal neurones projecting to the internal segment of the pallidum and to the substantia nigra contain G A B A - and P D Y N - d e r i v e d p e p t i d e s m. Inhibition of G A B A - T augments G A B A transmission in these p r o j e c t i o n neurones, which might lead to a r e d u c e d neuronal activity via feed-back inhibition, resulting in a r e d u c e d biosynthesis of opioid peptides. This m a y explain the decreased levels of P E N K and P D Y N m R N A in the striatum which contains the cell bodies of the p r o j e c t i o n neurones. F u r t h e r m o r e , G A B A and M E are k n o w n to exist in intrinsic neurones of the striatum, and activation of G A B A e r g i c neurones in striatal slices in vitro has been shown to inhibit M E release 2°.
questions on the basis of p e p t i d e levels, since the concentration of a p e p t i d e reflects the balance b e t w e e n release and biosynthesis. T h e r e f o r e , the release of opioid peptides has to be d e t e r m i n e d in the striatum and the various terminal fields of the striatal p r o j e c t i o n neurones in vivo using microdialysis techniques. This m e t h o d is presently established in o u r laboratory. G A B A - m i m e t i c drugs are used in the clinical treatm e n t o f H u n t i n g t o n ' s chorea (for review see Enna9). The levels of P E N K derived p e p t i d e s in the striatum of H u n t i n g t o n ' s chorea patients are u n c h a n g e d 1°. It remains to be d e t e r m i n e d w h e t h e r or n o t a t r e a t m e n t of these patients with G A B A e r g i c drugs m a y also cause a reduction in opioidergic activity in the striatum and if so, w h e t h e r this decrease is involved in the t h e r a p e u t i c effect of the drugs. In s u m m a r y , the d a t a p r e s e n t e d provide evidence for an inhibitory action of G A B A on striatal P D Y N and P E N K biosynthesis.
Increased G A B A e r g i c activity reduces striatal M E and D Y N p e p t i d e levels to a lesser degree than biosynthesis. This might indicate that the a u g m e n t e d G A B A e r g i c transmission also reduces the striatal release of the opioid peptides. It is also possible that G A B A differentially affects opioid p e p t i d e biosynthesis and/or release in striatal intrinsic neurones as c o m p a r e d to striatofugal p r o j e c t i o n neurones. It is difficult to answer these
Acknowledgements. We are very grateful to Dr. S. Sabol for the gift of the rat proenkephalin clone. We wish to thank Drs. W. Wuttke and H. Jarry, G6ttingen, F.R.G., for the determination of the GABA levels. We are very grateful to Merrel Dow Inc., Strassbourg, France for the gift of gammavinyl GABA and to Ms. Gabi Horn for excellent technical assistance. The animal experiments were performed in the year 1987 at the Max-Planck-Institute for Psychiatry, Martinsried, at the department of Dr. A. Herz. The work was supported by Deutsche Forschungsgemeinschaft (Schwerpunkt: Neuropeptide).
ABBREVIATIONS
GABA-T GVG ME PDYN PENK
AOAA DYN EOS GABA
aminooxyacetic acid dynorphin(1-8) ethanolamine O-sulphate y-aminobutyric acid
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