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Expression of glycine and other amino acid receptors by rat spinal cord mRNA in Xenopus oocytes
262
Neuroscience Letters, 95 (1988) 262-268 Elsevier Scientific Publishers Ireland Ltd.
NSL 05763
Expression of glycine and other amino acid receptors by rat spinal cord mRNA in Xenopus oocytes Hiroyuki Akagi and Ricardo Miledi Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, lrvine, CA 92717 (U.S.A.) (Received 16 June 1988; Revised version received 25 August 1988; Accepted 25 August 1988) Key words: Xenopus oocyte; Spinal cord; Glycine receptor; y-Aminobutyric acid receptor; Messenger RNA; Development; In vivo translation Xenopus oocytes were injected with mRNA from adult or neonatal rat spinal cord, or from adult cerebral cortex, and examined electrophysiologically to measure currents elicited by various receptor agonists. The mRNAs induced the oocytes to acquire similar types of amino acid receptors, albeit with different potencies. In oocytes injected with adult cord mRNA the responses to kainate and y-aminobutyrate were much smaller, whilst the currents elicited by glycine and fl-alanine were markedly larger, than those in oocytes injected with cortex mRNA. For these receptors, the expressional potency of neonatal spinal cord mRNA is similar to that of the adult cord mRNA, except for a lower sensitivity to fl-alanine.
The v e r t e b r a t e spinal c o r d serves to process a n d relay sensory i n f o r m a t i o n f r o m p e r i p h e r a l tissues to the brain, a n d also to regulate m o t o r a n d a u t o n o m i c functions. V a r i o u s n e u r o t r a n s m i t t e r s , including a m i n o acids, are t h o u g h t to t a k e p a r t in these n e u r o n a l functions (for a review see ref. 14). M a n y studies have s h o w n t h a t the distrib u t i o n o f some n e u r o t r a n s m i t t e r s in the spinal c o r d differs f r o m that in the b r a i n [4]. P r e s u m a b l y , there are also differences in the types a n d densities o f n e u r o t r a n s m i t ter receptors; but, in this respect, o n l y limited i n f o r m a t i o n is a v a i l a b l e a n d it derives m a i n l y f r o m studies on the b i n d i n g o f r a d i o l a b e l l e d iigands [18]. A n alternative app r o a c h to study the n e u r o t r a n s m i t t e r receptors o f the spinal c o r d is to isolate the p o l y ( A ) + messenger R N A ( m R N A ) f r o m the tissue, a n d inject it into Xenopus oocytes to ' t r a n s p l a n t ' the receptors f r o m the spinal c o r d into the o o c y t e m e m b r a n e , where they can be e x a m i n e d in m o r e detail [12, 13]. W e have used this a p p r o a c h to c o m p a r e the 'expressional potencies' o f m R N A s c o d i n g for a m i n o acid n e u r o t r a n s m i t t e r receptors e x t r a c t e d f r o m the a d u l t rat spinal c o r d a n d cerebral cortex, as well as from the n e o n a t a l spinal cord. Correspondence: R. Miledi, Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, CA 92717, U.S.A. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
263
Mature male rats (Sprague-Dawley, 12-14 weeks old) were anesthetized with pentobarbital (200-250 mg/kg, i.p.), and the cerebral cortex and whole spinal cord from the mid-cervical level down to the cauda equina were dissected on ice. The spinal cord was also dissected from 3- to 4-day-old rats anesthetized with ether. The tissues were immediately frozen in liquid nitrogen and stored at - 7 0 ° C until used. Isolation and purification of poly(A) + mRNA was essentially as used previously [12]. The mRNAs were dissolved in water to concentrations of 1.0-1.3 mg/ml (calculated from the optical density (1.0 = 50 pg/ml) at 260 nm); and 50 nl were injected into fully grown Xenopus laevis oocytes [12]. Injected and non-injected (native) oocytes were then cultured at 16°C, for 3-8 days in modified Barth's medium with antibiotics (cf. ref. 6). On the second or third day after injection most of the oocytes were treated with collagen° ase (Sigma, Type I, 0.5 to 1.0 mg/ml for about 1-2 h) to remove partially, and sometimes completely, follicular and other enveloping cells [11]. Electrophysiological recordings were as previously reported [11, 12]. The yields of poly(A) + mRNA in different preparations were; adult cortex, 35.2; adult spinal cord, 35.6 (preparation A in Table I); neonate spinal cord, 83.6 (pg mRNA/g wet wt. tissue). The recovery of the mRNAs from total RNA (including poly(A)- mRNA and ribosomal RNA) by oligo-dT cellulose column chromatography were approximately the same (3.5-3.9%) in the different preparations. Thus, the concentration of poly(A) + mRNA in neonatal cord was more than double that of adult cortex or spinal cord. As with mRNAs from other sources, different preparations of spinal cord mRNA varied considerably in their ability to express receptors in Xenopus oocytes. FurtherTABLE I AGONIST-ACTIVATED MEMBRANE CURRENTS IN OOCYTES INJECTED WITH mRNA FROM ADULT RAT SPINAL CORDS Messenger RNA preparations labelled A-D were obtained from 4 different groups of tissue. The concentration of the mRNA was calculated from the optical density at 260 nm, and 50 nl of the solution was injected into Xenopus oocytes. The oocytes were voltage-clamped at - 6 0 inV. Each value represents peak current amplitude (mean + S.E.M., in nA) obtained in oocytes (number in parenthesis) derived from 2 to 7 donors.
Preparation
Concentration of mRNA (mg/ml)
GABA (I mM)
fl-Alanine (1 mM)
Glycine (1 raM)
Kainate (0.1 raM)
A
1.23
B
1.32
C
1.30
D
1.05
21.3 _+ 4.2 (32) 26.9 + 6.3 (8) 15.7 __. 4.8 (ll) 13.3 + 3.7(11)
360 + 47 (36) 558 + 97 (9) 418 _ 64(11) 748 + 112(21)
1004 __+ 110 (36) 1616 + 246 (9) 894 _+ 95(11) 1658 _ 251 (21)
182 _+ 16 (31) 162 _+ 20 (9) 174 + 30(10) 191 + 23 (19)
264
more, the number of receptors expressed by a given mRNA preparation also varied greatly in oocytes from different donors, as well as in oocytes from the same donor. Nevertheless, the results obtained with the four preparations of spinal cord mRNA were essentially the same (Table I), suggesting that mRNA coding for amino acid receptors can be consistently isolated from different preparations. For the comparisons presented below we used the spinal cord mRNA preparation labelled A in Table I. Messenger RNA from both adult and neonatal spinal cord induced the oocytes to acquire the ability to generate 'smooth' inward currents in response to 7-aminobutyrate (GABA), fl-alanine, glycine and kainate (Fig. 1); while native oocytes used in the present study did not generate detectable currents in response to these drugs. The properties of these currents were similar to those already reported for oocytes injected with mRNAs from rat, human or chick brains [2, 7, 8, 15]. For example, the currents elicited by GABA, fl-alanine and glycine were all carried mainly by CIions, because they reversed direction at about - 2 0 mV, which corresponds to the equilibrium potential for C1- in Xenopus oocytes [11]. Preliminary pharmacological GABA
A
~-alanine glycine
",I
ka~nate
C-i
/~ - alanine glycfne
strychnine
C -il
2 0 0 nA
200s
Fig. 1. Sample records o f ' s m o o t h ' inward currents induced by receptor agonists in oocytes injected with m R N A derived from: adult rat cerebral cortex (A), adult rat spinal cord (B) and neonatal rat spinal cord (C). Each oocyte was injected with m R N A (55-60 ng in 50 nl) and the membrane currents were measured 3 8 days later. The oocytes were voltage-clamped at - 6 0 mV and during the periods indicated by the bars the bath was perfused with 1 mM GABA, fl-alanine or glycine; or kainate at 0.1 mM. In C,~, the responses to fl-alanine and glycine were recorded before (left side) and in the presence of 0.5/iM strychnine (right side). Traces in A, B and C~ were obtained from oocytes derived from a single donor. Downward deflections denote inward membrane currents.
265
studies showed that the responses to 1 mM glycine and fl-alanine were greatly depressed by strychnine (0.5/~M; Fig. 1Cii) and picrotoxin (20 pM). The cerebral cortex and spinal cord mRNAs expressed the various responses with different potencies (Fig. 2). For instance, kainate- and GABA-induced currents, which are usually quite large after injection of adult rat cortex mRNA, were rather small in oocytes injected with spinal cord mRNAs (for convenience, referred to as 'spinal cord oocytes', and so on). In contrast, the glycine-induced current, which was relatively small in cerebral cortex oocytes, was the most prominent current in spinal cord oocytes (Fig. 2 and Table I). Another interesting feature emerges by comparing the currents elicited by fl-alanine and glycine. In cerebral cortex oocytes the current elicited by fl-alanine is approximately the same, or larger than that elicited by glycine (Figs. 1A and 2). In contrast, in spinal cord oocytes, the glycine-induced current is much larger than the one generated by fl-alanine (Figs. 1B and 2). For the series of experiments illustrated in Fig. 2 the ratio of the current induced by glycine to that induced by fl-alanine (glycine/fl-alanine ratio) was 1.0 + 0.05 (mean +_S.E.M.) for cerebral cortex mRNA and 3.4 +_0.2 for adult spinal cord mRNA. The glycine/fl-alanine ratios obtained in individual oocytes injected with adult spinal cord mRNA was not constant and ranged from 1.6 to 6.8. In neonatal spinal cord oocytes the glycine/fl-alanine ratio was 12.1 + 0.8 (range 6.5-21.0); a value considerably different from that in adult spinal cord oocytes. The present results show that mRNA from the rat spinal cord induces the oocytes to acquire functional amino acid receptors that are essentially like those induced by 36 31
T 1000
40
c v >
E 0 i
50q 32
36
PLD
u
ii!i/~!i
o
31
27 a)
GABA
b)
/~ - Alanine
c)
Glycine
d]
Kainate
Fig. 2. Peak amplitudes of smooth inward currents induced by various agonists in oocytes injected with mRNA from adult rat cerebral cortex (dotted column), adult rat spinal cord (open column) and neonate rat spinal cord (solid column). Membrane potential was clamped at - 6 0 mV. GABA (a), fl-alanine (b), glycine (c) were all perfused at I mM, and kainate (d) at 0.1 mM. Each column represents the average value (with S.E.M.) obtained in oocytes (number above column) derived from 7 different donors.
266
cerebral cortex mRNA. However, cerebral cortex and spinal cord mRNAs have greatly different expressional potencies. These differences are substantially in parallel with those obtained in receptor binding experiments [9, 17, 18]. Assuming, for the time being, that the single channels of receptors expressed by cerebral cortex and spinal cord mRNAs have similar characteristics, then the peak amplitude of the evoked currents provides a measure of the number of receptors expressed, and of the relative amounts of their respective mRNAs in the cerebral cortex and the spinal cord. An alternative approach for quantifying the receptor mRNA levels in various tissues is to perform direct measurement using radiolabeled complementary DNA hybridizing with mRNA. However, information for making suitable hybridization probes is just becoming available and still only for some receptors (e.g. ref. 5). Moreover, even if a probe is available, interpretation of the results might be somewhat complicated, because the probes can hybridize also with precursor RNA and/or degradative fragments of the target mRNA, both of which may be unable to express functional receptors. In embryonic human and rat brains, the mRNA coding for glycine receptors appears to be more abundant than that coding for GABA receptors [2, 7, 8]. Moreover, during development of the rat cortex the amount of mRNA coding for glycine receptors reaches a peak near birth and then declines; whilst the GABA receptor mRNA increases progressively and reaches a maximum plateau several weeks after birth [2]. It is as if during development the production of mRNAs coding for inhibitory neurotransmitter receptors in the cortex switches from glycine to GABA. In the present study, we showed that the adult spinal cord mRNA was over 50 times more potent in expressing glycine, rather than GABA, receptors. This is the opposite of what is observed with adult cortex mRNA and made us wonder if during development the spinal cord began by having mainly mRNA coding for GABA receptors, and switched later to transcribe mainly glycine receptor mRNA. However, this does not seem to be the case, because we have already found that embryonic (18 days) and neonatal spinal cord mRNA also expressed poorly the GABA receptors (Figs. 1C and 2A, and unpublished observations). Another interesting outcome of these experiments concerns the responses to fl-alanine, which is thought to be an inhibitory neurotransmitter (see ref. 14 for a review). In oocytes injected with cerebral cortex mRNA, the responses to fl-alanine were smaller than those to GABA, but slightly larger than those to glycine; while in oocytes injected with adult spinal cord mRNA the currents elicited by fl-alanine were smaller than those evoked by glycine, but much larger than those to GABA. Moreover, in oocytes injected with neonatal spinal cord mRNA, the responses to fl-alanine were considerably smaller than those in adult spinal cord oocytes. The glycine/fl-alanine ratio in the oocytes injected with the different mRNA preparations was not constant, as would be expected if fl-alanine was simply acting on GABA, or glycine receptors. One explanation of these results is that there is a separate specific fl-alanine receptor which is coded by a message different from those coding for GABA and glycine receptors, and which is differentially expressed [15]. An alternative explanation is that there are multiple types of glycine receptors, to which fl-alanine binds
267 with different affinities. F o r instance, different glycine receptors could be encoded by different m R N A s transcribed f r o m one gene with alternative splicing, or f r o m two or more different genes. M a n y electrophysiological studies with n e w b o r n rat spinal cord preparations have shown that strychnine-sensitive glycine receptors are present in the rat spinal cord even at early postnatal stages [3, 10]. Moreover, some inhibitory postsynaptic potentials generated in m o t o n e u r o n s o f neonatal spinal cord are blocked by strychnine [16]. In agreement with these results, we have f o u n d that neonatal rat spinal cord is rich in m R N A coding for strychnine-sensitive glycine receptors, with an expressional p o t e n c y c o m p a r a b l e to that o f the adult cord. This contrasts with a report that neonatal cord has very little m R N A that hybridizes with a cloned c D N A encoding the strychnine binding subunit o f the glycine receptor f r o m 20-day-old rat spinal cord [5]. M a n y possibilities could a c c o u n t for this a p p a r e n t discrepancy. The m o s t likely explanation, already mentioned above, is that the neonatal spinal cord contains a glycine receptor m R N A , which is different f r o m that deduced f r o m the cloned c D N A . This hypothesis is strongly supported by our recent results [1, 19] showing that m o s t o f the glycine receptor m R N A in neonatal rat spinal cord differs f r o m that in adult spinal cord, in respect to its molecular size and to some electrophysiological properties o f the encoded receptor expressed in Xenopus oocytes. This w o r k was supported by a grant (RO1-NS23284) f r o m the U.S. Public Health Service. H.A. was partly supported by the Mitsubishi Kasei Institute o f Life Sciences, Tokyo. 1 Akagi, H. and Miledi, R., Two types of glycine receptors expressed in Xenopusoocytes injected with rat spinal cord mRNA, Soc. Neurosci. Abstr., 14 (1988) 877. 2 Carpenter, M.K., Parker, I. and Miledi, R., Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex, Proc. R. Soc. Lond. Ser. B, 234 (1988) 159-170. 3 Evans, R.H., The effects of amino acids and antagonists on the isolated hemisected spinal cord of the immature rat, Br. J. Pharmacol., 62 (1978) 171-176. 4 Fagg, G.E. and Foster, A.C., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuroscience, 9 (1983) 701-719. 5 Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E.D. and Betz, H., The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors, Nature (Lond.), 328 (1987) 215-220. 6 Gundersen, C.B., Miledi, R. and Parker, I., Glutamate and kainate receptors induced by rat brain messenger RNA in Xenopusoocytes, Proc. R. Soc. Lond. Ser. B, 221 (1984) 127-143. 7 Gundersen, C.B., Miledi, R. and Parker, I., Properties of human brain glycine receptors expressed in Xenopusoocytes, Proc. R. Soc. Lond. Ser. B, 221 (1984) 235-244. 8 Gundersen, C.B., Miledi, R. and Parker, I., Messenger RNA from human brain induces drug- and voltage-operated channels in Xenopusoocytes, Nature (Lond.), 308 (1984) 421-424. 9 Henke, H. and Cu6nod, M., Specific pH]kainic acid binding in the vertebrate CNS. In U.Z. Littauer, Y. Dudai, I. Silman, V.I. Teichberg and Z. Vogel (Eds.), Neurotransmitters and their Receptors, Wiley, New York, 1980, pp. 373-390. 10 Konishi, S., Saito, K. and Otsuka, M., Postsynaptic inhibition and glycine action in isolated spinal cord of newborn rats, Jpn. J. Pharmacol., 25 (1975) Suppl. 72-73P. 11 Kusano, K., Miledi, R. and Stinnakre, J., Cholinergic and cathecolaminergic receptors in the Xenopus oocyte membrane, J. Physiol. (Lond.), 328 (1982) 143-170.
268 12 Miledi, R. and Sumikawa, K., Synthesis of cat muscle acetylcholine receptors by Xenopus oocytes, Biomed. Res., 3 (1982) 390-399. 13 Miledi, R., Parker, I. and Sumikawa, K., Recording of single y-aminobutyrate- and acetylcholine-activated receptor channels translated by exogenous mRNA in Xenopus oocytes, Proc. R. Soc. Lond. Ser. B, 218 (1982) 481-484. 14 Nistri, A., Spinal cord pharmacology of GABA and chemically related amino acid. In R.A. Davidoff (Ed.), Handbook of the Spinal Cord, Vol. 1. Pharmacology, Dekker, New York 1983, pp. 45-104. 15 Parker, I., Sumikawa, K. and Miledi, R., Responses to GABA, glycine and fl-alanine induced in Xenopus oocytes by messenger RNA from chick and rat brain, Proc. R. Soc. Lond. Ser. B, 233 (1988) 201216. 16 Takahashi, T., Inhibitory miniture synaptic potentials in rat motoneurons, Proc. R. Soc. Lond. Ser. B, 221 (1984) 103-109. 17 Wilkin, G.P., Hudson, A.L., Hill, D.R. and Bowery, N.G., Autographic localization of GABAB receptors in rat cerebellum, Nature (Lond.), 294 (1981) 584-587. 18 Young, A.B. and Snyder, S.H., Strychnine binding associated with glycine receptors of the central nervous system, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 2832-2836. 19 Akagi, H. and Miledi, R., Heterogeneity of glycine receptor and their mRNAs in rat brain and spinal cord, Science, in press.
Neuroscience Letters, 95 (1988) 262-268 Elsevier Scientific Publishers Ireland Ltd.
NSL 05763
Expression of glycine and other amino acid receptors by rat spinal cord mRNA in Xenopus oocytes Hiroyuki Akagi and Ricardo Miledi Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, lrvine, CA 92717 (U.S.A.) (Received 16 June 1988; Revised version received 25 August 1988; Accepted 25 August 1988) Key words: Xenopus oocyte; Spinal cord; Glycine receptor; y-Aminobutyric acid receptor; Messenger RNA; Development; In vivo translation Xenopus oocytes were injected with mRNA from adult or neonatal rat spinal cord, or from adult cerebral cortex, and examined electrophysiologically to measure currents elicited by various receptor agonists. The mRNAs induced the oocytes to acquire similar types of amino acid receptors, albeit with different potencies. In oocytes injected with adult cord mRNA the responses to kainate and y-aminobutyrate were much smaller, whilst the currents elicited by glycine and fl-alanine were markedly larger, than those in oocytes injected with cortex mRNA. For these receptors, the expressional potency of neonatal spinal cord mRNA is similar to that of the adult cord mRNA, except for a lower sensitivity to fl-alanine.
The v e r t e b r a t e spinal c o r d serves to process a n d relay sensory i n f o r m a t i o n f r o m p e r i p h e r a l tissues to the brain, a n d also to regulate m o t o r a n d a u t o n o m i c functions. V a r i o u s n e u r o t r a n s m i t t e r s , including a m i n o acids, are t h o u g h t to t a k e p a r t in these n e u r o n a l functions (for a review see ref. 14). M a n y studies have s h o w n t h a t the distrib u t i o n o f some n e u r o t r a n s m i t t e r s in the spinal c o r d differs f r o m that in the b r a i n [4]. P r e s u m a b l y , there are also differences in the types a n d densities o f n e u r o t r a n s m i t ter receptors; but, in this respect, o n l y limited i n f o r m a t i o n is a v a i l a b l e a n d it derives m a i n l y f r o m studies on the b i n d i n g o f r a d i o l a b e l l e d iigands [18]. A n alternative app r o a c h to study the n e u r o t r a n s m i t t e r receptors o f the spinal c o r d is to isolate the p o l y ( A ) + messenger R N A ( m R N A ) f r o m the tissue, a n d inject it into Xenopus oocytes to ' t r a n s p l a n t ' the receptors f r o m the spinal c o r d into the o o c y t e m e m b r a n e , where they can be e x a m i n e d in m o r e detail [12, 13]. W e have used this a p p r o a c h to c o m p a r e the 'expressional potencies' o f m R N A s c o d i n g for a m i n o acid n e u r o t r a n s m i t t e r receptors e x t r a c t e d f r o m the a d u l t rat spinal c o r d a n d cerebral cortex, as well as from the n e o n a t a l spinal cord. Correspondence: R. Miledi, Laboratory of Cellular and Molecular Neurobiology, Department of Psychobiology, University of California, Irvine, CA 92717, U.S.A. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.
263
Mature male rats (Sprague-Dawley, 12-14 weeks old) were anesthetized with pentobarbital (200-250 mg/kg, i.p.), and the cerebral cortex and whole spinal cord from the mid-cervical level down to the cauda equina were dissected on ice. The spinal cord was also dissected from 3- to 4-day-old rats anesthetized with ether. The tissues were immediately frozen in liquid nitrogen and stored at - 7 0 ° C until used. Isolation and purification of poly(A) + mRNA was essentially as used previously [12]. The mRNAs were dissolved in water to concentrations of 1.0-1.3 mg/ml (calculated from the optical density (1.0 = 50 pg/ml) at 260 nm); and 50 nl were injected into fully grown Xenopus laevis oocytes [12]. Injected and non-injected (native) oocytes were then cultured at 16°C, for 3-8 days in modified Barth's medium with antibiotics (cf. ref. 6). On the second or third day after injection most of the oocytes were treated with collagen° ase (Sigma, Type I, 0.5 to 1.0 mg/ml for about 1-2 h) to remove partially, and sometimes completely, follicular and other enveloping cells [11]. Electrophysiological recordings were as previously reported [11, 12]. The yields of poly(A) + mRNA in different preparations were; adult cortex, 35.2; adult spinal cord, 35.6 (preparation A in Table I); neonate spinal cord, 83.6 (pg mRNA/g wet wt. tissue). The recovery of the mRNAs from total RNA (including poly(A)- mRNA and ribosomal RNA) by oligo-dT cellulose column chromatography were approximately the same (3.5-3.9%) in the different preparations. Thus, the concentration of poly(A) + mRNA in neonatal cord was more than double that of adult cortex or spinal cord. As with mRNAs from other sources, different preparations of spinal cord mRNA varied considerably in their ability to express receptors in Xenopus oocytes. FurtherTABLE I AGONIST-ACTIVATED MEMBRANE CURRENTS IN OOCYTES INJECTED WITH mRNA FROM ADULT RAT SPINAL CORDS Messenger RNA preparations labelled A-D were obtained from 4 different groups of tissue. The concentration of the mRNA was calculated from the optical density at 260 nm, and 50 nl of the solution was injected into Xenopus oocytes. The oocytes were voltage-clamped at - 6 0 inV. Each value represents peak current amplitude (mean + S.E.M., in nA) obtained in oocytes (number in parenthesis) derived from 2 to 7 donors.
Preparation
Concentration of mRNA (mg/ml)
GABA (I mM)
fl-Alanine (1 mM)
Glycine (1 raM)
Kainate (0.1 raM)
A
1.23
B
1.32
C
1.30
D
1.05
21.3 _+ 4.2 (32) 26.9 + 6.3 (8) 15.7 __. 4.8 (ll) 13.3 + 3.7(11)
360 + 47 (36) 558 + 97 (9) 418 _ 64(11) 748 + 112(21)
1004 __+ 110 (36) 1616 + 246 (9) 894 _+ 95(11) 1658 _ 251 (21)
182 _+ 16 (31) 162 _+ 20 (9) 174 + 30(10) 191 + 23 (19)
264
more, the number of receptors expressed by a given mRNA preparation also varied greatly in oocytes from different donors, as well as in oocytes from the same donor. Nevertheless, the results obtained with the four preparations of spinal cord mRNA were essentially the same (Table I), suggesting that mRNA coding for amino acid receptors can be consistently isolated from different preparations. For the comparisons presented below we used the spinal cord mRNA preparation labelled A in Table I. Messenger RNA from both adult and neonatal spinal cord induced the oocytes to acquire the ability to generate 'smooth' inward currents in response to 7-aminobutyrate (GABA), fl-alanine, glycine and kainate (Fig. 1); while native oocytes used in the present study did not generate detectable currents in response to these drugs. The properties of these currents were similar to those already reported for oocytes injected with mRNAs from rat, human or chick brains [2, 7, 8, 15]. For example, the currents elicited by GABA, fl-alanine and glycine were all carried mainly by CIions, because they reversed direction at about - 2 0 mV, which corresponds to the equilibrium potential for C1- in Xenopus oocytes [11]. Preliminary pharmacological GABA
A
~-alanine glycine
",I
ka~nate
C-i
/~ - alanine glycfne
strychnine
C -il
2 0 0 nA
200s
Fig. 1. Sample records o f ' s m o o t h ' inward currents induced by receptor agonists in oocytes injected with m R N A derived from: adult rat cerebral cortex (A), adult rat spinal cord (B) and neonatal rat spinal cord (C). Each oocyte was injected with m R N A (55-60 ng in 50 nl) and the membrane currents were measured 3 8 days later. The oocytes were voltage-clamped at - 6 0 mV and during the periods indicated by the bars the bath was perfused with 1 mM GABA, fl-alanine or glycine; or kainate at 0.1 mM. In C,~, the responses to fl-alanine and glycine were recorded before (left side) and in the presence of 0.5/iM strychnine (right side). Traces in A, B and C~ were obtained from oocytes derived from a single donor. Downward deflections denote inward membrane currents.
265
studies showed that the responses to 1 mM glycine and fl-alanine were greatly depressed by strychnine (0.5/~M; Fig. 1Cii) and picrotoxin (20 pM). The cerebral cortex and spinal cord mRNAs expressed the various responses with different potencies (Fig. 2). For instance, kainate- and GABA-induced currents, which are usually quite large after injection of adult rat cortex mRNA, were rather small in oocytes injected with spinal cord mRNAs (for convenience, referred to as 'spinal cord oocytes', and so on). In contrast, the glycine-induced current, which was relatively small in cerebral cortex oocytes, was the most prominent current in spinal cord oocytes (Fig. 2 and Table I). Another interesting feature emerges by comparing the currents elicited by fl-alanine and glycine. In cerebral cortex oocytes the current elicited by fl-alanine is approximately the same, or larger than that elicited by glycine (Figs. 1A and 2). In contrast, in spinal cord oocytes, the glycine-induced current is much larger than the one generated by fl-alanine (Figs. 1B and 2). For the series of experiments illustrated in Fig. 2 the ratio of the current induced by glycine to that induced by fl-alanine (glycine/fl-alanine ratio) was 1.0 + 0.05 (mean +_S.E.M.) for cerebral cortex mRNA and 3.4 +_0.2 for adult spinal cord mRNA. The glycine/fl-alanine ratios obtained in individual oocytes injected with adult spinal cord mRNA was not constant and ranged from 1.6 to 6.8. In neonatal spinal cord oocytes the glycine/fl-alanine ratio was 12.1 + 0.8 (range 6.5-21.0); a value considerably different from that in adult spinal cord oocytes. The present results show that mRNA from the rat spinal cord induces the oocytes to acquire functional amino acid receptors that are essentially like those induced by 36 31
T 1000
40
c v >
E 0 i
50q 32
36
PLD
u
ii!i/~!i
o
31
27 a)
GABA
b)
/~ - Alanine
c)
Glycine
d]
Kainate
Fig. 2. Peak amplitudes of smooth inward currents induced by various agonists in oocytes injected with mRNA from adult rat cerebral cortex (dotted column), adult rat spinal cord (open column) and neonate rat spinal cord (solid column). Membrane potential was clamped at - 6 0 mV. GABA (a), fl-alanine (b), glycine (c) were all perfused at I mM, and kainate (d) at 0.1 mM. Each column represents the average value (with S.E.M.) obtained in oocytes (number above column) derived from 7 different donors.
266
cerebral cortex mRNA. However, cerebral cortex and spinal cord mRNAs have greatly different expressional potencies. These differences are substantially in parallel with those obtained in receptor binding experiments [9, 17, 18]. Assuming, for the time being, that the single channels of receptors expressed by cerebral cortex and spinal cord mRNAs have similar characteristics, then the peak amplitude of the evoked currents provides a measure of the number of receptors expressed, and of the relative amounts of their respective mRNAs in the cerebral cortex and the spinal cord. An alternative approach for quantifying the receptor mRNA levels in various tissues is to perform direct measurement using radiolabeled complementary DNA hybridizing with mRNA. However, information for making suitable hybridization probes is just becoming available and still only for some receptors (e.g. ref. 5). Moreover, even if a probe is available, interpretation of the results might be somewhat complicated, because the probes can hybridize also with precursor RNA and/or degradative fragments of the target mRNA, both of which may be unable to express functional receptors. In embryonic human and rat brains, the mRNA coding for glycine receptors appears to be more abundant than that coding for GABA receptors [2, 7, 8]. Moreover, during development of the rat cortex the amount of mRNA coding for glycine receptors reaches a peak near birth and then declines; whilst the GABA receptor mRNA increases progressively and reaches a maximum plateau several weeks after birth [2]. It is as if during development the production of mRNAs coding for inhibitory neurotransmitter receptors in the cortex switches from glycine to GABA. In the present study, we showed that the adult spinal cord mRNA was over 50 times more potent in expressing glycine, rather than GABA, receptors. This is the opposite of what is observed with adult cortex mRNA and made us wonder if during development the spinal cord began by having mainly mRNA coding for GABA receptors, and switched later to transcribe mainly glycine receptor mRNA. However, this does not seem to be the case, because we have already found that embryonic (18 days) and neonatal spinal cord mRNA also expressed poorly the GABA receptors (Figs. 1C and 2A, and unpublished observations). Another interesting outcome of these experiments concerns the responses to fl-alanine, which is thought to be an inhibitory neurotransmitter (see ref. 14 for a review). In oocytes injected with cerebral cortex mRNA, the responses to fl-alanine were smaller than those to GABA, but slightly larger than those to glycine; while in oocytes injected with adult spinal cord mRNA the currents elicited by fl-alanine were smaller than those evoked by glycine, but much larger than those to GABA. Moreover, in oocytes injected with neonatal spinal cord mRNA, the responses to fl-alanine were considerably smaller than those in adult spinal cord oocytes. The glycine/fl-alanine ratio in the oocytes injected with the different mRNA preparations was not constant, as would be expected if fl-alanine was simply acting on GABA, or glycine receptors. One explanation of these results is that there is a separate specific fl-alanine receptor which is coded by a message different from those coding for GABA and glycine receptors, and which is differentially expressed [15]. An alternative explanation is that there are multiple types of glycine receptors, to which fl-alanine binds
267 with different affinities. F o r instance, different glycine receptors could be encoded by different m R N A s transcribed f r o m one gene with alternative splicing, or f r o m two or more different genes. M a n y electrophysiological studies with n e w b o r n rat spinal cord preparations have shown that strychnine-sensitive glycine receptors are present in the rat spinal cord even at early postnatal stages [3, 10]. Moreover, some inhibitory postsynaptic potentials generated in m o t o n e u r o n s o f neonatal spinal cord are blocked by strychnine [16]. In agreement with these results, we have f o u n d that neonatal rat spinal cord is rich in m R N A coding for strychnine-sensitive glycine receptors, with an expressional p o t e n c y c o m p a r a b l e to that o f the adult cord. This contrasts with a report that neonatal cord has very little m R N A that hybridizes with a cloned c D N A encoding the strychnine binding subunit o f the glycine receptor f r o m 20-day-old rat spinal cord [5]. M a n y possibilities could a c c o u n t for this a p p a r e n t discrepancy. The m o s t likely explanation, already mentioned above, is that the neonatal spinal cord contains a glycine receptor m R N A , which is different f r o m that deduced f r o m the cloned c D N A . This hypothesis is strongly supported by our recent results [1, 19] showing that m o s t o f the glycine receptor m R N A in neonatal rat spinal cord differs f r o m that in adult spinal cord, in respect to its molecular size and to some electrophysiological properties o f the encoded receptor expressed in Xenopus oocytes. This w o r k was supported by a grant (RO1-NS23284) f r o m the U.S. Public Health Service. H.A. was partly supported by the Mitsubishi Kasei Institute o f Life Sciences, Tokyo. 1 Akagi, H. and Miledi, R., Two types of glycine receptors expressed in Xenopusoocytes injected with rat spinal cord mRNA, Soc. Neurosci. Abstr., 14 (1988) 877. 2 Carpenter, M.K., Parker, I. and Miledi, R., Expression of GABA and glycine receptors by messenger RNAs from the developing rat cerebral cortex, Proc. R. Soc. Lond. Ser. B, 234 (1988) 159-170. 3 Evans, R.H., The effects of amino acids and antagonists on the isolated hemisected spinal cord of the immature rat, Br. J. Pharmacol., 62 (1978) 171-176. 4 Fagg, G.E. and Foster, A.C., Amino acid neurotransmitters and their pathways in the mammalian central nervous system, Neuroscience, 9 (1983) 701-719. 5 Grenningloh, G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger, E.D. and Betz, H., The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors, Nature (Lond.), 328 (1987) 215-220. 6 Gundersen, C.B., Miledi, R. and Parker, I., Glutamate and kainate receptors induced by rat brain messenger RNA in Xenopusoocytes, Proc. R. Soc. Lond. Ser. B, 221 (1984) 127-143. 7 Gundersen, C.B., Miledi, R. and Parker, I., Properties of human brain glycine receptors expressed in Xenopusoocytes, Proc. R. Soc. Lond. Ser. B, 221 (1984) 235-244. 8 Gundersen, C.B., Miledi, R. and Parker, I., Messenger RNA from human brain induces drug- and voltage-operated channels in Xenopusoocytes, Nature (Lond.), 308 (1984) 421-424. 9 Henke, H. and Cu6nod, M., Specific pH]kainic acid binding in the vertebrate CNS. In U.Z. Littauer, Y. Dudai, I. Silman, V.I. Teichberg and Z. Vogel (Eds.), Neurotransmitters and their Receptors, Wiley, New York, 1980, pp. 373-390. 10 Konishi, S., Saito, K. and Otsuka, M., Postsynaptic inhibition and glycine action in isolated spinal cord of newborn rats, Jpn. J. Pharmacol., 25 (1975) Suppl. 72-73P. 11 Kusano, K., Miledi, R. and Stinnakre, J., Cholinergic and cathecolaminergic receptors in the Xenopus oocyte membrane, J. Physiol. (Lond.), 328 (1982) 143-170.
268 12 Miledi, R. and Sumikawa, K., Synthesis of cat muscle acetylcholine receptors by Xenopus oocytes, Biomed. Res., 3 (1982) 390-399. 13 Miledi, R., Parker, I. and Sumikawa, K., Recording of single y-aminobutyrate- and acetylcholine-activated receptor channels translated by exogenous mRNA in Xenopus oocytes, Proc. R. Soc. Lond. Ser. B, 218 (1982) 481-484. 14 Nistri, A., Spinal cord pharmacology of GABA and chemically related amino acid. In R.A. Davidoff (Ed.), Handbook of the Spinal Cord, Vol. 1. Pharmacology, Dekker, New York 1983, pp. 45-104. 15 Parker, I., Sumikawa, K. and Miledi, R., Responses to GABA, glycine and fl-alanine induced in Xenopus oocytes by messenger RNA from chick and rat brain, Proc. R. Soc. Lond. Ser. B, 233 (1988) 201216. 16 Takahashi, T., Inhibitory miniture synaptic potentials in rat motoneurons, Proc. R. Soc. Lond. Ser. B, 221 (1984) 103-109. 17 Wilkin, G.P., Hudson, A.L., Hill, D.R. and Bowery, N.G., Autographic localization of GABAB receptors in rat cerebellum, Nature (Lond.), 294 (1981) 584-587. 18 Young, A.B. and Snyder, S.H., Strychnine binding associated with glycine receptors of the central nervous system, Proc. Natl. Acad. Sci. U.S.A., 70 (1973) 2832-2836. 19 Akagi, H. and Miledi, R., Heterogeneity of glycine receptor and their mRNAs in rat brain and spinal cord, Science, in press.