Camp. Biochem. Physiol.Vol.
103C,No. 1,pp. 189-193,1992
0306~4492/92 $5.00 + 0.00
Printed in Great Britain
0
1992 Pergamon Press Ltd
TIME COURSE OF GLUTAMATE RECEPTOR EXPRESSION IN INDIVIDUAL OOCYTES OF XENOPUS LAEVLS AFTER INJECTION OF RAT BRAIN RNA U. MUSSHOFF,*M. MADEJA,*P. BLOMS*and E.-J. SPECKMANN*~ *Institut fiir Physiologie, Robert-Koch-Str. 27a, 4400 Miinster, F.R.G. and TInstitut fiir Experimentelle Epilepsieforschung, Hiifferstr. 68, 4400 Miinster, F.R.G. (Received 2 January 1992)
Abstract-l.
The receptor for the neurotransmitter glutamate was functionally expressed in oocytes of Xenopus luevis after microinjection of rat brain RNA. The functional differentiation of this receptor type was further analyzed. 2. The development of the sensitivity to the agonists showed a time course which was differential for the various ligands in individual oocytes. 3. Sensitivity appeared after one day for kainate (KA), after two days for alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionate (AMPA) and quisqualate (QA), and after five days for N-methyl-D-aspartate (NMDA). 4. The KA response was markedly reduced by simultaneous application of AMPA. This was even found on the first day when an agonistic AMPA reaction was not detectable. 5. NMDA and non-NMDA receptors can clearly be differentiated by their delay of expression.
MATERIALAND
INTRODUCTION The excitatory amino acid glutamate is described to
act on four pharmacologically dis,tinct receptors: three ionotropic receptors, which are labelled according to their relatively selective agonists as the N-methyl-Daspartate (NMDA), kainate (KA) and alpha-amino3-hydroxy-S-methyl-4-isoxazolepropionate (AMPA) receptor, and a fourth metabotropic receptor, which is preferentially activated by the agonist quisqualate (QA; cf. Nicoll et al., 1990). The well-defined NMDA receptor shows pharmacological and electrophysiological characteristics which differ markedly from the other glutamate subreceptors (cf. Lambert and Heinemann, 1986). For this reason the ionotropic KA and AMPA receptors are referred to as nonNMDA receptors. However, evidences for two separated ionotropic non-NMDA receptors are not yet established because there are no specific antagonists available which are able to discriminate between KA and AMPA receptors (Nicoll ef al., 1990). Moreover, some biochemical and pharmacological investigations support the concept of a unitary non-NMDA receptor complex (Henley et al., 1989; Ambrosini et af., 1991; Patneau and Mayer, 1991). Nevertheless, the question is still unanswered (Mayer, 1990). To get further information about the functional differentiation of the receptors in question, the time needed for the expression of glutamate subreceptors was studied. To this purpose RNA isolated from rat brain tissue was microinjected into individual oocytes of Xenopus laevis. Since injected oocytes showed a variability in their translation efficiency, the same cells were tested daily for sensitivity to the ligands.
METHODS
Oocytes of Xenopus laevis (stage V and VI; Dumont, 1972) were prepared mechanically without removing the follicle cells. RNA was extracted from neocortical tissue of adult rat brains using a guanidine/LiCl method (Cathala et al., 1983). For injection total RNA was dissolved in distilled water at a final concentration of I .5mg/ml. The solution was injected into the occytes by pressure via a glass capillary. The total volume injected was 50 nl per cell. The occytes were maintained in a modified Barth medium composed- of (in mmol/l): NaCl88, CaCl, 1.5, KC1 1, NaHCO, 2.4. MeSO, 0.8. The nH was adiusted to 7.4 bv HEPES (5 mmoljl). genicillin (COOW/ml) &d streptomycin (lOOpg/ml) were added to the solution. The temperature was kept constant at 20°C. The medium was exchanged every second day. Under these conditions the oocytes were kept for up to 9 days. For recording transmembraneous ion currents conven-
tional two-electrode voltage-clamp technique was applied. The holding potential was -50 mV. Substances were administered for 60s each using a concentration-clamp device (Madeja ef al., 1991).The concentrations of the tested ligands were: GLU 200 pmol/l, KA 50 pmol/l, AMPA 1OO~mol/l, QA SO~mol/l and NMDA lOO~mol/l (glycine added, 1 pmol/l).
RESULTS The oocytes were injected with RNA from 3 separate batches. The results were obtained from 24 oocytes of 7 different donors. The same oocytes were daily examined electrophysiologically for 5 to 9 days after injection. The examination started 24 hr after RNA injection except for 2 oocytes which were 189
U. MUSSHOFFet al.
190
A KA
NMDA
AMPA
151 nA
B GLU
QA
Fig. 1. Membrane currents of an individual oocyte (Xenopus hois) elicited by kainate (KA; 50 fimol/l), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 pmol/l), N-methyl-D-aspartate (NMDA; 100 pmol/l), glutamate (GLU; 200 pmol/l) and quisqualate (QA; 50 pmol/l). Administration is marked by horizontal bars. Holding potential: - 50 mV. Inward current: downward deflection. Recordings 5 days after injection of RNA from rat neocortex. studied already after 6 hr. Resting membrane potential and input resistance of the oocytes ,showed maximal variations of 10 mV and 0.1 Mn during the period of examination. Injected oocytes developed sensitivity to all excitatory amino acids tested, i.e. to GLU, KA, AMPA, QA and NMDA (Fig. 1). Application of KA and
AMPA elicited current reactions in all and NMDA in 13 of 24 oocytes. The responses consisted in smooth inward currents (Fig. 1A). Administration of GLU and QA evoked oscillatory inward currents superimposed on a smooth component (Fig. 1B) in 19 oocytes and in 5 oocytes only smooth inward currents. In case that oscillations were present, the wash-out of the agonists was associated with the appearance of an inward current of high amplitude (Fig. 1B; MuDhoff et al., 1991). Water-injected (n = 5) or native oocytes (n = 30) showed no responses to the tested ligands. The NMDA response could be selectively blocked by the antagonist 2-amino-S-phosphonovaleric acid (APV) and the KA and AMPA response by the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione
WQW.
The development of the sensitivity to the agonists tested here showed a time course which was different for the various agonists (Fig. 2). A reaction to the application of an agonist was determined to be present when its amplitude exceeded 5 nA and when it could be blocked by the corresponding antagonist. On the basis of these criteria no detectable response to KA was observed 6 hr after injection of RNA (n = 2). The first KA response occurred after 24 hr (n = 24). The KA reaction increased with time of incubation and reached a maximum after 8 days (n = 24 for 5 days; n = 5 for 8 days). At this point of time the KAactivated current had an amplitude which was about
100-fold that of the first day after injection. With further incubation of the oocyte the KA-activated current amplitude re-decreased (n = 5). The response to AMPA (n = 24) and to QA (n = 24) appeared after 2 days (Fig. 2) and that to NMDA after 5 days (Fig. 3) of incubation. The AMPA and NMDA reactions reached their maximum amplitude after 8 (n = 5) and after 7 days (n = 3), respectively. The AMPA reaction re-decreased after 9 days (n = 5) whereas the NMDA response persisted up to this point of time. With QA application the smooth current response reached its maximum after 8 days (n = 5) and the oscillations (peak to peak) after 4 days (n = 15). The smooth component redecreased after 9 days (n = 4) and the oscillations after 5 days of incubation (cf. Fig. 2; n = 15). Comparing the development of the KA and AMPA response two relations became apparent: in one group of oocytes the KA response increased in amplitude with the AMPA reaction having already reached a plateau level (Fig. 4A) and in another group of oocytes the KA response and the AMPA response increased in parallel (Fig. 4B). Therewith in the first case the ratio of the amplitudes of the KA- and AMPA-currents increased and in the latter remained nearly constant. The size of the agonist responses varied between oocytes from different donors, but was relatively consistent in oocytes taken from the same frog. However, the latency of the functional expression of the different subreceptors had the same characteristics for all investigated oocytes. Since it has been reported in literature (Rassendren et al., 1989; Verdoorn and Dingledine, 1989) that the KA response was reduced markedly by the simultaneous application of AMPA, it was tested whether
Glutamate receptors in
KA 1st Day u--
Xenopus
191
QA
AMPA -
oocytes
’
2nd Day
?r 3rd Day
1’ 4th Day
li 5th Day
30 s
Fig. 2. Different latencies of appearance of membrane currents of an individual oocyte (Xenopus laeuis) elicited by kainate (KA; 50 pmol/l), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; lOO~mol/l), and quisqualate (QA; 50pmol/l). Days after injection of RNA from rat neocortex are marked at the left margin. Administration is marked by horizontal bars. Holding potential: -50 mV. Inward current: downward deflection. Each row represents the recordings at the day indicated.
or not AMPA is able to exert such an influence at a point of time when AMPA itself had no effect, i.e. after the first day. As can be seen from Fig. 5, the interaction between KA and AMPA was the same in principle after 1 and 5 days of incubation. This demonstrates that the effect of AMPA on the KA reaction proved to he independent of whether an AMPA current was present (after 5 days) or missing (after 1 day). DISCUSSION
Injection of mRNA into oocytes led, with different delays, to the functional expression of glutamate sub-
receptors sensitive to KA, AMPA, QA and NMDA. Such a temporal sequence of receptor expression has not yet been demonstrated in individual oocytes. A survey on the pertinent literature reveals that many groups were also able to investigate KA, AMPA and
QA reactions already on the second day after injection (Randle et al., 1988; Rassendren et af., 1989; Verdoom and Dingledine, 1989; Bowie and Smart, 199 1). These investigations, however, were performed with various aims and not in individual but in different oocytes. In contrast to the present findings the NMDA receptor response existed after an expression latency which is similar to the non-NMDA receptors (Kleckner and Dingledine, 1988; Leonard and Kelso, 1990; Watson et al., 1990). As a whole, with respect to the nonNMDA receptors the findings in literature show rough agreement with the present results, whereas there are discrepancies regarding the expression time of NMDA receptors. For the differential latencies of expression of receptors the following mechanisms can in principle be taken into account: a first hypothesis is based on the prerequisite that the different receptors are encoded by separate mRNA molecules. In this case there are
U.
192
MUSSHOFF
e&
NMDA
NMDA
NMDA
al.
II
1st
Day -_rY
2nd
Day
‘...
3rd
Day
1
4th
Day -_+-
-
k
,
-
:.
70
-I
nA 30 s
Fig. 3. Latencies of appearance of membrane currents of 3 individual oocytes (Xenopus laeuis) elicited by N-methyl-D-aspartate (NMDA; lOO~mol/l). Days after injection of RNA from rat neocortex are marked at the left margin. Administration is marked by horizontal bars. Holding potential: - 50 mV. Inward current: downward deflection. Each row represents the recordings at the day indicated. basically
two possibilities:
(i) Different
amounts
of
separate mRNA molecules may lead to differential latencies of detectable receptor expressions if the bind-
A,d,(pP* 20
10
100
0
0 2nd Day
3rd Day
4th Day
5th Day
6th Day
B @Al) 503 20
402 m
10
MO 103
0
0 2nd Day
3rd Day
4th Day
5th Day
6th Day
Fig. 4. Ratios between membrane currents elicited by alphaamino-3-hydroxy-S-methyl&isoxazolepropionate (AMPA; 100 pmol/l) and kainate (KA; 50 pmol/l) at different days after injection of RNA from rat neocortex into individual oocytes (Xenopuslaeuis).A and B: Individual oocytes with increasing (A) and constant (B) ratio of KA- and AMPAcurrents. Holding potential: - 50 mV. AMPA and KA: Amplitudes of currents elicited by the substances. KA}AMPA: Ratio of the amplitudes of currents elicited by KA and AMPA. Abscissa: Days after injection.
ing to the ribosomes occurs in a stochastic manner. (ii) Identical amounts of separate mRNA molecules, provided that the binding to the ribosomes occurs in a stochastic manner, may lead to differential latencies of receptor expression if the post-translational processes involved need different periods of time until the receptors are incorporated into the membrane. A second hypothesis is based on the prerequisite that all or at least some receptors are encoded by the same mRNA molecules. In this case the different latencies of receptor expression may be due to the sequential functional maturation after protein synthesis and incorporation into the membrane. In this context many processes may be involved, e.g. phosphorylation, glycosylation, changes in the lipid environment of the plasma membrane and others. As to the aim of the present paper the following conclusions may be drawn. The temporal sequence of appearance of currents elicited by KA and AMPA (Figs 2 and 4A) and the missing parallelism of time course of current augmentation (Fig. 4A) found in one part of experiments are in favour of the idea of separated KA- and AMPA-receptors. The interaction of KA and AMPA (Fig. 5) found already on the first day after injection and the parallelism in the time-course of current augmentation (Fig. 4B) found in the other part of experiments are in favour of a unique KA/AMPA receptor. The relatively long time taken for the expression of the NMDA receptor supports the assumption that the NMDA receptor represents a receptor type separated from the other ionotropic receptors. The results allow no reliable conclusion as to the differentiation of KA and AMPA
Glutamate receptors in Xenopus oocytes
AMPA
5thDay-
KA
193
KA+AMPA
-L&r
-I
90 nA 30 s
Fig. 5. Interaction of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 pmol/l) and kainate (KA; 50 pmol/l) in an individual oocyte (Xenopus lueuis) at the first and fifth day after injection of RNA from rat neocortex. Administration is marked by horizontal bars. Holding potential: - 50 mV. Inward current: downward deflection. KA + AMPA: simultaneous application of both substances.
receptors. However, taking all findings together one may assume that the receptors coexist in separated as well as in combined types (cf. Egebjerg et al., 1991; Hollmann et al., 1991; Pruss et al., 1991). REFERENCES
Ambrosini A., Barnard E. A. and Prestipino G. (1991) AMPA and kainate-operated channels reconstituted in artificial bilayers. FEBS I&t. 281, 27-29. Bowie D. and Smart T. G. (1991) Interaction of S-bromowillardiine with non-NMDA receptors expressed in Xenopus laeuis oocytes injected with chick brain mRNA. Neurosci. L.ett. 121, 68-72.
Cathala C., Savouret J.-F., Mendez B., West B. L., Karin M., Martial J. A. and Baxter J. D. (1983) A method for isolation of intact, translationally active ribonucleic acid. DNA 2, 329-335.
Dumont J. N. (1972) Oogenesis in Xenopus laeuis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morph. 136, 153-180. Egebjerg J., Bettler B., Hermans-Borgmeyer I. and Heinemann S. (1991) Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature 351, 745-748.
Henley J. M., Ambrosini A., Krogsgaard-Larsen P. and Barnard E. A. (1989) Evidence for a single glutamate receptor of the ionotropic kainate/quisqualate type. The New Biologist 1, 153-158. Hollmann M., Hartley M. and Heineman S. (1991) Ca2+ permeability of KA- AMPA- gated glutamate receptor channels depends on subunit composition. Science 252, 851-853.
Kleckner N. W. and Dingledine R. (1988) Requirement for glycine in activation of NMDA-receptors expressed in Xenopus oocytes. Science 241, 835-837. Lambert J. D. C. and Heinemann U. (1986) Extracellular calcium changes accompanying the action of excitatory amino acids in area CA1 of the hippocampus. Possible implications for the initiation and spread of epileptic discharges. In Epilepsy and Calcium (Edited by Speckmann E.-J., Schulze H. and Walden J.), pp. 35-61. Urban and Schwarzenberg, Miinchen-Wien-Baltimore.
Leonard J. P. and Kelso S. R. (1990) Apparent desensitization of NMDA responses in Xenopus oocytes involves calcium-dependent chloride current. Neuron 2, 53-60. Madeja M., MuDhoff U. and Speckmann E.-J. (1991) A concentration+lamp system allowing two-electrode voltage-clamp investigations in oocytes of Xenopus luevis. Neurosci. Meth. 38, 267-269.
Mayer M. L. (1990) Glutamate: three meetings but how many receptors? The New Biologist 2, 865869.
MuDhoff U., Madeja M. Kuhlmann D. and Speckmann E.-J. (1991) Inward currents elicited by stream of fluid during transmitter-induced current oscillations in RNAinjected oocytes of Xenopus laevis. Neurosci. Lett. 125, 212-214.
Nicoll R. A., Malenka R. C. and Kauer J. A. (1990) Functional comparison of neurotransmitter receptor subtypes in mammalian central nervous system. Physiol. Rev. 70, 513-565.
Patneau D. K. and Mayer M. L. (1991) Kinetic analysis of interactions between kainate and AMPA: evidence for activation of a single receptor in mouse hippocampal neurons. Neuron 6, 785-798. Pruss R. M., Akeson R. L., Racke M. M. and Wilburn J. L. (1991) Agonist-activated cobalt uptake identifies divalent cation-permeable kainate receptors on neurons and glial cells. Neuron 7, 509-518. Randle J. C. R., Vernier P., Garrigues A.-M. and Brault E. (1988) Properties of the kainate channel in rat brain mRNA injected Xenopus oocytes: ionic selectivity and blockage. Mol. Cell. Biochem. 80, 121-132. Rassendren F.-A., Lory P., Pin J.-P., Bockaert J. and Narageot P. (1989) A specific quisqualate agonist inhibits kainate responses induced in Xenopus oocytes injected with rat brain RNA. Neurosci. l&f. 99, 333339.
Verdoorn T. A. and Dingledine R. (1989) Excitatory amino acid receptors expressed in Xenopus oocytes: agonist apharmacology. Mol. Pharmacol. 34, 298-307. Watson G. B., Bolanowski M. A., Baganoff M. P., Deppeler C. L. and Lanthom T. H. (1990) D-cycloserine acts as a partial agonist at the glycine modulatory site of the NMDA receptor expressed in Xenopus oocytes. Brain Rex 510, 158-160.
103C,No. 1,pp. 189-193,1992
0306~4492/92 $5.00 + 0.00
Printed in Great Britain
0
1992 Pergamon Press Ltd
TIME COURSE OF GLUTAMATE RECEPTOR EXPRESSION IN INDIVIDUAL OOCYTES OF XENOPUS LAEVLS AFTER INJECTION OF RAT BRAIN RNA U. MUSSHOFF,*M. MADEJA,*P. BLOMS*and E.-J. SPECKMANN*~ *Institut fiir Physiologie, Robert-Koch-Str. 27a, 4400 Miinster, F.R.G. and TInstitut fiir Experimentelle Epilepsieforschung, Hiifferstr. 68, 4400 Miinster, F.R.G. (Received 2 January 1992)
Abstract-l.
The receptor for the neurotransmitter glutamate was functionally expressed in oocytes of Xenopus luevis after microinjection of rat brain RNA. The functional differentiation of this receptor type was further analyzed. 2. The development of the sensitivity to the agonists showed a time course which was differential for the various ligands in individual oocytes. 3. Sensitivity appeared after one day for kainate (KA), after two days for alpha-amino-3-hydroxy-5methyl-4-isoxazolepropionate (AMPA) and quisqualate (QA), and after five days for N-methyl-D-aspartate (NMDA). 4. The KA response was markedly reduced by simultaneous application of AMPA. This was even found on the first day when an agonistic AMPA reaction was not detectable. 5. NMDA and non-NMDA receptors can clearly be differentiated by their delay of expression.
MATERIALAND
INTRODUCTION The excitatory amino acid glutamate is described to
act on four pharmacologically dis,tinct receptors: three ionotropic receptors, which are labelled according to their relatively selective agonists as the N-methyl-Daspartate (NMDA), kainate (KA) and alpha-amino3-hydroxy-S-methyl-4-isoxazolepropionate (AMPA) receptor, and a fourth metabotropic receptor, which is preferentially activated by the agonist quisqualate (QA; cf. Nicoll et al., 1990). The well-defined NMDA receptor shows pharmacological and electrophysiological characteristics which differ markedly from the other glutamate subreceptors (cf. Lambert and Heinemann, 1986). For this reason the ionotropic KA and AMPA receptors are referred to as nonNMDA receptors. However, evidences for two separated ionotropic non-NMDA receptors are not yet established because there are no specific antagonists available which are able to discriminate between KA and AMPA receptors (Nicoll ef al., 1990). Moreover, some biochemical and pharmacological investigations support the concept of a unitary non-NMDA receptor complex (Henley et al., 1989; Ambrosini et af., 1991; Patneau and Mayer, 1991). Nevertheless, the question is still unanswered (Mayer, 1990). To get further information about the functional differentiation of the receptors in question, the time needed for the expression of glutamate subreceptors was studied. To this purpose RNA isolated from rat brain tissue was microinjected into individual oocytes of Xenopus laevis. Since injected oocytes showed a variability in their translation efficiency, the same cells were tested daily for sensitivity to the ligands.
METHODS
Oocytes of Xenopus laevis (stage V and VI; Dumont, 1972) were prepared mechanically without removing the follicle cells. RNA was extracted from neocortical tissue of adult rat brains using a guanidine/LiCl method (Cathala et al., 1983). For injection total RNA was dissolved in distilled water at a final concentration of I .5mg/ml. The solution was injected into the occytes by pressure via a glass capillary. The total volume injected was 50 nl per cell. The occytes were maintained in a modified Barth medium composed- of (in mmol/l): NaCl88, CaCl, 1.5, KC1 1, NaHCO, 2.4. MeSO, 0.8. The nH was adiusted to 7.4 bv HEPES (5 mmoljl). genicillin (COOW/ml) &d streptomycin (lOOpg/ml) were added to the solution. The temperature was kept constant at 20°C. The medium was exchanged every second day. Under these conditions the oocytes were kept for up to 9 days. For recording transmembraneous ion currents conven-
tional two-electrode voltage-clamp technique was applied. The holding potential was -50 mV. Substances were administered for 60s each using a concentration-clamp device (Madeja ef al., 1991).The concentrations of the tested ligands were: GLU 200 pmol/l, KA 50 pmol/l, AMPA 1OO~mol/l, QA SO~mol/l and NMDA lOO~mol/l (glycine added, 1 pmol/l).
RESULTS The oocytes were injected with RNA from 3 separate batches. The results were obtained from 24 oocytes of 7 different donors. The same oocytes were daily examined electrophysiologically for 5 to 9 days after injection. The examination started 24 hr after RNA injection except for 2 oocytes which were 189
U. MUSSHOFFet al.
190
A KA
NMDA
AMPA
151 nA
B GLU
QA
Fig. 1. Membrane currents of an individual oocyte (Xenopus hois) elicited by kainate (KA; 50 fimol/l), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 pmol/l), N-methyl-D-aspartate (NMDA; 100 pmol/l), glutamate (GLU; 200 pmol/l) and quisqualate (QA; 50 pmol/l). Administration is marked by horizontal bars. Holding potential: - 50 mV. Inward current: downward deflection. Recordings 5 days after injection of RNA from rat neocortex. studied already after 6 hr. Resting membrane potential and input resistance of the oocytes ,showed maximal variations of 10 mV and 0.1 Mn during the period of examination. Injected oocytes developed sensitivity to all excitatory amino acids tested, i.e. to GLU, KA, AMPA, QA and NMDA (Fig. 1). Application of KA and
AMPA elicited current reactions in all and NMDA in 13 of 24 oocytes. The responses consisted in smooth inward currents (Fig. 1A). Administration of GLU and QA evoked oscillatory inward currents superimposed on a smooth component (Fig. 1B) in 19 oocytes and in 5 oocytes only smooth inward currents. In case that oscillations were present, the wash-out of the agonists was associated with the appearance of an inward current of high amplitude (Fig. 1B; MuDhoff et al., 1991). Water-injected (n = 5) or native oocytes (n = 30) showed no responses to the tested ligands. The NMDA response could be selectively blocked by the antagonist 2-amino-S-phosphonovaleric acid (APV) and the KA and AMPA response by the antagonist 6-cyano-7-nitroquinoxaline-2,3-dione
WQW.
The development of the sensitivity to the agonists tested here showed a time course which was different for the various agonists (Fig. 2). A reaction to the application of an agonist was determined to be present when its amplitude exceeded 5 nA and when it could be blocked by the corresponding antagonist. On the basis of these criteria no detectable response to KA was observed 6 hr after injection of RNA (n = 2). The first KA response occurred after 24 hr (n = 24). The KA reaction increased with time of incubation and reached a maximum after 8 days (n = 24 for 5 days; n = 5 for 8 days). At this point of time the KAactivated current had an amplitude which was about
100-fold that of the first day after injection. With further incubation of the oocyte the KA-activated current amplitude re-decreased (n = 5). The response to AMPA (n = 24) and to QA (n = 24) appeared after 2 days (Fig. 2) and that to NMDA after 5 days (Fig. 3) of incubation. The AMPA and NMDA reactions reached their maximum amplitude after 8 (n = 5) and after 7 days (n = 3), respectively. The AMPA reaction re-decreased after 9 days (n = 5) whereas the NMDA response persisted up to this point of time. With QA application the smooth current response reached its maximum after 8 days (n = 5) and the oscillations (peak to peak) after 4 days (n = 15). The smooth component redecreased after 9 days (n = 4) and the oscillations after 5 days of incubation (cf. Fig. 2; n = 15). Comparing the development of the KA and AMPA response two relations became apparent: in one group of oocytes the KA response increased in amplitude with the AMPA reaction having already reached a plateau level (Fig. 4A) and in another group of oocytes the KA response and the AMPA response increased in parallel (Fig. 4B). Therewith in the first case the ratio of the amplitudes of the KA- and AMPA-currents increased and in the latter remained nearly constant. The size of the agonist responses varied between oocytes from different donors, but was relatively consistent in oocytes taken from the same frog. However, the latency of the functional expression of the different subreceptors had the same characteristics for all investigated oocytes. Since it has been reported in literature (Rassendren et al., 1989; Verdoorn and Dingledine, 1989) that the KA response was reduced markedly by the simultaneous application of AMPA, it was tested whether
Glutamate receptors in
KA 1st Day u--
Xenopus
191
QA
AMPA -
oocytes
’
2nd Day
?r 3rd Day
1’ 4th Day
li 5th Day
30 s
Fig. 2. Different latencies of appearance of membrane currents of an individual oocyte (Xenopus laeuis) elicited by kainate (KA; 50 pmol/l), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; lOO~mol/l), and quisqualate (QA; 50pmol/l). Days after injection of RNA from rat neocortex are marked at the left margin. Administration is marked by horizontal bars. Holding potential: -50 mV. Inward current: downward deflection. Each row represents the recordings at the day indicated.
or not AMPA is able to exert such an influence at a point of time when AMPA itself had no effect, i.e. after the first day. As can be seen from Fig. 5, the interaction between KA and AMPA was the same in principle after 1 and 5 days of incubation. This demonstrates that the effect of AMPA on the KA reaction proved to he independent of whether an AMPA current was present (after 5 days) or missing (after 1 day). DISCUSSION
Injection of mRNA into oocytes led, with different delays, to the functional expression of glutamate sub-
receptors sensitive to KA, AMPA, QA and NMDA. Such a temporal sequence of receptor expression has not yet been demonstrated in individual oocytes. A survey on the pertinent literature reveals that many groups were also able to investigate KA, AMPA and
QA reactions already on the second day after injection (Randle et al., 1988; Rassendren et af., 1989; Verdoom and Dingledine, 1989; Bowie and Smart, 199 1). These investigations, however, were performed with various aims and not in individual but in different oocytes. In contrast to the present findings the NMDA receptor response existed after an expression latency which is similar to the non-NMDA receptors (Kleckner and Dingledine, 1988; Leonard and Kelso, 1990; Watson et al., 1990). As a whole, with respect to the nonNMDA receptors the findings in literature show rough agreement with the present results, whereas there are discrepancies regarding the expression time of NMDA receptors. For the differential latencies of expression of receptors the following mechanisms can in principle be taken into account: a first hypothesis is based on the prerequisite that the different receptors are encoded by separate mRNA molecules. In this case there are
U.
192
MUSSHOFF
e&
NMDA
NMDA
NMDA
al.
II
1st
Day -_rY
2nd
Day
‘...
3rd
Day
1
4th
Day -_+-
-
k
,
-
:.
70
-I
nA 30 s
Fig. 3. Latencies of appearance of membrane currents of 3 individual oocytes (Xenopus laeuis) elicited by N-methyl-D-aspartate (NMDA; lOO~mol/l). Days after injection of RNA from rat neocortex are marked at the left margin. Administration is marked by horizontal bars. Holding potential: - 50 mV. Inward current: downward deflection. Each row represents the recordings at the day indicated. basically
two possibilities:
(i) Different
amounts
of
separate mRNA molecules may lead to differential latencies of detectable receptor expressions if the bind-
A,d,(pP* 20
10
100
0
0 2nd Day
3rd Day
4th Day
5th Day
6th Day
B @Al) 503 20
402 m
10
MO 103
0
0 2nd Day
3rd Day
4th Day
5th Day
6th Day
Fig. 4. Ratios between membrane currents elicited by alphaamino-3-hydroxy-S-methyl&isoxazolepropionate (AMPA; 100 pmol/l) and kainate (KA; 50 pmol/l) at different days after injection of RNA from rat neocortex into individual oocytes (Xenopuslaeuis).A and B: Individual oocytes with increasing (A) and constant (B) ratio of KA- and AMPAcurrents. Holding potential: - 50 mV. AMPA and KA: Amplitudes of currents elicited by the substances. KA}AMPA: Ratio of the amplitudes of currents elicited by KA and AMPA. Abscissa: Days after injection.
ing to the ribosomes occurs in a stochastic manner. (ii) Identical amounts of separate mRNA molecules, provided that the binding to the ribosomes occurs in a stochastic manner, may lead to differential latencies of receptor expression if the post-translational processes involved need different periods of time until the receptors are incorporated into the membrane. A second hypothesis is based on the prerequisite that all or at least some receptors are encoded by the same mRNA molecules. In this case the different latencies of receptor expression may be due to the sequential functional maturation after protein synthesis and incorporation into the membrane. In this context many processes may be involved, e.g. phosphorylation, glycosylation, changes in the lipid environment of the plasma membrane and others. As to the aim of the present paper the following conclusions may be drawn. The temporal sequence of appearance of currents elicited by KA and AMPA (Figs 2 and 4A) and the missing parallelism of time course of current augmentation (Fig. 4A) found in one part of experiments are in favour of the idea of separated KA- and AMPA-receptors. The interaction of KA and AMPA (Fig. 5) found already on the first day after injection and the parallelism in the time-course of current augmentation (Fig. 4B) found in the other part of experiments are in favour of a unique KA/AMPA receptor. The relatively long time taken for the expression of the NMDA receptor supports the assumption that the NMDA receptor represents a receptor type separated from the other ionotropic receptors. The results allow no reliable conclusion as to the differentiation of KA and AMPA
Glutamate receptors in Xenopus oocytes
AMPA
5thDay-
KA
193
KA+AMPA
-L&r
-I
90 nA 30 s
Fig. 5. Interaction of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA; 100 pmol/l) and kainate (KA; 50 pmol/l) in an individual oocyte (Xenopus lueuis) at the first and fifth day after injection of RNA from rat neocortex. Administration is marked by horizontal bars. Holding potential: - 50 mV. Inward current: downward deflection. KA + AMPA: simultaneous application of both substances.
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