l -Glutamate binding sites in the cerebellum and electric lobes of Torpedo marmorata

Camp. Biochem. Physiof. Vol. 9OC, No. 1,

0306-4492/88$3.00+ 0.00 0 1988Pergamon Press plc

pp. 281-284, 1988

Printed in Great Britain

L-GLUTAMATE BINDING SITES IN THE CEREBELLUM AND ELECTRIC LOBES OF ~~~~~~U ~~~~0~~~ A. MITSACOS* and P. E. GIOMPRESQ *Department of Physiology, Medical School, University of Patras, Patra, Greece, and tAbteilung Neurochemie, Max-Planck Institute fur Biophysikalische Chemie, Giittingen, Federal Republic of Germany (Receioed 30 June 1987) Abstract-l. The Cl-/Ca*+ independent r$H]glutamate binding sites have been studied in frozen sections of cerebellum and electric lobes of the tish Torpedo marmorura. 2. in vitro autoradiography showed that labelling was #n~ntra~ in the molecular layer of cerebellum and homogeneously distributed in the electric lobes. 3. Kinetics of t.-[3H]glutamate binding revealed a specific and saturable binding in both regions. 4. The pharmacological characterization showed that quisqualate was the most potent displacer of the t.-[3H]glutamate binding in both regions of Torpedo marmoruta brain.

been examined, glutamate had the highest content of

INTRODUCTION

all amino acids next to taurine in the cerebellum and next to taurine and /I-alanine in the electric lobes (Davies and Dowe, 1979). The present study reports the kinetic and preliminary pharmacological characteristics of L-[‘HIglutamate binding sites in the electric lobe of Torpedo marmorata. These binding sites were also studied in the fish cerebellum, a region where considerable evidence is available that L-glutamate serves as a neurotransmitter in mammals (Crepe1 et al., 1981; Flint et al., 1981; Freeman et al., 1983; Hudson et al., 1976). In vitro autoradiography was used to visualize the distribution of L-glutamate binding sites in the studied regions of Torpedo brain.

In higher vertebrates, receptors for excitatory amino

acids have been extensively investigated and it has recently become apparent that multiple receptors exist for these substances (Foster and Fagg, 1984). However, a limited amount of information is available in lower vertebrates (Nistri and Constanti, 1979). In lamprey spinal cord interneurons and Miiller cells, L-glutamate has fullfilled several of the neurotrans~tter criteria (Homma, 1983). Activation of N-methyl-D-aspartate (NMDA) and kainate receptors, but not quisqualate, have been recently shown to elicit fictive locomotion in the lamprey spinal cord (Brodin et al., 1985). In goldfish, glutamate has been impli~ted as the neurotransmitter in the projections from the torus longitudinalis to the optic tectum (Poli et al., 1984) and from the olfactory bulbs to the telencephalon (Bissoli et al., 1985). Specific binding of L-glutamate has been described in goldfish brain membranes where it is enriched in the crude synaptosomal fraction and elevated in the cerebellum (Francis et al., 1981). In isolated amphibian spinal cord preparations the excitatory action of glutamate and its analogues has been studied on motorneurons and several lines of evidence suggest that kainate and NMDA might act via different receptors (Nistri and Constanti, 1979). The elasmobranch tish Torpedo marmorata possesses bilateral electric organs which receive extremely dense innervation from the electric lobes in the brain stem. The available evidence suggests that the electric lobes are innervated by fibers from the oval nuclei which lie beneath the anterior end of each lobe and use a yet unknown chemical transmitter (Fox, 1977; Richardson and Fox, 1982). In a study where the amino acid content of various parts of the central nervous system of Torpedo m~rmorata has SPresent address: Laboratory of Human and Animal Physiology, Department of Biology, University of Patras, Patra, Greece.

MATERIALS AND METHODS The cerebellum and electric lobes were removed from lightly anaesthetized fishes with MS 222 (Sandoz, 0.5 g/l. sea water), mounted onto cryostat chucks and frozen under powdered dry ice. Briefly, frozen tissue sections were thawmounted onto microscope slides; pre-incubated with 50 mM Tris-citrate buffer, pH 7.1, at 4°C for 1 hr; incubated with the same buffer containing t$H]glutamate (44.1 Ci/mmol, Amersham) at 22°C for 30min; ouickly rinsed in 50mM Tris-citrate buffer, pH 7.1 at 4°C for a total of 1 min and imm~ately dried with a stream of cool air. Sections were finally scraped off the slides, placed in 1 N NaOH and dissolved with heating at 60°C. Bound radioactivity was determined by the addition of 6 ml scintillant (Beckman HP) followed by liquid scintillation counting with correction for quenching and machine efficiency. In order to generate autoradiographs, the sections were opposed to tritium sensitive film (ultorlilm, LKB) for 8-10 hr.

RESULTS The distribution of r$H]glutamate binding sites in the cerebellum and electric lobes of Torpedo marmorata was revealed by autoradiography. In the cerebellum these sites were remarkably more concentrated in the molecular than in the granule cell layer (Fig. l(A), (C)). In the electric lobes autoradiography

281

A. MITSACOS and P. E. GIOMPRE~

282

binding in both regions of Torpedo marmorata brain. The maximum number of L-[3H]glutamate binding sites (B,,,) was remarkably higher in the cerebellum than in the electric lobes (81.06 + 3.72, N = 4 and 25.31 + 0.29, N = 3, pmol/mg of protein, respectively). The ability of unlabelled L-glutamate and L-glutamate analogues to displace L-[3H]glutamate from its binding sites was assessed by liquid scintillation counting in both cerebellar and electric lobe sections at a ligand concentration of 175 nM (Tables 1, 2). Both regions exhibited a similar pharmacological profile of the Cl-/Ca*+ independent L-glutamate binding as appeared from the concentrations of the drugs tested. Quisqualic acid exhibited the strongest displacement of the L-glutamate binding in both fish cerebellum and electric lobe regions. Noticeably, at all concentrations tested quisqualic acid was a more potent inhibitor than glutamic acid itself. L-glutamate acid was a potent displacer of the L-glutamate binding with an apparent ICY,,value in the nanomolar range. Ibotenic acid was a moderate displacer with an apparent IC,, value of approximately 10 p M. Amino-phosphonobutyric acid (APB) was ineffective in displacing L-glutamate binding in the cerebellum, while N-methyl-D-aspartate acid (NMDA) and amino-phosphonovaleric acid (APV) were intermediate displacers. Fig. 1. Photographs of autoradiographs (left) and Nisslstained sections (right) of cerebellum (A), (B), (C), (D) and electric lobes (C), (D). Experiments were performed in 50mM Tris-citrate buffer at a ligand concentration of 250 nM. g-granule cell; m-molecular layer.

revealed that the L-[3H]glutamate binding sites were homogeneously distributed (Fig. l(C)). The binding of L-[3H]glutamate to both cerebellar and electric lobe frozen sections was specific and saturable (Fig. 2(A), (B)). Mean Kd values of 286.93 + 23.14 nM (N = 4) and 265.09 f 15.45 nM (N = 3) for cerebellum and electric lobes, respectively, indicated similar affinities of the L-[3H]glutamate Table

1. Competition

Compound _~ L-Glutamic acid Ibotenic acid Quisqualic acid NMDA DL-APV DL-APB

of L-[3H]glutamate glutamate analogues 1kJM 73.1 f 5.8 33.9 f 4.7 94.6 i 0.2 23.8 f 2.2

DISCUSSION

Our finding of L-[3H]glutamate binding sites in the cerebellum of the elasmobranch fish Torpedo marmorata is in accordance to a number of evidence supporting the role of glutamate as a neurotransmitter in this region (Crepe1 et al., 1981; Flint et al., 1981; Freeman et al., 1983; Hudson et al., 1976). These Na+, Cl- and Ca*+-independent binding sites in the cerebellum, as well as in the electric lobes of the fish, appeared to have similar affinity to the glutamate binding described in rat brain sections (Greenamyre et al., 1984, 1985; Monaghan et al., 1985). The maximum number (B,,) of L-glutamate binding sites in the fish cerebellum was much higher binding by unlabelled in the cerebellum

Percentage 1OpM 93.5 50.6 99.0 27.3

f k * f

glutamate

of inhibition at 100pM

0.4 1.1 0.1 1.6

99.7 86.9 100.1 40.6 15.4 12.0

f + f k i i

0.1 3.7 0.2 9.2 8.3 1.8

and

1mM 100 98.2 f 0.6 100.3 f 0.3 58.6 k 8.5 46.6 k 1.1 28.8 f 3.8

Data represent the mean + SEM (N = 4). NMDA: N-methyl-D-aspartate acid; acid; DL-APB: tx-2-Amino-C DL-APV: DL-2-Amino-5-phosphonovaleric phosphonobutyric acid.

Table

2. Competition

Compound L-Glutamic acid Ibotenic acid Ouisaualic acid NMCA

Data represent

of L-[‘HIglutamate glutamate &alogues

IpM 68.9 29.6 81.1 25.2

+ 5.3 + 4.9 +4.4 + 5.1

binding by unlabelled in the electric lobe

Percentage 1ObM 85.2 48.5 98.1 30.0

+ f f +

glutamate

of inhibition at lOO/lM

9.6 8.3 1.4 8.6

the mean k SEM (N = 4). NMDA:

97.6 69.9 100.5 52.7

f + + k

0.8 1.7 0.4 6.4

and

1mM 100 94.1 f 3.9 100.0 + 0.6 54.1 f 12.7

N-methyl-D-aspartate

acid.

L-glutamate binding in Torpedo brain

283

A

Kd \

.

= 240.25 nu B tnox = 72.66 p mol/mg pmt. .

10 30 50 70 L-PHI glutamate bound (p mollmg prot.)

lb0

Xi0

560

L-[‘HI glutamate

760

LnMl

nM Kd z2L9.65 B max: 25.02 p mollmg prot.

0

5 L-i3H] glutamate

I

0

300 L-PHI glutamate

500

IO 15 20 bound (p mollmg pmt.)

I

25

I

700

( nM1

Fig. 2. Representative saturation and Scatchard plots of Lj3H]glutamate binding in Torpedo marmoruta cerebellar (A) and electric lobe (B) sections. The experiments were performed in 50 mM Tris-citrate buffer with ligand concentrations between 50nM and 800nM. Specific binding was measured by liquid scintillation counting. Mean values are given in the text.

compared to different mammalian brain regions (Greenamyre et al., 1984; Halpain et nl., 1984; Monaghan and Cotman, 1985; Monaghan et al., 1985). It is interesting to point out that, in contrast to the rat cerebellum the distribution of Cl-/Ca2+ independent L-glutamate binding sites in the fish is more prominent in the molecular than in the granule cell layer (Monaghan and Cotman, 1985). The homogeneous distribution of glutamate binding sites in the electric lobes of the fish Torpedo marmorata could be attributed to the single neuronal cell type, the electromotor neuron, that consists the electric lobes (Richardson and Fox, 1982). The maximum number of binding sites in the lobes was lower than in the cerebellum, but higher than in mammalian brain regions. The high number of Cl-/Ca*+ independent glutamate binding sites in the fish brain and the difference in the distribution of these sites in the cerebellum between fish and mammals brought up the question of which subtype(s) exist in the fish

brain. Three physiologically identified subtypes of excitatory amino acid receptors have been described in previous studies, the quisqualate, kainate and N-methyl-D-aspartate-sensite sites. Based on the effect of chloride and calcium, glutamate binding sites have been distinguished into two distinct populations (Foster and Fagg, 1984). The pharmacological study of the Cl-/Ca*+ independent glutamate binding sites presented in this work can give some preliminary characterization of the subtype(s) in the fish brain. Further detailed characterization and a comparison to the Cl-/Ca*+ dependent sites is still needed. Three observations could be pointed out from the pharmacological study: (1) both cerebellum and electric lobes exhibited a similar pharmacological profile; (2) NMDA, at a concentration of 1 mM, reached an inhibition of L-glutamate binding of only 50% in both regions of the Torpedo brain studied. Monaghan and Cotman (1985) report that, under similar experimental conditions to ours in rat brain sections, NMDA reached a

284

A. MITSACO~and P. E. GIOMPRES

75% inhibition and exhibited an ICY value close to 10pM. A lower potency of the NMDA inhibition was thus observed in Torpedo cerebellum and electric lobes which was also supported by the rather similar potency of APV, an NMDA antagonist (Foster and Fagg, 1984); (3) quisqualic acid exhibited a potent inhibition of L-[‘HIglutamate binding in Torpedo cerebellum and electric lobes with an apparent IC, value in the nanomolar range. In mammalian cerebellum, quisqualate inhibition of L-[3H]glutamate binding has been only studied in the presence of Cl- and Ca2+ ions. In Torpedo brain, at a quisqualate concentration of lOOpM, the inhibition of L-[‘HIglutamate binding reached lOO%, while in rat cerebellar layers in the presence of Cl- and CaZ+ ions an inhibition of 65-80% has been reported (Greenamyre et al., 1985). Our results indicate that in cerebellum and electric lobes of Torpedo marmorata quisqualic acid is the most potent displacer of the L-[‘HIglutamate binding in the absence of Cl- and Ca2+ ions. It has been previously reported that projections to the electric lobes of Torpedo marmorata arise from neurons located in the oval nuclei of the anterior medulla which might serve either a “command” or a “pacemaker” function (Fox, 1977). The neurotransmitter used by these oval nuclei neurons is yet unknown. Our results on the kinetics, pharmacology and high specific binding of L-[3H]glutamate in the electric lobes provide an indication of a possible neurotransmitter role of L-glutamate in this region. However, uptake-release experiments of L-[‘Hlglutamate and further pharmacological study, are needed to support this notion. Acknowledgements-The

authors would like to thank Professor V. P. Whittaker for helpful advice and discussion and the Max-Planck-Gesellschaft for the award of fellowships.

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