64 \curosciem'c i.c~tcr.~. J l~2 f l 9~9) ~4 ,,.~

Elsevier Scientific Publishcr~ Ireland [ t ~

NSL 06172

Extracellular taurine increase in rat hippocampus evoked by specific glutamate receptor activation is

related to the excitatory potency of glutamate agonists

Nieves Men6ndez, Oscar Herreras, Jos6 M. Solis, Antonio S. Herranz and Rafael Martin del Rio

Departamento de Investigaci6n, Hospital "Rambn y Cqjal', Madrid (Spare)

(Received 8 February 1989; Revised version received 14 March 1989; Accepted 15 March 1989)

Key wor&." Taurine; Glutamate agonist; Glutamate antagonist; Hippocampus; Brain dialysis; Neurotox- icity

Taurine increases in brain extracellular space due to glutamate agonists were studied in vivo in the rat hippocampus using a dialysis technique, both in the absence and in the presence of glutamate receptor antagonists. Extracellular taurine levels increased during perfusions of agonists, listed in descending order of potency: kainate (KA), N-methyI-D-aspartate (NMDA), and quisqualate (QA). While taurine increases due to KA or QA perfusions were inhibited by 6,7-dinitro-quinoxaline-2,3-dione (DNQX), those induced by NMDA were abolished in the presence of 3-(carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP). These results indicate that increases in extracellular taurine levels evoked by NMDA, KA or QA in the rat hippocampus are caused by activation of their specific receptors. Field potentials, concomitantly recorded, were quickly abolished during NMDA or KA perfusions (0.1 mM), while QA (0.25 raM) induced the appearance of bicuculline-like evoked responses. Since taurine has been proposed as an osmoregulatory substance in the rat brain, and cell swelling is known to be an early component of gluta- mate agonists neurotoxicity, the increases in extracellular taurine reported here could be due to taurine released through an osmoregulatory process, counteracting the neurotoxic cellular oedema induced by glu- tamate agonists.

Increases of extracellular taurine levels in several brain regions due to the action of excitatory amino acids such as kainate (KA) and N-methyl-D-aspartate (NMDA) have been reported before [3, 10, l 1]. It has also been observed that 2-amino-5-phos- phonovalerate (APV), an NMDA antagonist, counteracts the effect of NMDA on extracellular taurine levels [10], suggesting that this effect is probably mediated by activation of NMDA-specific receptors. With the recent discovery of potent and se-

Correspondence: R. Martin del Rio, Departamento de Investigaci6n, Hospital 'Rambn y Cajal', Ctra. Col- menar, km. 9, 28034 Madrid, Spain.

0304-3940/89/$ 03.50 ~t') 1989 Elsevier Scientific Publishers Ireland Ltd.

65

C 4 21

160

140

UJ Z ~20

,~ 100 I,,-

8o

,_1 ' ~ 60 ,,-I ,,_1 LLI t J 40-

tr t,',- 20 X I,,I,,I

0 c

".t, /

q,/ ~ o

i

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2~

S A M P L E N U M B E R

Fig. 1. Effects of KA perfusion on both extracellular taurine levels and CAl-evoked lield potentials in the absence or presence of glutamate antagonists. Filled bar represents KA 0.1 mM perfusion period. Open bar represents perfusion period of antagonist either CPP 0.1 mM (dotted line) or DNQX 0.1 mM (dashed line). No antagonist was perfused in control experiments (solid line). Each point is the mean + S. E.M. obtained from 3 rats. Averages of basal levels obtained in each rat were taken as a simple experi- mental obserwltion, and their mean is depicted as C. KRB was perfused in samples 16 21. Records at the top of the figure are the average of 6 field potentials evoked by single pulses and taken during perl'usion of samples C, 4 and 21 in one of the control experiments. Similar averages were obtained in experiments in which CPP or DNQX were perfused. Calibration: 10 ms, 5 mV.

lective antagonis ts of n o n - N M D A receptors [8] it is now possible to determine

whether increases in extracellular taur ine levels induced by n o n - N M D A agonists are

also related to act ivat ion of their specific receptors. Using a cerebral microdialysis

device, the in vivo changes in extracellular taur ine levels were determined in the pres-

ence or absence of specific antagonis ts dur ing perfusions of N M D A , quisqualate

(QA) and KA.

Adult Sprague Dawley rats were studied while main ta ined under urethane anaes-

thesia (1.2 g/kg, i.p.). Brain dialysis probes, constructed as previously described [7],

were implanted stereotaxically and bilaterally in the h ippocampal CA1 Iield, with the

tip of each probe located in the molecular layer. Ten rain perfusate samples (20/d)

were obta ined and analyzed for amino acid content as previously reported [12].

Extracellular concent ra t ion of taur ine was calculated as in Lerma et al. [12]. Field

potentials were recorded by a metallic 30 ~m electrode glued to the dialysis probe.

Or thodromica l ly evoked popula t ion spikes (PSs) of CA! were obta ined by electrical

66

stimulation (0. I ms, 0.1 0.5 tnA, 0.03 Hz, square pulses) of the CA3 pyramidal layer. The control perfusion liquid, which was also used to dissolve the drugs, was standard Krebs- Ringer bicarbonate (KRB). At 90 rain after probe implantation, control sam- ples were taken for 1 h in both hippocampi. In each animal, effects evoked by NMDA and QA were tested in the right hippocampus using the left one to study these effects in the presence of antagonists. Perfusion sequence is indicated in figures.

Although 2 h perfusions of 0.1 mM NMDA, 0.25 mM QA or 0.1 mM KA in one hippocampus did not affect extracellular taurine levels in the contralateral structure, KA decreased PSs evoked in the contralateral hippocampus (data not shown). Then, to avoid any possible misinterpretation of results, experiments of KA perfusions were performed in only one hippocampus.

KA evoked a biphasic increase in extracellular taurine levels which was maximum in the second sample (Fig. 1). Simultaneous perfusion of KA and 3-(carboxypipera- zin-4-yl)-propyl-l-phosphonic acid (CPP), a potent and specific antagonist of

8O

~z 60

40

3 ~< so

0

c 9

¸, . . . . . . . .

c 4

21 ^ 14

21

2 3 4 5 6 7 8 9 10 11 12 13 14 15 1~ 17 18 19 20 2~

S A M P L E N U M B E R

Fig. 2. Effects of QA perfusion on both extracellular taurine levels and CAI-evoked field potentials in the absence or presence of glutamate antagonists. Filled bar represents QA 0.25 mM perfusion period. As in Fig. 1, open bar represents antagonist perfusion period: CPP 0.1 mM in dotted line, DNQX 0. l mM in dashed line, or none in solid line. KRB was perfused in samples 16 21. Field potentials at the upper row were evoked by paired pulses and recorded during perfusion of samples C, 9 and 21 of one of the control experiments. In the lower row are depicted averages of field potentials evoked by single pulses and recorded during samples C, 4 and 21 of one experiment in which DNQX was perfused. Cali- bration: I0 ms, 5 inV.

67

N MDA receptors [6], did not alter the KA effect on taurine levels, whereas 6,7-dini- troquinoxaline-2,3-dione (DNQX), a competitive antagonist of both KA and QA receptors [8], blocked it almost totally and reversibly (Fig. 1). QA (0.25 raM) evoked increases in extracellular taurine levels which were markedly lower than those elicited by KA, completely inhibited by DNQX in a reversible manner and again not affected by CPP (Fig. 2). N M D A perfusions also caused a biphasic increase in extracellular taurine levels, as shown in Fig. 3. This effect was totally abolished by CPP (Fig. 3). Perfusion of a solution containing both 0.! mM DNQX and 0.1 mM N MD A induced an increase of extracellular taurine levels that was slightly lower compared to that evoked by 0.1 mM NMDA alone (Fig. 3). It has been reported in in vitro experiments that DNQX can act as a non-competitive antagonist of NMDA recep- tors in the baby rat spinal cord [1]. To determine whether the DNQX concentration used in our experiments was sufficient to elicit a similar effect in hippocampal neu-

I00 -

l[ 8 0 -

ill g 221

60- I-"

, , . ,140- ,m

,.,.i w

lag x UJ o

c 4 b ' ) 4 (a') 21

. ",, '.I... • 4-:_. e t

2 3 ,4 5 ~ 7 8 9 10 11 '~2 1~ 14 15 16 17 18 19 20 21

S A M P L E N U M B E R

Fig. 3. Effects of N M D A perfusion on both extracellular taurine levels and CAI-evoked lield potentials in the absence or presence of glutamate antagonists. Filled bar represents N M D A 0.1 mM perfusion peri- od. ()pen bar represents antagonist perfusion period: CPP 0.1 mM in dotted line, DNQX 0.1 mM in

dashed line or none in solid line. KR B was perfused in samples 16 to 21. Field potentials at the upper row were evoked by single pulses and recorded during perfusion of samples C, 4 ( ls t and 3rd rain) and 21 of one of the control experiments. Lower row: averages of tield potentials evoked by single pulses and recorded during samples C, 9, 15 and 20 of one experiment in which CPP was perfused. Calibration: 10

IllS, 5 m Y .

68

rons, a 50/IM solution of DNQX was used instead of 100/zM. This dose proved to be enough to counteract the KA (0.1 mM) evoked taurine increase without affect- ing that evoked by 0.1 mM NMDA (data not shown).

As could be expected from their excitatory potency, KA and N MD A abolished PS in less than 3 min (Figs. I and 3), probably because of a great depolarization that inactivated the action potential generation [14]. QA increased the PS amplitude and also elicited the appearance of multiple PSs, concomitantly diminishing the potency of recurrent inhibition on pyramidal cells, as determined with the twin-pulse stimula- tion paradigm (Fig. 2). Since it is known that QA is able to modify the response of GABAA receptors [15], the diminished potency of recurrent inhibition could be explained by decreased GABAA channel currents. On the other hand, DNQX perfu- sions abolished all evoked field potentials as previously reported [7], while those of CPP blocked the decrease in PS amplitude evoked by NMDA (Fig. 3).

The present study shows that taurine releases caused by NMDA, KA or QA are mediated by their specific receptors. CPP was unable to diminish either the KA- or QA-evoked release of taurine, indicating that N MD A receptors do not participate, directly or indirectly, in the action of these two agonists. Since non-NMDA receptors are expressed not only in neurons but also in glial cells [2, 9], at least part of taurine increases elicited by KA and QA may be of glial origin. A possible neuronal origin for taurine increases evoked by NMDA should be postulated, because no NMDA receptors have been described in glial cells [2, 9].

KA, NMDA and QA promote neurodegenerative effects on the mammalian cen- tral nervous system [13, 16] and a close correlation exists between the neurotoxic po- tency of these agents and their ability to depolarize neurons (KA > N MD A > > QA) [5, 13]. Our results suggest that a similar correspondence may exist between inactiva- tion of PS generation and increase of extracellular taurine levels in response to these substances. Since cell swelling is an early component of neurotoxicity events induced by glutamate analogs [4, 13] and taurine has been proposed as an osmoregulatory substance in the rat brain [16, 17], we suggest that in the present experiments KA, NMDA and QA elicit cellular swelling which, in turn, triggers taurine release. Extra- cellular taurine levels may therefore be used as an index of the early neurotoxic com- ponent of excitatory amino acid action in vivo.

We thank Mrs. A. Latorre for technical assistance and Mrs. C. Stoddard for edi- torial help. This study was supported in part by the Fondo de Investigaciones Sani- tarias (F.I.S.s.s., 89/0158).

1 Birch, P.J., Grossman, C.J. and Hayes, A.G., Kynurenate and FG9041 have both competitive and non-competitive antagonist actions at excitatory amino acid receptors, Eur. J. Pharmacol., 151 (1988) 313 315.

2 Bowman, C.L. and Kimelberg, H.K., Excitatory amino acids directly depolarize rat brain astrocytes in primary culture, Nature (Lond.), 311 (1984) 656-659.

3 Butcher, S.P., Lazarewicz, J.W. and Hamberger, A., In vivo microdialysis studies on the effects of decortication and excitotoxic lesions on kainic acid-induced calcium fluxes, and endogenous amino acid release, in the rat striatum, J. Neurochem., 49 (1987) 1355 1360.

69

4 Choi, D.W., Ionic dependence of glutamate neurotoxicity, J. Neurosci., 7 (1987) 369 379. 5 Garthwaite, J., Garthwaite, G. and Hajos, F., Amino acid neurotoxicity: relationship to neuronal

depolarizarion in rat cerebellar slices, Neuroscience, 18 (1986) 449 460. 6 Harris, F.W., Ganong, A.H., Monaghan, D.T., Watkins, J.C. and Cotman, C.W., Action of

3-(( + )-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid (CPP): a new and highly potent antagonist of N-methyl-D-aspartate receptors in the hippocampus, Brain Res., 382 (1986) [74 177.

7 Herreras, O., Mendndez, N., Herranz, A.S., Soils, J.M. and Martin del Rio, R., Synaptic transmission at the Schaffer-CAl synapse is blocked by 6,7-Dinitro-quinoxaline-2,3-dione. An in vivo brain dialysis study in lhe rat, Neurosci. Lett., 99 (1989) 119 124.

8 Honord, T., Davies, S.N., Drejer, J., Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, F.E., Qinoxa- linedioncs: potent competitive non-NMDA glutamate receptors antagonists, Science, 241 (1988) 701 703.

9 Kcttenmann, H. and Schachner, M., Pharmacological properties of ?,-aminobutyric acid-, glutamate-, and aspartate-induced depolarizations in culture astrocytes, J. Neurosci., 5 (1985) 3295 3301,

10 kehmann, A., Hagberg, H., Nystr6m, B., Sandberg, M. and Hamberger, A., In vivo regulation o1" extracellular taurine and other neuroactive amino acids in the rabbit hippocampus. In S.S. Oja, k. Ahtec, P., Kontro and M.K. Paasonen (Eds.)., Taurine: Biological Actions and Clinical Perspectives, Liss, New York, 1985, pp. 289 311.

11 Lchmann, A., lsacsson, H. and Hamberger, A., Effects of in vivo administration of kainic acid on the extracellular amino acid pool in the rabbit hippocampus, J. Neurochem., 40 (1983) 1314 1320.

12 Lerma, J., Herranz, A.S., Herreras, O., Abraira, V. and Martin del Rio, R., In vivo determination of exlracellular concentration of amino acids in the rat hippocampus. A method based on brain dialysis and computerized analysis, Brain Res., 384 (1986) 145 155.

13 Olney, J.W.. Excitotoxins: an overview. In: K. Fuxe, P. Roberts and R. Schwartz (Eds.), Excitotoxins, Macmillan, London, 1984, pp. 82 96.

14 Segal, M., The actions of glutamic acid on neurons in the rat hippocampal slice. In G. di Chiara and G.L. Gessa (Eds.), Glutamate as a Neurotransmitter, Raven, New York, 1981, pp. 217 225.

15 Sigel, E. and Baur, R., Quisqualate receptors differentially modulate neuronal Na +, Ca z ~ and GABA.x channels via protein kinase C, Eur. J. Neurosci., Suppl. 1 (1989) 341P.

16 Soils, J.M., Herranz, A.S., Herreras, O., kerma, J. and Martin del Rio, R., Does taurine act as an osmoregulatory substance in the rat brain?, Neurosci. Lett., 91 (1988) 53 58.

17 Wade, J.V., Olson, J.P., Samson, F.E., Nelson, S.R. and Pazdernik, T.L., A possible role for taurinc in osmoregulation within the brain, J. Neurochem., 51 (1988) 740 745.