Effects ofl -cysteine-sulphinate andl -aspartate, mixed excitatory amino acid agonists, on the membrane potential of cat caudate neurons

330

Brain Research, 414 (1987) 330-338 Elsevier

BRE 12666

Effects of L-cysteine-sulphinate and L-aspartate, mixed excitatory amino acid agonists, on the membrane potential of cat caudate neurons Waldemar A. Turski 1., Paul L. Herrling I and Kim Quang Do 2 l Sandoz Research Institute, Berne (Switzerland) and 2Brain Research Institute, University of Zurich, Zurich (Switzerland)

(Accepted 18 November 1986) Key words: L-Cysteine-sulphinate; L-Aspartate; N-Methyl-D-aspartate; Quisqualate; Kainate; Iontophoresis; Membrane potential; Caudate; Excitatory amino acid; Cat

Responses evoked by L-cysteine-sulphinate (L-CSA) and L-aspartate (L-Asp) were recorded with intracellular electrodes from caudate neurons in halothane anesthetized cats. L-CSA and L-Asp were applied microiontophoretically to caudate cells and their effects on membrane and action potentials, as well as on cortically evoked synaptic potentials were evaluated. L-CSA and L-Asp induced depolarizations accompanied by regular firing resembling kainate (KA)- or quisqualate (QUIS)-induced excitation patterns (type 1) in 82% and 72% of the recorded neurons, respectively, and a mixed pattern consisting of a N-methyl-o-aspartate (NMDA)-like excitation (type 2) followed by a regular type 1 pattern in the remaining cells. In about a quarter of the cells the effects of L-CSA and L-Asp, but not those of KA or QUIS, were partially antagonized by 2-amino-7-phosphonoheptanoate (AP-7), a specific NMDA receptor antagonist. Kynurenate, a broad spectrum excitatory amino acid antagonist, blocked responses elicited by either L-CSA or QUIS. The actions of L-CSA and L-Asp on the firing pattern and membrane potential of cat caudate neurons in situ provide evidence in favor of their mixed agonist nature with respect to NMDA and non-NMDA excitatory amino acid receptors. INTRODUCTION Distinct r e c e p t o r types subserve postsynaptic actions of excitatory amino acid neurotransmitter candidates L-glutamate (L-Glu) and L-aspartate (L-Asp) in the m a m m a l i a n central nervous system_ (CNS) 33'34. These receptors have been defined on the basis of preferential affinities for agonists such as N-methylD-aspartate ( N M D A ) , kainate ( K A ) and quisqualate ( Q U I S ) . The characteristics of excitatory amino acid-induced conductances in cultured spinal cord 2°-22 or cortical 1°'11 neurons and the analysis of the effects induced by N M D A , K A and Q U I S in whole cell p a t c h - c l a m p experiments 26 show that each of these receptors subtypes appears to be linked to different ionic mechanisms. L-GIu, L-Asp and the sulphur-containing amino acids, e.g. L-cysteine-sulphinate (L-CSA), are potent

neuronal excitants in the m a m m a l i a n CNS 4'25"30'34. A possible transmitter role for C S A in the CNS has been suggested by d e m o n s t r a t i o n of its release after preloading and uptake by synaptosomal fractions of rat cerebral cortex 1'18, by experiments showing its release from e n d o g e n o u s stores in rat brain slices 7 and by the demonstration of specific binding sites in cortical m e m b r a n e preparations 19'3°. A possible functional role of C S A in the brain is also s u p p o r t e d by the observation of convulsant effects of this drug following intracerebroventricular application in rodents 17. Neurotoxic and excitatory actions of systemically given e-cysteine in mice are partly explained by the excitatory action of its immediate metabolite, LC S A 27. Previous studies using both electrophysiological and pharmacological techniques have established that both N M D A and n o n - N M D A excitatory amino

* On leave from the Department of Pharmacology, Medical School, Jaczewskiego 8, PL-20-090 Lublin, Poland. Correspondence: P.L. Herding, Sandoz Research Institute, P.O. Box 2173, CH-300I Berne, Switzerland. 0006-8993/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

331 acid receptor systems exist in the cat caudate nucleus and that the excitatory corticocaudate pathway involves non-NMDA excitatory amino acid receptors 13,14. Because of these reports, the present study was aimed at determining the effects of L-CSA on membrane and synaptic potentials of cat caudate neurons, extending previous observations with L-Asp 13 and comparing them to well-known selective agonists of excitatory amino acid receptors. Some of these results have been reported previously in abstract formS,15,16. MATERIALS AND METHODS

Intracellular recordings and iontophoresis The surgical methods and the procedures for intracellular recording in the caudate with simultaneous microiontophoretic application of the drugs during stimulation of the corticocaudate pathway have previously been described in detail 13. Briefly, mongrel cats of either sex, weighing 3.0-4.0 kg, were anesthetized for surgery by i.v. injection of an ultra-shortacting barbiturate (methohexital sodium, Brietal, Lilly) and the saphenous vein and femoral artery were cannulated for drug injections and blood pressure measurements, respectively. Tracheotomy for artificial respiration, a bilateral pneumothorax and opening of the cisterna magna were performed in order to reduce cardiovascular and respiratory pulsations. The skull was exposed and windows (ca. 1.5 x 1.5 cm) opened vertically above the lateral ventricles. The cortical tissue overlying the caudate was removed by suction until the lateral ventricles were reached. Stimulation electrodes were implanted bilaterally through the opened frontal sinuses into the anterior sigmoid gyrus. The positions of the recording electrodes were verified in frozen tissue sections. At the end of surgery, anesthesia was switched to 0.5-1% halothane (Fluothane, ICI) in pure oxygen. The body temperature was kept at 37-38 °C and endtidal CO 2 in the tracheal cannula at 3.6-4.4%. Cell recordings were included in the present study only if blood pressure was above 80 mm Hg. One channel of the iontophoretic and recording electrode assemblies was filled with a physiological NaCI solution for occasional balance controls, and one channel with the same NaC1 solution but at pH

9.2 as an approximate pH and current control. All resuits included in this study were controlled for current effects. The inter-tip distance between recording and iontophoretic electrodes was around 50 pm. Recording electrodes were filled with 1.6 M K-citrate adjusted to pH 7.2 with citric acid and had a resistance of 80-150 MQ measured in the cerebrospinal fluid just before penetration of the tissue. The signals from the recording electrode was amplified and displayed on commercially available equipment and stored on audiotape (FM) for later analysis. Data were analyzed by digitizing the membrane potential recordings during the period around cortical stimulation (500 points, programm BASAL, written by Dr. P. Linscheid) and averaging consecutive sequences to allow detection of drug-induced changes of the evoked responses. Statistical analysis was achieved by performing a point-by-point Student's t-test between the averages of control and test responses.

Materials The following drugs were used in the iontophoretic experiments and ejected as anions: DL-2-amino-5phosphonovaleric acid, 0.1 M, (kindly donated by Dr. J.C. Watkins, Bristol); DL-2-amino-7-phosphonoheptanoic acid, 0.1 M, Sandoz; L-aspartic acid, 0.1 M, Sigma; L-cysteinesulfinic acid, 0.1 M, Sigma; Lhomocysteic acid, 0.1 M, Sigma; kainic acid, 0.1 M, Sigma; kynurenic acid, 0.2 M, Sigma; N-methyl-DEaspartic acid, 0.2 M, Tocris Chemicals; quisqualic acid, 0.1 M, Sigma. All drugs were dissolved in 100 or 200/A of 1 N NaOH and made up in i ml with distilled water. The pH was adjusted to 8-9 with HCI. All solutions were kept frozen between experiments. RESULTS

Characteristics of recorded cells Stable intracellular recordings were obtained from 192 neurons impaled in the caudate nucleus of 68 cats at depths ranging from 500 to 4500~m below the dorsal surface at AP +16.5 to +17.5 (ref. 2). The impaled cells were presumably of the medium spiny type since reports from intracellular staining studies using horseradish peroxidase filled electrodes indicate that the majority of caudate cells stained were of this type 3. These cells were silent unless stimulated

332 electrically, synaptically or by excitatory amino acids. The mean amplitude of action potentials measured from spike threshold was 41 + 8 mV (mean + S.D.), ranging from 30 to 70 mV, with durations of 0.5-1 ms at the half-maximal amplitude. The mean resting potential, estimated from the potential drop that occurred at the end of the intracellular recordings, had a value of 49 + 8 mV. The amplitude of resting potentials ranged from 40 to 70 inV. A discussion of the possible reasons for the relatively small amplitude of action potentials is to be found in ref. 13.

Effects of L-CSA and L-Asp on the firing pattern and membrane potential of caudate neurons Depolarizations induced by all excitatory amino acids tested elicited repetitive firing in all recorded caudate neurons. The pattern of action potential firing in response to excitatory amino acids fell into two types according to the characteristics described by Herrling et al/3. In most cases (90% of 146 neurons), N M D A induced a firing pattern with action potentials superimposed on large depolarizations of fast

on- and offset and long duration (100 to >1000 ms), the previously described type 2 pattern 13 (e.g. Figs. lc, 3a, Table I). In 10% of the cells, N M D A evoked the steady firing of action potentials referred to as type 1 excitations (Table I). KA (19 cells) and QUIS (80 cells) elicited exclusively type 1 depolarization patterns (Table I). L-CSA and L-Asp-induced responses were examined in 110 and 32 caudate neurons, respectively. Fig. 1 illustrates the predominant firing pattern evoked by these two agonists. In the majority of neurons, L-CSA (82%) and LAsp (72%) induced the type 1 pattern (Table I). Type 2 pattern was less reliably induced by L-CSA (18%) and L-Asp (28%) (Table I). Fig. 2 shows that in some cases a 'bursty' (type 2) firing pattern preceded the more regular type 1 pattern usually elicited by both L-CSA (Fig. 2a, b) and L-Asp (Fig. 2c, d). Interestingly, even prolonged iontophoresis of N M D A onto the same cell did not result in a type l firing pattern, even at high doses (Fig. 2e, f). Since the possibility existed that some of the ejected agonists might be sucked up during iontophoretic ejection by the retaining current of a neighbor-

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Fig. 1. Comparison of the effects of iontophoretically applied excitatory amino acids on the membrane potential of a cat caudate neuron. a: L-Asp applied at -265 nA evoked a depolarization with a rather regular firing pattern (type 1 excitation), b: L-CSA applied at -200 nA elicited qualitatively similar effects, but the excitation was more pronounced, c: NMDA applied at- 120 nA evoked a bursty firing pattern with abrupt de- and repolarizations (type 2 excitation). In this and all followingintracellular recordings depolarization is upward. Dots indicate the time of cortical stimulation. In most cases iontophoretic applications are indicated by horizontal bars below the records.

333 TABLE I

Firing patterns elicited by iontophoreticaUy applied excitatory amino acids in cat caudate neurons Type 1: for example see Fig. la, b; Type 2: for example see Fig. 3a; Mixed: for example see Fig. 2a, b.

Firing type (number of cells)

Agonists KA QUIS L-CSA L-Asp NMDA

1

2

Mixed

19 75 90 23 15

0 0 0 0 135

0 5 20 9 0

ing channel, 'electrical diversion '3t, we conducted a series of experiments with electrode assemblies either containing or not containing N M D A agonists. In electrodes not containing N M D A , L-CSA induced transient type 2 patterns in 8 of 31 cells, L-Asp in 3 of 10 cells. With assemblies containing N M D A agonists, L-CSA evoked transient type 2 patterns in 12 of 79 and L-Asp in 6 of 22 cells. This indicates that type 2 patterns seen with these two agonists are not due to sucking up of N M D A agonists into their ejection barrel.

a

Effects of mixtures o f excitatory amino acids on the firing pattern and membrane potential The observations described above, where L-CSA and L-Asp evoked two types of firing patterns in the same cell, led to the assumption that these two agents might interact with both N M D A and n o n - N M D A excitatory amino acid receptors present on the same caudate neuron. To further examine this possibility we recorded and analyzed the pattern of firing that was elicited by application of mixtures of agonists applied from the same micropipette. Fig. 3 and Table II show the results from such experiments. It was apparent that increasing the concentration of N M D A in the mixtures containing K A or Q U I S converted type 1 to type 2 firing patterns (Table II). However, at certain concentration ratios both types of firing pattern could be seen during the same iontophoretic application (Fig. 3). These observations suggested that iontophoresis of appropriate mixtures of N M D A and n o n - N M D A excitatory amino acids could elicit mixed excitation patterns, similar to those seen with L-CSA and L-Asp. Fig. 3 indicates that such a mixed firing pattern can be converted to a pure type 1 pat-

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Fig. 3. Effects of the application of an NMDA/QUIS mixture from the same iontophoretic electrodes and their sensitivity to an NMDA antagonist, a: control, a pure type 2 pattern, was elicited by NMDA alone, b: an excitation where elements of type 1 and 2 patterns occur simultaneously was elicited by a mixture of QUIS and NMDA (1:10), applied from the same iontophoretic barrel, c: additional application of the selective NMDA antagonist AP-7 changed the composite pattern shown in b into a pure, regular type 1 pattern. d: control, a pure type 1 pattern, was induced by application of QUIS alone onto the same cell.

TABLE II tern by the additional application of a selective N M D A antagonist, AP-7 (ref. 29). However, there is a difference between the mixture effects and those induced by L-CSA and L-Asp: while with the latter agonists type 2 firing patterns occur predominantly at the beginning of iontophoretic applications (Fig. 2), in the mixture experiments, both patterns coexist in time (Fig. 3). Fig. 4 and Table III demonstrate the effect of AP-7 on depolarization patterns elicited by iontophoresis of L-CSA, L-Asp and reference excitatory amino acids, N M D A , K A and QUIS. AP-7 was an effective antagonist of N M D A - i n d u c e d depolarizations at low

Firing patterns of cat caudate cells elicited by iontophoretic application of mixtures of excitatory amino acids

Symbols as in Table I. Type and ratio of mixtures

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Fig. 4. Interactions of AP-7, a selective NMDA-antagonist, with NMDA-, L-CSA- and L-Asp-elicitedexcitations of a caudate neuron. a: control applications of the 3 agonists. Note that NMDA caused the largest depolarization amplitude and duration, b: application of the same agonists in the presence of a lower iontophoretic current of AP-7. The NMDA-induced excitation was abolished, whereas LCSA and L-Asp excitations were less affected, &, 97 s after beginning of AP-7 current, c: recovery, &, 159 s after end of AP-7 current. d: same experiment as in b, but at a higher AP-7 current. Excitations induced by all 3 agonists are strongly inhibited, &, 12 s after beginning of AP-7 current, e: recovery, &, 261 s after end of AP-7 current. The agonists were applied in regular cycles throughout the experiment.

TABLE III

A: The inhibitory effect of AP-7 on excitations of cat caudate neurons elicited by iontophoresis of single excitatory amino acids. B: The inhibitory effect of AP-7 on excitations of caudate neurons elicited by iontophoresis of mixtures of excitatory amino acids For an example of A, see Fig. 4.

Sensitivity to AP-7 (number of cells) Sensitive

Not sensitive

0 3 14 4 76

11 47 42 11 0

B. Type and ratio of mixtures NMDA/QUIS 2:1 4 10:1 8

2 0

A. Agonists KA QUIS L-CSA L-Asp NMDA

NMDA/KA 2:1 5:1 10:1

0 2 3

3 2 0

application currents, while only partially inhibiting responses evoked by L-CSA and L-Asp. A t higher currents of AP-7, however, strong inhibitions of the effects of these two agents were achieved, but not of Q U I S or KA. Table III shows that the sensitivity of excitations elicited by mixtures of agonists to AP-7 correlates with the relative quantitity of N M D A in the mixtures. Kynurenic acid, a broad spectrum excitatory amino acid antagonist 12'14'28, clearly inhibited L-CSA- and Q U I S - i n d u c e d excitations to about the same extent in 14 of 14 cells.

Effects o f L-CSA and L-Asp on cortically evoked synaptic potentials Since results of a previous study 9 indicated that N M D A but not Q U I S receptor agonists are capable of potentiating cortically evoked EPSPs, L-CSA and L-Asp were applied to caudate n e u r o n s during stimulation of the corticocaudate pathway. The resulting EPSPs were digitized, averaged and compared to EPSPs elicited in the same cell in the presence of QUIS. Iontophoretic currents were adjusted so as to cause an equal depolarization during application of

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Fig. 5. Effects of excitatory amino acid agonists on cortically evoked EPSPs. a: thick trace, averaged (n = 20), cortically evoked EPSPs during a control application of C1- ions through an NaCl-containing iontophoretic barrel. Thin trace, the application of QUIS (-20 nA) resulted in firing on averaged (n = 10) EPSPs, but the relative EPSP amplitude was decreased. The QUIS-induced depolarization was 15 mV. b: comparison of the effects of QUIS (-30 nA, n = 20, thin trace) and L-CSA (- 110 hA, n = 15, thick trace) on averaged EPSPs. There was no significant difference, and in both cases, the depolarization induced by the agonists was 12 mV. c: comparison of t.he effects of QUIS (-30 nA, n = 15, thin trace) and L-Asp (- 150 nA, n = 10, thick trace) on averaged EPSPs. Again, there was no significant difference. The cell was depolarized by 12 mV during QUIS and by 10 mV during L-Asp. d: occasional potentiation of averaged, cortically evoked EPSPs by L-CSA (thin trace, -140 nA, n = 20). Thick trace, effect of QUIS (-40 nA, n = 33) as a control. Depolarization induced by the agonists was 20 mV in both cases, e: occasional potentiation of cortically evoked EPSPs by L-Asp, thin trace L-Asp (-90 nA, n = 30). Thick trace, effect of QUIS for comparison (-40 nA, n = 30). In both cases agonist-induced depolarizations were 20 mV. Sweep length was 150 ms for a-d.

all agonists. A s previously described 9, Q U I S did n o t p o t e n t i a t e cortically e v o k e d E P S P s (Fig. 5a). In the m a j o r i t y of cells tested, L-Asp (4/6) a n d LC S A (6/8) affected cortically e v o k e d E P S P s in a m a n n e r indistinguishable from Q U I S (Fig. 5b, c). In the r e m a i n i n g cells the E P S P s were larger t h a n during Q U I S (Fig. 5d, e), b u t p o t e n t i a t i o n of the m a g n i t u d e o b s e r v e d d u r i n g application of L-homocysteic (L-HCA) 9 was n e v e r reached.

DISCUSSION T h e p r e s e n t results show that the e n d o g e n o u s sulp h u r - c o n t a i n i n g excitatory a m i n o acid L-CSA 7 is cap a b l e of reversibly exciting cat c a u d a t e n e u r o n s , confirming earlier o b s e r v a t i o n s o n its n e u r o e x c i t a t o r y p r o p e r t i e s in a m p h i b i a n a n d m a m m a l i a n spinal cords4-6, 25. The

analysis

of

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337 recorded after iontophoresis of L-CSA shows that this agonist induces QUIS/KA-Iike type 1 firing patterns in most cells tested. In the remaining cells it induces a mixed pattern, where type 2 precedes type 1. The pattern observed with L-Asp is also predominantly of type 1, although the mixed type is seen in about a third of tested neurons. The fact that the type 2 pattern always preceded type 1 in all cases where mixed patterns occurred, might indicate that L-Asp and L-CSA have affinities for both NMDA and nonNMDA excitatory amino acid receptors, albeit with a higher affinity for the NMDA receptor. Thus, at low concentrations, i.e. at the beginning of iontophoretic application, activation of NMDA receptors results in a 'bursty' firing pattern. When the agonist concentration increases during prolonged iontophoresis, the non-NMDA receptors are also activated, producing a regular firing pattern which then predominates. This concept is similar to the one proposed for Glu and Asp by Mayer and Westbrook 23'35 and Nowak et al. 26, following observations that while low concentrations of these agonists induce N-shaped I - V curves in neurons in vitro (NMDA-like), they become linear (QUIS-like) at higher concentrations. Excitations produced by L-CSA and L-Asp were clearly less sensitive to AP-7 than NMDA excitations tested on the same cells, i.e. depolarizations induced by NMDA could be easily abolished, while L-CSA and L-Asp induced depolarizations persisted at least partially during AP-7 iontophoresis (e.g. Fig. 4b). Application of AP-7 inhibited L-CSA- or L-Asp-induced depolarization in only about one quarter of cells at relatively high currents. The finding that in all cases tested L-CSA was inhibited to the same extent as QUIS on the same neurons by the broad spectrum, but specific antagonist for excitatory amino acids, kynurenate, indicates that L-CSA interacts with excitatory amino receptors. Kynurenate does not affect acetylcholine-induced excitations or action potentials elicited by intraceUular current injections 12'14'28. Depolarizations of type 1 produced by KA or QUIS in caudate cells are highly resistant to the inhibitory action of AP-7 (ref. 14). This is consistent with the reREFERENCES 1 Baba, A., Okumura, S., Mizuo, H. and Iwata, H., Inhibition by diazepam and gamma-aminobutyricacid of depolarization-induced release of [14C]cysteinesulfinate and

suits of Mewett and colleagues 25 who reported that LCSA was affected by NMDA-antagonists in an in vitro spinal cord preparation but much less than NMDA itself. Our results in cat caudate neurons are consistent with the hypothesis that both L-CSA and L-Asp are interacting with NMDA as well as with nonNMDA excitatory amino acid receptors. In an in vitro study which appeared during the course of this work, results obtained with L-CSA and L-Asp in cortical slices led the author to a very similar conclusion 32. The concept of mixed agonist action of excitatory amino acids originated from Watkins et al. 33. The resuits of extracellular recordings from Renshaw cells and non-identified cat spinal cord neurons following iontophoretic application of L-GIu and L-Asp have suggested that L-GIu acts mainly on QUIS and L-Asp on NMDA receptors in that structure 6. To explain the voltage dependence of L-GIu responses recorded from mouse spinal cord neurons under voltage clamp, two groups 23'24"26,35postulated that L-GIu and L-Asp act at two receptors linked to different membrane conductances. The co-iontophoresis experiments where NMDA and QUIS or NMDA and KA were applied from the same pipette show that NMDA and QUIS/KA receptors can be activated simultaneously on the same neuron. Our observations indicate that L-Asp and L-CSA have predominantly QUIS-like effects on cortically evoked EPSPs, in contrast to the previously described 9 clear potentiating effect of L-HCA. In conclusion, the present observations suggest that L-CSA and L-Asp interact in mammalian caudate neurons with both NMDA and non-NMDA excitatory amino acid receptors, and are thus mixed excitatory amino acids in this region of the vertebrate nervous system. ACKNOWLEDGEMENT The authors thank Mr. Ch. Durtschi for excellent technical assistance. [3H]glutamate in rat hippocampai slices, J. Neurochem., 40 (1983) 280-284. 2 Berman, A.L. and Jones, E.G., The Thalamus and Basal Telencephalon of the Cat, University of Wisconsin Press, 1982.

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