Br. J. Pharmacol.

Br.

J.

Pharmacol.

(1991), 104, 873-878 (1991),

104,

(D Macmillan Press Ltd, 1991

873-878

©

Macmillan

Press

Ltd,

Novel kainate derivatives: potent depolarizing actions on spinal motoneurones and dorsal root fibres in newborn rats M. Ishida & 1H. Shinozaki The Tokyo Metropolitan Institute of Medical Science, 3-18-22, Honkomagome, Bunkyo-ku, Tokyo 113, Japan 1 Neuropharmacological actions of several kainate derivatives (kainoids) were examined for electrophysiological effects in the isolated spinal cord and the dorsal root fibre of the newborn rat. 2 Some kainoids caused depolarization of the motoneurone much more effectively than kainic acid or domoic acid and others were weaker. The rank order of the depolarizing activities of the kainoids tested here is as follows: 442-methoxyphenyl)-2-carboxy-3-pyrrolidineacetic acid (MFPA) > acromelic acid A > domoic acid _ 4-(2-hydroxyphenyl)-2-carboxy-3-pyrrolidineacetic acid (HFPA) _ acromelic acid B > kainic acid. 3 In the isolated dorsal root fibre, domoic acid caused the most significant depolarization. There were distinct differences with regard to the rank order of the depolarizing activity between the motoneurone and the dorsal root fibre. The rank order in the dorsal root fibre is domoic acid > acromelic acid B > 5-bromowillardiine _ MFPA > acromelic acid A > HFPA > kainic acid. 4 Significant desensitization of kainate receptors was observed in the isolated dorsal root fibre during prolonged application of L-glutamate, kainate and its derivatives. Cross desensitization was also observed among these excitatory amino acids. Receptors desensitized by kainate did not respond to MFPA, HFPA and acromelic acids, suggesting that these kainate derivatives activated common kainate receptors in the dorsal root fibre. 5 In both motoneurones and dorsal root fibres, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) effectively depressed the depolarization induced by kainoids, and neither 3-[(±)-2-carboxypiperazin-4-yl]propyl-1phosphonic acid (CPP) nor picrotoxin blocked or affected the depolarization, but there were some differences in pharmacological potencies of glutamate antagonists between both preparations. 6 MFPA, HFPA and acromelic acids should provide valuable pharmacological tools for analysis of physiological functions of excitatory amino acids, in particular, as specific agonists for some subtypes of kainate receptors. Keywords: Kainate derivatives; excitatory amino acid; depolarizing activity; spinal motoneurone; dorsal root C fibre; desensitization; electrophysiology

Introduction Kainic acid is one of the most potent excitants in the mammalian central neurone and its powerful excitatory actions gave rise to the excitotoxic concept that glutamate destroys neurones by excessive activation of excitatory receptors. A potent kainate analogue, acromelic acid A, isolated from a poisonous mushroom, Clitocybe acromelalga (Konno et al., 1983; 1988), causes depolarization more markedly than kainic acid or domoic acid in the newborn rat spinal motoneurone (Ishida & Shinozaki, 1988), and is also one of the most potent excitatory amino acids in both vertebrates and invertebrates (Shinozaki et al., 1986; 1991; Shinozaki, 1988; 1991; Shinozaki & Ishida, 1988a,b). Acromelic acid has two structural isomers, acromelic acids A and B. From the structural and pharmacological similarities of acromelic acids to kainic acid or domoic acid, it seems reasonable to predict kainate-type excitotoxic actions of acromelate in the mammalian central nervous system. However, single systemic injection of acromelic acid A caused behavioural and pathological effects quite different from those seen after systemic administration of kainate in the rat (Shinozaki et al., 1989a; 1991; Kwak et al., 1990; 1991). It is of great interest to examine these pharmacological differences between kainate and acromelate. In the course of the study on chemical synthesis of acromelic acids and their derivatives (Shirahama et al., 1988; Hashimoto et al., 1990; Hashimoto & Shirahama, 1990), we were able to obtain some kinds of kainate related compounds (kainoids), which were expected to provide a useful tool for analysing the mechanism underlying kainate functions. Author for correspondence.

At present, highly specific antagonists for kainate receptors are not yet available, therefore, it is difficult to classify electrophysiologically these derivatives as pure kainate agonists, but it has been reported that dorsal root C fibres of immature rats are directly depolarized by kainic acid, whereas quisqualate and (± )-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) are much less active than kainate, and Nmethyl-D-aspartate (NMDA) does not cause depolarization of the dorsal root even at higher concentrations (Davies et al., 1979; Agrawal & Evans, 1986; Huettner, 1990), although the pharmacological properties of the dorsal root C fibre depolarization have been reported to be not always identical to those of spinal motoneurones (Evans et al., 1987; Shinozaki, 1991). Therefore, we have compared the neuropharmacological properties of depolarizations induced by kainoids in spinal motoneurones and dorsal root fibres of the newborn rat.

Methods The methods used for the electrophysiological experiments in the isolated newborn rat spinal cord were essentially similar to those described previously (Shinozaki et al., 1989b). The spinal cords of 1-7 day old Wistar rats were used for the experiments. Under ether anaesthesia, the spinal cord below the thoracic part was isolated, hemisected sagitally and placed in a 0.15 ml bath perfused at a fixed flow rate of 5-6 ml min- 1 with artificial cerebrospinal fluid (in mM: NaCl 138.6, KCI 3.4, CaCl2 1.3, NaHCO3 21.0, NaH2PO4 0.58, glucose 10.0) which was oxygenated with a gas mixture of 95% 02 and 5% CO2. Tetrodotoxin (TTX, 0.5 pM) was added to the bathing solution in order to block spontaneous depolarization and indirect

M. ISHIDA & H. SHINOZAKI

874

drug effects. In some cases, MgCl2 was added to the perfusing fluid in a concentration of 1.Omm to block the depolarizing action of N-methyl-D-aspartate (NMDA-type agonists. The potential changes generated in the motoneurones were recorded extracellularly from the L3-L. ventral root with a suction electrode. For the recording of depolarization induced in the isolated dorsal root fibre (L3-L.), the central end was placed inside the suction electrode which was positioned 3-4mm from the peripheral end bathed with Ringer and drug solutions. Excitatory amino acids and other test compounds were applied to the preparation either by perfusion or by briefpulse injection into the perfusion system at a constant duration. The temperature of the perfusing fluid was kept at 270C.

Drugs The following compounds were used: acromelic acids A and B generously donated by Prof. H. Shirahama, Hokkaido University), N-acetylkainate (a generous gift from the late Prof. T. Takemoto), (± )-2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, Tocris), 5-bromowillardiine and willardiine (Tocris), 3-[( ± )-2-carboxypiperazine-4-yl]propyl1-phosphonic acid (CPP, Tocris), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, Tocris), dihydroxykainic acid (Tocris), domoic acid (Sigma), sodium L-glutamate mono (Wako), L-a-kainic acid (Sigma), N-methyl-D-aspartic acid (NMDA, Sigma), picrotoxin (Tokyo Kasei), L-quisqualic acid (purity > 99.7%) isolated from the plant, Quisqualic indica L. The following 4-substituted derivatives of 2-carboxy-3-pyrrolidineacetic acid were tested: 4-(2-carboxypyridine)2carboxy-3-pyrrolidineacetic acid (CPPA), 4-(2-carboacid xypyridine N-oxide)-2-carboxy-3-pyrrolidineacetic (CNOPA), 4-(2-hydroxymethylpyridine)-2-carboxy-3-pyrrolidineacetic acid (HMPPA), 4-(2-hydroxyphenyl)-2-carboxy-3pyrrolidineacetic acid (HFPA), 4-(2-methoxyphenyl)-2carboxy-3-pyrrolidineacetic acid (MFPA), 4-methylketone-2carboxy-3-pyrrolidineacetic acid (MKPA, a generous gift from the late Prof. T. Takemoto), 4-(2-methylpyridine)-2-carboxy-3pyrrolidineacetic acid (MPPA) and 4-phenyl-2-carboxy-3-pyrrolidineacetic acid (FPA). These derivatives except for MKPA were all generous gifts from Prof. H. Shirahama, Hokkaido University.

Results Various depolarizing activities of kainate derivatives Spinal motoneurones When kainate derivatives tested in the present study (Figure 1) were added to the bathing solution in various concentrations, almost all derivatives demonstrated a depolarization of the spinal motoneurones with a large variation in their depolarizing activities. Some derivatives caused X,, N *

HOOC^

Kain

COOH

Domo 0O

O

H N

COON V--,,

N

A~Q9 Ho

N

Acro B

MPPA

HOOC

N

U.-

'O~~~~~

x

Acro A

HMPPA

CPPA

0 HOOC

U.1

j A

A

A

A

0.3 Rm 1

b 3 .Lm

3M

3M

A

tLm

0.5 mj

1

mM

~ ~min ~~~1

A

A

j0.2 mV 1min 0.1 mm

MFPA Acro A HFPA Domo Acro B Kain

Glu

Figure 2 (a) Sample records of depolarizing responses to various kainate derivatives (3pgm) and L-glutamate (1 mm) in the newborn rat spinal motoneurone. (b) Sample records of the depolarization of the dorsal root fibre. The concentration is shown near each trace. Test samples were applied at the point of solid triangles for a period of l0 s. MFPA: 4-(2-methoxyphenyl)-2-carboxy-3-pyrrolidineacetic acid; HFPA: 4-(2-hyrodoxyphenyl)-2-carboxy-3-pyrrolidineacetic acid; Acro A: acromelic acid A; Acro B: acromelic acid B; Domo: domoic acid; Kain: kainic acid; Glu: L-glutamate.I the depolarization much more effectively than kainic acid. 4 -(2 - Methoxyphenyl)- 2 -carboxy'. 3 -pyrrolidineacetic acid (MFPA) was the most potent among the kainoids tested here, when they were compared in terms of peak amplitudes of their depolarizing responses at the same concentration (Figure 2a). The depolarizing responses to excitatory amino acids were generally reproducible in the present spinal preparation, and could be roughly divided into two groups showing fast and slow responses (Figure 2a). Kainic acid, domoic acid, 4-2hydroxyphenyl)-2-carboxy-3-pyrrolidineacetic acid (HFPA) and MFPA produced the relatively prolonged time course of action (slow responses), but acromelic acids A and B, quisqualate and L-glutamate gave relatively fast responses. The time course of depolarizing responses to MFPA closely resembled that of kainic acid and domoic acid. Thus, kainate derivatives tested in the present study did not always show slow responses. Figure 3 shows dose-response curves for the kainoids, and relative potencies were tentatively calculated from concentrations producing the same depolarization as kainate 5ym (Table 1). The rank order was as follows: MFPA > acromelic acid A > domoic acid . (HFPA) . acromelic acid B > HMPPA -. CPPA'. kainic acid > MPPA'. FPA > CNOPA > MKPA. This order did not change even when the duration of drug application was prolonged to 2 mm. It was difficult to determine the relative potency of excitation induced by these kanoids and excitatory amino acids in terms of the peak amplitude, because the slopes of dose-response curves for these excitatory amino acids differed significantly (Figure 3), and the peak amplitudes of depolarizing responses to excitatory amino acids sometimes varied in accordance with the duration of drug application (Shinozaki et al., 1989b). This could lead to an underestimate of the more slowly developing responses relative to the fast Dorsal roots The isolated dorsal root fibre of the newborn rat demonstrated a large variety of depolarizing responses to

H

HOOCN

,LM

responses.

H

MKPA Di-kain

All 3

a

N

0 Q-

-OICH,(3

,"

CNOPA FPA MFPA HFPA 1 of kainate derivatives tested in the structure Chemical Figure present study. Acro A: acromelic acid A; Acro B: acromelic acid B; Domo: domoic acid; Di-kain: dihydrokainic acid; Kain: kainic acid; for other abbreviations, see text.

the kainoids tested in the present study (Figure 2b). These were concentration-dependent, and almost all the kainoids tested here and L-glutamate had a very short time course of action; this may be attributed in part to rapid development of receptor desensitization (see below). For the experiment shown in Figure 4, peak amplitudes of depolarizing responses to these kainoids were plotted against their concentrationsun quitsimilrt those ofn_ Figure 3. It was possible to determine the relative potency of their depolarizing activities in the dorsal root fibre, because dose-response curves had almost similar slopes, except for Lglutamate, in which peak amplitudes did not increase further at concentrations greater than 100pM (Figure 4), probably

depolarizing responses

NEW KAINATE DERIVATIVES

875

Table I Mean equimolar potency ratios of various kainate derivatives in the dorsal root fibres and motoneurones of the newborn rat

Compound Domoate Acro B Br-Willardiine MFPA Acro A HFPA Kainate CPPA HMPPA CNOPA FPA MKPA Glutamate MPPA Quis AMPA DihydroKA N-acetylKA NMDA Willardiine

Dorsal root fibr es Mean + n s.e.mean 34 + 2.4 13 + 0.7 5.9 + 0.57 4.2 + 0.29 1.7 + 0.08 1.3 + 0.13 1.0 0.91 + 0.12 0.45 + 0.04 0.29 + 0.06 0.22 + 0.03 0.11 + 0.02 0.11 + 0.01 0.045 + 0.006 0.035 + 0.005 0.014 + 0.005 CPPA > HMPPA > CNOPA FPA _ MKPA = L-glutamate > MPPA. Table 1 presents their relative depolarizing activities in the dorsal root fibre as compared with those in spinal motoneurones. When L-glutamate and the kainoids tested here were added to the perfusion fluid for a period of more than 30s, the amplitude of their depolarizing responses was not maintained, but rapidly declined despite the presence of these excitatory amino acids. The decrease in amplitude of depolarizing responses to these kainoids during their application appears to be due to desensitization. Significant desensitization seemed to be one of the characteristics of kainate receptors on the dorsal root fibres of the newborn rat. L-Glutamate induced desensitization of the receptor more markedly than kainate and other kainoids and onset of desensitization induced by L-glutamate seemed more rapid than that by the kainoids tested here (Figure 5). On the other hand, in the newborn rat spinal motoneurones, desensitization was hardly detectable even after prolonged application of high concentrations of kainic acid or L-glutamate. After prolonged application of kainic acid to the spinal motoneurones, the amplitude of depolarization induced by acromelic acid A did not decrease but rather increased in an additive manner.

0 10-6

lO-5

1o-4

Concentration (M)

Figure 3 Concentration-depolarization relationships for various kainate derivatives in the newborn rat spinal motoneurone. Peak amplitudes of depolarizing responses to agonists (10s application) were plotted against their concentrations. Responses from individual preparations have been normalized, so that results are expressed as a percentage of the control depolarization to SAM kainic acid. Vertical lines represent standard error of means (s.e.mean; n at least 4), MFPA: 4-(2-methoxyphenyl)-2-carboxy-3-pyrrolidineacetic acid; Acro A: acromelic acid A; Acro B: acromelic acid B; Domo: domoic acid; HFPA: 442-hydroxyphenyl)-2-carboxy-3-pyrrolidineacetic acid; HMPPA: 4-(2-hydroxymethylpyridine)-2-carboxy-3-pyrrolidineacetic acid; CPPA: 4-(2-carboxypyridine)-2-carboxy-3-pyrrolidineacetic acid; Kain: kainic acid; MPPA: 4{2-methylpyridine)-2-carboxy-3pyrrolidineacetic acid; FPA: 4-phenyl-2-carboxy-3-pyr-rolidineacetic acid; CNOPA: 4-(2-carboxypyridine N-oxide)-2-carboxy-3-pyrrolidineacetic acid; MKPA: 4-methylketone-2-carboxy-3-pyrroli- dineacetic acid.

Cross desensitization between kainate derivatives Since significant desensitization of kainate receptors developed in the dorsal root fibres after prolonged application of Lglutamate or kainate derivatives, it seemed relatively easy to test the development of cross desensitization. Cross desensitization induced by various excitatory agonists would provide evidence that they activate common receptors. As shown in Figure 5, cross desensitization was observed between Lglutamate (1 mM) and kainic acid (5,M), and between kainic acid (100pM) and acromelic acid A (5puM). Similar results were obtained between domoic and kainic acids and between MFPA and kainic acid; however, even after the development of receptor desensitization induced by kainate or L-glutamate, y-aminobutyric acid (GABA) (5 pM) still caused normal responses of the dorsal root fibre. At high concentrations, quisqualate and AMPA caused a slight depolarization of the dorsal root fibre, but after the receptor was desensitized by

M. ISHIDA & H. SHINOZAKI

876

b

a

I0.2

Kain

AcroA

5p.M

5 1M

Kain

Glu d

c

2

0.1

p.m

Acro A 5 RM

5

RM

mA tM 1

AcroA

5

1mM

Glu

MFPA

Kain 5pRm

mnV

1 min

A

A

A

mmf A

Kain 100 RM

Domo

0.3 RM

t MPFA

Glu

0.1 mM 5 Rm Domo Acro A 0.3 jiM 5 RM

Figure 5 Desensitization of receptors on the dorsal root fibre. (a) Control responses to kainic acid and acromelic acid A (lOs application, 5pM). (b) No further depolarization occurred by kainate or acromelic acid A after desensitization induced by L-glutamate. (c) Control responses to various kainate derivatives and L-glutamate (10 s application). (d) No responses after development of kainate-induced desensitization. The concentration was shown near each response. Abbreviations are the same as in Figure. 3.

kainate, they did not cause any further depolarization. Therefore, quisqualate, AMPA and all kainoids seemed to act at the same receptor on the dorsal root fibre. When MFPA or HFPA was applied immediately after development of kainateind ced desensitization of kainate receptors, further additional responses were not observed suggesting that MFPA and HFPA activated receptors in common with kainate in the dorsal root fibre.

Depression of kainoid-induced depolarization by CNQX but not by CPP It has been shown that CNQX depresses depolarizing kainate- and quisqualate-type agonists (Honore et al., 1988), and CPP is a selective NMDA antagonist (Davies et al., 1986). Therefore, it was predicted that the kainoidinduced depolarization would not be affected by CPP but depressed effectively by CNQX. In fact, CPP, in concentrations ranging from 2 to 100pM, hardly depressed amplitudes of depolarization induced by the kainoids tested here in the newborn rat spinal motoneurones, and magnesium ions (1 mM) also did not affect the response to these kainoids. On the other hand, CNQX was quite effective in reducing responses to

400 0)

Acro A

AMPA

MF

C

o

300

a

Domo

Kain

0) 200 C

100

C.) ...

1

10

1

10

1

lo

1

10

1

10

Concentration (jiM) Figure 6 Dose-response curves for various kainate derivatives in the absence and presence of CNQX in the newborn rat spinal motoneurones. Peak amplitudes of depolarizing responses to AMPA, kainate, acromelic acid A, domoic acid and MFPA were plotted against their logarithmic concentrations in the absence and presence of 1 and 5 M CNQX (results were normalized to that of kainate 5 PM). Vertical lines represent s.e.mean (n at least 4). Control (0); CNQX 1 fiM (A); CNQX 5 pM (0). Abbreviations are the same as in Figure 3.

kainoid-induced depolarization (Figure 6), but there were some differences in the inhibitory action of CNQX on depolarizing responses to the kainoids, particularly in the case of relatively low concentrations of CNQX. CNQX at a concentration of 1 or 5juM depressed more effectively depolarizing responses to acromelic acids A and B, AMPA and 5bromowillardiine than those to kainic acid, domoic acid, MFPA and HFPA in the newborn rat spinal motoneurone (Figure 6). The order of depression of agonist-induced responses with CNQX as an antagonist was as follows: AMPA (1) 5-bromowillardiine (1) > acromelate A (1.2) > acromelate B (2.0) > kainate (2.3) > MFPA (3.2) * . HFPA (3.4) _ domoate (3.7). The numbers in parentheses represent the dose of CNQX, relative to AMPA, for causing the same depression of depolarization (N at least 4, calculated from the pA2 value). On the other hand, the dorsal root fibre, depolarizing responses to 5-bromowillardiine and the kainoids tested here were almost equally depressed by CNQX, but quite insensitive to selective NMDA blockers, suggesting a single receptor type on dorsal root fibres.

Discussion In the present study, both HFPA and MFPA showed potent depolarizing activities in the dorsal root fibre and the spinal motoneurone. In our previous examinations, the rank order of in the adult rat IC_0 for displacement of [3H]-kainate binding spinal cord was as followed: domoate > HFPA > kainate*. MFPA > quisqualate > acromelic acid B > L-glutamate > acromelic acid A > NMDA = AMPA, and that of [3H]AMPA was quisqualate > AMPA > MFPA . acromelic acid A > L-glutamate > HFPA > domoate > kainate > NMDA. MFPA and HFPA demonstrated high binding affinities for kainate receptors in the rat spinal cord, substantially comparable to kainate or domoate, but acromelic acid A showed a considerably lower affinity for kainate receptors than kainate, and it was of particular interest that acromelic acid A and MFPA possessed valuable affinities for AMPA receptors. The AMPA receptor may exist in two forms, one of which has high affinity for AMPA and quisqualate, while the other has higher affinity for kainate but is distinct from the kainate receptor (Watkins et al., 1990a). The difference in depolarizing activities between dorsal root C fibres and motoneurones of the newborn rat would provide some useful information for elucidating the mechanism of activation of kainate receptors (Agrawal & Evans, 1986; Evans et al., 1987; Shinozaki, 1991). Dorsal root fibres of the newborn rat are depolarized selectively by kainate and its derivatives (Agrawal & Evans, 1986; Shinozaki, 1991). Some excitatory amino acids, however, that are not related structurally to kainate, including 5-bromowillardiine (Agrawal & Evans, 1986) and some derivatives of 2-(carboxycyclopropyl)glycine (CCG) (Ishida et al., 1991), also cause considerable depolarization of the dorsal root fibre, and 5bromowillardiine is much more potent than kainate in causing depolarization of the dorsal root fibre. Therefore, they have been regarded as kainate-type agonists (Watkins et al.,

1990a; Ishida et al., 1991). The present work revealed that MFPA was so far the most potent among the kainoids in the newborn rat spinal motoneurones; however, it was not superior to domoic acid in the dorsal root fibre. The depolarizing activity of MFPA in the dorsal root fibre was almost equal to that of 5bromowillardiine. Acromelic acid B was significantly more potent than acromelic acid A in the dorsal root fibre unlike the spinal motoneurone. Domoic acid and MFPA were almost equally sensitive to CNQX in both the dorsal root fibre and the motoneurone, and their pharmacological properties seemed quite similar in quality in both preparations. However, there was a difference in the depolarizing activity of the kainoids between both preparations; depolarizing

NEW KAINATE DERIVATIVES

responses to acromelic acids A and B were significantly depressed by CNQX in the spinal motoneurone in a manner similar to that of AMPA, while kainate and domoate were a little more resistant to CNQX. Watkins et al. (1990b) have obtained similar results using acromelic acid A, AMPA, kainate and domoate in the neonatal rat spinal motoneurones. Significant desensitization of kainate receptors was observed in the dorsal root fibre (Agrawal & Evans, 1986) and the dorsal root ganglion (Huettner, 1990) while it did not occur in the spinal motoneurone. Therefore, the difference in degree of development of receptor desensitization between both preparations may play a key role for a difference in the rank order of the depolarizing activity, but the existence of different types of kainate receptors is also strongly suggested. Multiplicity of kainate receptors based on their permeability to Ca2+ is also proposed from electrophysiological evidence on cultured rat hippocampal neurones (Iino et al., 1990). In addition, recent studies on cDNA for glutamate receptors suggest the presence of a family of kainate receptor subunits with regional difference in the rat brain and with differential potencies of some non-NMDA agonists (Boulter et al., 1990; Keinanen et al., 1990; Sommer et al., 1990; Egebjerg et al., 1991; Hollmann et al., 1991; Werner et al., 1991). Systemic administration of acromelic acid A to the rat causes persistent spastic paraplegia after demonstrating tonic

877

extension of the hindlimbs followed by transient flaccid paralysis, without limbic seizures or 'wet-dog-shakes' (WDS), and induces specific lesions of interneurones in the lower spinal cord with little or no damage in the hippocampal neurones (Shinozaki et al., 1989a; 1991; Shinozaki, 1991; Kwak et al., 1990; 1991). Kainate never causes tonic extension of the hindlimbs or spastic paraplegia. Thus, acromelic acid A and kainic acid demonstrate quite distinct behavioural signs and regional differences in neuronal damage when administered systemically. In our preliminary experiments in the rat, MFPA induced interesting behavioural signs, both tonic extension of the hindlimbs and limbic seizures, which were characteristic of acromelic acid A and kainic acid respectively, suggesting at least two types of kainate receptors (Shinozaki et al., 1990; 1991). Therefore, the kainoids tested here would be expected to provide a useful addition to known kainoids as a valuable tool for examination of supposed subtypes of kainate receptors. The authors wish to thank Prof. H. Shirahama for the generous gift of kainate derivatives. This work was supported in part by Grants-inAid for Scientific Research, for Developmental Scientific Research, and for Scientific Research on Priority Area from the Ministry of Education, Science and Culture of Japan, by Seijin-byo Institute Memorial Foundation, and by the Epilepsy Foundation.

References AGRAWAL, S.G. & EVANS, R.H. (1986). The primary afferent depolarizing action of kainate in the rat. Br. J. Pharmacol., 87, 345-355.

KEINANEN, K., WISDEN, W., SOMMER, B., WERNER, P., HERB, A., VERDOORN, T.A., SAKMANN, B. & SEEBURG, P.H. (1990). A family

BOULTER, J., HOLLMANN, M., O'SHEA-GREENFIELD, A., HARTLEY, M., DENERIS, E., MARON, C. & HEINEMANN, S. (1990). Molecular

KONNO, K., HASHIMOTO, K., OHFUNE, Y., SHIRAHAMA, H. & MAT-

cloning and functional expression of glutamate receptor subunit genes. Sciences, 249, 1033-1037. DAVIES, J., EVANS, R.H., FRANCIS, A.A. & WATKINS, J.C. (1979). Excitatory amino acid receptors and synaptic excitation in the mammalian central nervous system. J. Physiol. (Paris), 75, 641-654. DAVIES, J., EVANS, R.H., HERRLING, P.L., JONES, A.W., OLVERMAN,

H.J., POOK, P.C. & WATKINS, J.C. (1986). CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for 3H-D-AP5 binding sites on brain membranes and anticonvulsant activity. Brain Res., 382, 169-173. EGEBERG, J., BETTLER, B., HERMANS-BORGMEYER, I. & HEINE-

MANN, S. (1991). Cloning of a cDNA for a glutamate receptor subunit activated by kainate but not AMPA. Nature, 351, 745748. EVANS, R.H., EVANS, S.J., POOK, P.C. & SUNTER, D.C. (1987). A comparison of excitatory amino acid antagonists acting at primary afferent C fibres and motoneurones of the isolated spinal cord of the rat. Br. J. Pharmacol., 91, 531-537. HASHIMOTO, K., HIROKAWA, M. & SHIRAHAMA, H. (1990). Simple analogs of acromelic acid, which are highly active agonists of kainate type neuroexcitant. Tetrahed. Lett., 31, 7047-7050. HASHIMOTO, K. & SHIRAHAMA, H. (1990). Synthesis and neuroexcitatory activity of new kainoids. In Amino Acids: Chemistry, Biology and Medicine. ed. Lubec, G. & Rosenthal, G.A., pp. 566572. Leiden: ESCOM. HOLLMANN, M., HARTLEY, M. & HEINEMANN, S. (1991). Ca2+ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition. Science, 252, 851-853. HONORE, T., DAVIES, S.N., DREJER, J., FLETCHER, E.J., JACOBSEN, P., LODGE, D. & NIELSEN, F. (1988). Quinoxalinediones: potent competitive non-NMDA glutamate receptor antagonists. Science, 241, 701-703. HUETTNER, J.E. (1990). Glutamate receptor channels in rat DRG neurons: Activation by kainate and quisqualate and blockade of desensitization by Con A. Neuron, 5, 255-266. IINO, M., OZAWA, S. & TSUZUKI, K. (1990). Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones. J. Physiol., 424, 151-165. ISHIDA, M., OHFUNE, Y., SHIMADA, Y., SHIMAMOTO, K. & SHINOZAKI, H. (1991). Changes in preference for receptor subtypes of

configurational variants of a glutamate analog: conversion from the NMDA-type to the non-NMDA type. Brain Res., 550, 152156. ISHIDA, M. & SHINOZAKI, H. (1988). Acromelic acid is a much more potent excitant than kainic acid or domoic acid in the isolated rat spinal cord. Brain Res., 474, 386-389.

of AMPA-selective glutamate receptors. Science, 249, 556-560. SUMOTO, T. (1988). Acromelic acids A and B. Potent neuroexcitatory amino acids isolated from Clitocybe acromelalga. J. Am. Chem. Soc., 110, 4807-4815. KONNO, K., SHIRAHAMA, H. & MATSUMOTO, T. (1983). Isolation and structure of acromelic acid A and B, new kainoids of Clitocybe acromelalga. Tetrahed. Lett., 24, 939-942. KWAK, S., AIZAWA, H., ISHIDA, M., GOTOH, Y. & SHINOZAKI, H.

(1990). Small neurons in the lower spinal cord are selectively damaged in the spastic rat by acromelic acid. In Amino Acids: Chemistry, Biology and Medicine. ed. Lubec, G. & Rosenthal, G.A., pp. 318-326. Leiden: ESCOM. KWAK, S., AIZAWA, H., ISHIDA, M. & SHINOZAKI, H. (1991). Acromelic acid, a novel kainate analogue, induces long-lasting paraparesis with selective degeneration of interneurons in the rat spinal cord. Exp. Neurol., (in press). SHINOZAKI, H. (1988). Pharmacology of the glutamate receptor. Prog. Neurobiol., 30, 399-435. SHINOZAKI, H. (1991). Kainic acid receptor agonists. In Excitatory Amino Acid Receptors. ed. Krogsgaard-Larsen, P. & Jansen, J.J. Chichester: Ellis Horwood (in press). SHINOZAKI, H. & ISHIDA, M. (1988a). Novel excitatory amino acid related compounds of natural origin. In Neurotox '88: Molecular Basis of Drugs and Pesticide Action. ed. Lunt, G.G. pp. 91-104. Amsterdam: Elsevier. SHINOZAKI, H. & ISHIDA, M. (1988b). Glutamate agonists and excitotoxicity: Pharmacology of new potent agonists. In Neurotransmitters: Focus on Excitatory Amino Acids. ed. Kanazawa, I. pp. 17-30. Tokyo: Excerpta Medica. SHINOZAKI, H., ISHIDA, M., GOTOH, Y. & KWAK, S. (1989a). Specific lesions of rat spinal interneurons induced by systemic administration of acromelic acid, a new potent kainate analogue. Brain Res., 503, 330-333. SHINOZAKI, H., ISHIDA, M., GOTOH, Y. & KWAK, S. (1990). Acromelic acid as a tool for the study of specific neurone damages. In Amino Acids: Chemistry, Biology and Medicine. ed. Lubec, G. & Rosenthal, G.A. pp. 281-293. Leiden: ESCOM. SHINOZAKI, H., ISHIDA, H., KWAK, S. & NAJAJIMA, T. (1991). Use of acromelic acid for production of rat spinal lesions. In Methods in Neuroscience. ed. Conn, P.M. Volume 7, pp. 38-57. New York: Academic Press. SHINOZAKI, H., ISHIDA, M. & OKAMOTO, T. (1986). Acromelic acid, a novel excitatory amino acid from a poisonous mushroom: effects on the crayfish neuromuscular junction. Brain Res., 399, 395-398. SHINOZAKI, H., ISHIDA, M., SHIMAMOTO, K. & OHFUNE, Y. (1989b). Potent NMDA-like actions and potentiation of glutamate

878

M. ISHIDA & H. SHINOZAKI

responses by conformational variants of a glutamate analogue in the rat spinal cord. Br. J. Pharmacol., 98, 1213-1224. SHIRAHAMA, H., KONNO, K., HASHIMOTO, K. & MATSUMOTO, T.

(1988). Acromelic acid A and B, very potent excitatory amino acids from a poisonous mushroom. In Neurotox '88: Molecular Basis and Pesticide Action. ed. Lunt, G.G. pp. 105-122. Amsterdam: Elsevier. SOMMER, B., KEINANEN, K., VERDOORN, T.A., WISDEN, W., BUNASHEV, N., HERB, A., KOHLER, M., TAKAGI, T., SAKMANN, B. &

SEEBURG, P.H. (1990). Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS. Science, 249, 1580-1585. WATKINS, J.C., KROGSGAARD-LARSEN, P. & HONORE, T. (1990a).

Structure-activity relationships in the development of excitatory amino acid receptor agonists and competitive antagonists. Trends Pharmacol. Sci., 11, 25-33. WATKINS, J.C., POOK, P.C.K., SUNTER, D.C., DAVIES, J. & HONORE, T.

(1990b). Experiments with kainate and quisqualate agonists and antagonists in relation to the sub-classification of non-NMDA receptors. In Excitatory Amino Acids and Neuronal Plasticity. ed. Ben-Ari, Y., Advances in Experimental Medicine and Biology, Vol. 268, pp. 49-55. New York: Plenum Publishing Corp. WERNER, P., VOIGT, M., KEINANEN, K., WISDEN, W. & SEEBURG,

P.H. (1991). Cloning of a putative high-affinity kainate receptor expressed predominantly in hippocampal CA3 cells. Nature, 351, 742-744.

(Received May 28,1991 Revised July 24, 1991 Accepted August 13, 1991)