Heuring Research, 25 (1987) 77-81

Elsevier

HRR 00840

77

Kynurenic acid and y-D-glutamylaminomethylsulfonic acid suppress the compound action potential of the auditory nerve *

Richard P. Bobbin and Gail Ceasar Kresge Hearing Research Laboratory of the South, Department of Otorhinolmygology and Biocommunication,

Louisiana State University Medical Center, New Orleans. LA 70119-J 799, U.S.A.

(Received 25 April 1986; accepted 8 July 1986)

Kynurenic acid and y-D-glutamylaminomethylsulfonic acid, two excitatory amino acid antagonists, were perfused through the

guinea pig cochlea while monitoring various cochlear potentials. Both drugs (0.6-10 mM) reduced the magnitude of the compound

action potential (CAP) of the cochlear nerve without much effect on other potentials. The results are consistent with the hypothesis

that the hair cell transmitter is an excitatory amino acid, possibly L-glutamate.

kynurenic acid, y-D-glutamylaminomethylsulfonic acid, cochlea, hair cell. neurotransmitter

Introduction

Several workers have examined the hypothesis that the excitatory amino acid, L-glutamate, is a neurotransmitter released by hair cells of the mammalian cochlea (see reviews by Bobbin et al., 1984, 1985) and other hair cell systems (see discus-

sion by Jenison et al., 1986). Three criteria of transmitter identification seem to have been met by L-glutamate: (1) it is excitatory to all hair cell

afferents tested (as cited in Jenison et al., 1986); (2) it is released into the fluid that bathes the hair cells of the cochlea (Jenison et al., 1985) and the lateral line (Bledsoe et al., 1980); and (3) L- glutamine, a L-glutamate precursor, is taken up by the hair cells of the cochlea (Eybalin and Pujol, 1983; Ryan and Schwartz, 1984). However an additional criterion, antagonism of both exoge- nously administered L-glutamate and hair cell transmission with a specific glutamate blocker, has

Correspondence address: Kresge Hearing Research Lab.,

LSUMC, 1100 Florida Ave., Bldg. 124, New Orleans, LA 70119-2799, U.S.A.

* A preliminary report of these findings was presented

February, 1986, at the 9th Midwinter Research Meeting of

the Association for Research in Otolaryngology, Clearwater Beach, Florida.

not been demonstrated in the cochlea, although it seems to have been shown in two other hair cell systems (Bledsoe and Bobbin, 1982; Annoni et al., 1984).

At present there are thought to be three or more types of excitatory amino acid receptors that may be activated by an endogenous compound such as L-glutamate (Fagni et al., 1983; Bobbin et al., 1985). One classification names three types according to the exogenous substances thought to activate them: N-methyl-D-aspartate (NMDA), quisqualate, and kainate. NMDA and excitatory amino acid antagonists known to antagonize

NMDA such as a-aminoadipate and 2-amino-5 phosphonovalerate are notably without effect in the cochlea (Fex and Martin, 1980; Bobbin et al., 1981, 1984; Jenison et al., 1986). In contrast, NMDA antagonists are effective in blocking both NMDA and afferent transmission in a few other

hair cell systems, such as the lateral line of Xeno- pus (Bledsoe and Bobbin, 1982). The non-NMDA antagonists that have been tested in the cochlea have little effect on the compound action potential (CAP) of the auditory nerve or have additional actions on the cochlear microphonic potential (CM) (Bobbin et al., 1984, 1985).

Therefore we have continued searching for

IX

compounds which might be effective in the cochlea by testing chemicals which have antagonistic

properties on the non-NMDA types of excitatory amino acid receptors. This study describes our

results with two new antagonists, kynurenic acid (kynurenate) and y-D-glutamylaminomethylsul- fonic acid (GAMS), which have significant block- ing effects at non-NMDA receptors in other neu- ronal systems (Ganong et al., 1983; Croucher et al., 1984; Davies and Watkins, 1985; Herrling, 1985; Jahr and Jessell, 1985; Stone and Connick, 1985).

Methods

Experiments were performed on 13 pigmented guinea pigs of both sexes. The methods used are

similar to those previously described (Bobbin and Konishi, 1974; Bledsoe et al., 1981). The animals

were anesthetized with chlorprothixene (Taractan@, 25 mg/kg, i.p.) and pentobarbital (Nembutal@, 30 mg/kg, i.p.), artificially respired, and maintained at a rectal temperature of 37-39°C. During ex-

periments, additional pentobarbital was adminis- tered (25 mg/kg, i.p.) to assure a deep level of anesthesia. The right cochlea of each animal was exposed ventrolaterally and both middle ear muscles were sectioned.

The CAP (Nl-Pl), CM and summating poten- tial (SP) were recorded from a 200 pm enamel-in- sulated silver wire placed in a hole in the Scala

vestibuli of the basal turn, amplified and stored on computer. The measured potentials were average responses (20 samples) obtained to 10 kHz tone bursts having 0.25 ms exponential rise-fall times, 10 ms durations and 100 ms interstimulus inter- vals. Intensity functions were obtained by varying

tone burst intensities (2-104 dB SPL, in 6 dB

steps). The endocochlear potential (EP) and the crossed olivocochlear potential (COCP), the change in the EP evoked by stimulating the cochlear efferents in the midline of the fourth ventricle, were recorded from animals which had received gallamine triethiodide (Flaxedila, Davis and Geck, 8 mg/kg, i.p.) as previously described (Bobbin and Konishi, 1974).

Artificial perilymph solutions were infused into the basal turn Scala tympani and allowed to flow out of the basal turn Scala vestibuli at approxi-

mately 2 pl/min for 10 min through holes made in the cochlea. Two artificial perilymph composi- tions were used. One, a phosphate/bicarbonate buffered solution, has been used previously (Konishi and Kelsey, 1973: Jenison et al., 1986)

and the other was a modification containing a Hepes buffer (Takada and Schacht, 1982). The modified solution had a composition of (mM): NaCl, 137; KCl, 5; CaCl,, 2; NaH,PO,, 0.1; MgCl,, 1; Hepes, 10; glucose, 11; and the pH was adjusted with 1 N NaOH to 7.4. The GAMS (Tocris) and kynurenate (Sigma) were initially dis- solved to make a 10 mM concentration and the pH adjusted to 7.4 with NaOH. Dilutions were then made to yield the concentrations tested (mM): 10, 5, 2.5, 1.25 and 0.625. In all animals, artificial perilymph without drug was the first perfusion.

This was then followed by successive perfusions with increasing concentrations of the drug from 0.625 to 10 mM, except in the animals where the

l Pre KYNA + GAMS

o Post GAMS IlOmM)

A Post KYNA t10mM)

40 80 g0 lb0

dB SPL

Fig. 1. Effect of 10 mM kynurenate (KYNA) and 10 mM

GAMS on the intensity function of the CAP. Because there

was no difference between the prekynurenate-postartificial per-

ilymph function and the preGAMSpostartificia1 perilymph

function. they were combined. Shown are functions obtained

after perfusing with artificial perilymph alone (Pre KYNA+

GAMS; n =10), after perfusing with GAMS (Post GAMS;

tt = 5) and after perfusing with kynurenate (Post KYNA:

?I = -5).

EP and COCP were monitored when only one concentration was tested. All drug perfusions were followed by another perfusion with artificial per- ilymph without drug. Intensity functions to the

tone bursts were recorded before any perfusions and immediately (within 2 min) after each perfu- sion. It is known empirically that the CAP inten-

sity’ function has a drug sensitive low intensity portion and a drug insensitive high intensity por- tion (Bobbin and Kisiel, 1981). Therefore, to quantify the effects of the drugs, we carried out statistical tests on CAP, CM, and SP data evoked by one intensity from each of these areas, 74 and 104 dB SPL, respectively. Analysis of variance and Newman-Keuls multiple-range tests were used to determine significance (P < 0.05). The data are expressed as mean k S.E.

19

Results

The first perfusion of either artificial perilymph increased the magnitude of all cochlear potentials

(e.g., Hepes, n = 3-5: CAP, 39%; CM, 4%; SP, 56%; EP, 9% and COCP, 17%). The effect on the

CAP, CM, EP, and COCP has been reported previously (Bobbin and Konishi, 1974; Bobbin and Thompson, 1978). In three animals, multiple perfusions of artificial perilymph without drug were carried out (phosphate/ bicarbonate solu- tion, n = 2; Hepes solution, n = 1). Perfusions subsequent to the first produced very little ad- ditional change in the cochlear potentials. There- fore, all drug effects are compared to potentials recorded after the first perfusion with artificial

perilymph.

200 CM74dB SPL

1 75 150 j 1 T

JJV

50 100

25 50

0 R 0.6 1.2 2.5 5 10 R P R 0.6 1.2 2.5 5 10 R 0

P R 0.6 1.2 2.5 5 10 R

CAP74dB SPL

CAP104dB SPL SP104dB SPL CM104dB SPL

P - R 0.6 1.2 2.5 6 10 R ” P R 0.6 1.2 2.6 6 10 R P R 0.6 1.2 2.6 6 10 R

Fig. 2. Effect of increasing concentrations of kynurenate on the CAP. SP and CM in five animals (n = 5). Shown are the data evoked

by tone bursts of 74 dB SPL and 104 dB SPL before any perfusions (P) and after perfusion with artificial perilymph alone before

drug (R, on the left in each frame), successive perfusions of increasing concentrations of kynurenate (0.6, 1.2, 2.5, 5, and 10 mM) and a post drug artificial perilymph (R, on the right in each frame).

X0

Artificial perilymph solutions containing kyn-

urenate (n = 5) or GAMS (n = 5) suppressed the CAP, but did not affect the CM or SP. The suppression induced by both drugs was readily reversed upon perfusing with artificial perilymph without drug. The concentration which caused approximately 50% of the maximum suppressive response (measured at 74 dB SPL) for kynurenate

and GAMS was 1.25 and 2.5 mM, respectively. The greatest suppression occurred near the maxi- mum dosage utilized; therefore only the intensity functions generated before any drug and after 10 mM concentrations of each drug are shown in Fig. 1. The CAP suppression was equal to a lo-20 dB

reduction in sound intensity for GAMS and greater than 40 dB for kynurenate (Fig. 1). Statistically, GAMS did not produce a significant (P r 0.05) change in the CM or SP at 74 or 104 dB SPL and

its effect on CAP was only significant (P < 0.05) at 5 and 10 mM for 74 dB SPL. Since kynurenate gave the largest response on the CAP, its dose response relationship on CAP, CM and SP evoked by tone bursts on the CAP plateau (74 dB SPL) and at the maximal CAP value (104 dB SPL) are shown in Fig. 2. Statistical examination revealed that kynur’enate significantly (P < 0.05) reduced the CAP measured at both 74 and 104 dB SPL, in

concentrations of 1.25 mM and greater, but had no significant effect (P > 0.05) on the CM or SP measured at these intensities.

The effects of kynurenate on the EP and COCP were descriptively evaluated in three animals to examine further its selectivity for the CAP. We

chose to test a concentration of 5 mM since it was at the top of the CAP dose response curve (Fig. 2). After perfusing artificial perilymph, which was the first perfusion in each animal, neither the EP (pre: 82 _rt 7 mV> nor the COCP (pre: 1.5 & 0.3 mV)

were changed (post: EP, 85 Ifr 4 mV; COCP, 1.7 f 0 mV). After a total of five subsequent perfusions with kynurenate (5 mM) alternated with artificial perilymph, the EP (pre: 80 + 7 mV) was decreased slightly and reversibly (post: 70 & 4 mV), but the COCP (pre: 1.7 f 0.2 mv) was not changed (post: 1.6 i 0.1 mV). In each animal, the COCP was readily abolished by a final perfusion of strych- nine sulfate (10 PM), as reported by others (Bob- bin and Konishi, 1974).

Discussion

Our current hypothesis is that the guinea pig cochlea hair cells release L-glutamate as an excita- tory neurotransmitter (Bobbin et al., 1984, 1985). Furthermore, because NMDA and blockers of NMDA have no effect in the cochlea, we have proposed that if L-glutamate is the hair cell neuro- transmitter, then it acts on non-NMDA receptors (Bobbin et al., 1985; Jenison et al., 1986). There- fore we examined two relatively new antagonists which have prominent actions at non-NMDA sites, kynurenate and CAMS.

GAMS has been reported to antagonize kainate

> quisqualate > NMDA > L-glutamate (Croucher et al., 1984; Davies and Watkins, 1985). Kynure-

nate appears to be more of a general ‘broad spectrum antagonist’ that competitively blocks NMDA > kainate > L-glutamate 2 quisqualate (Ganong et al., 1983; Herrling, 1985; Jahr and Jesse& 1985; Stone and Connick, 1985; Ganong and Cotman, 1986). In the present study, GAMS appears selective for the CAP by its lack of effect on CM and SP. Likewise, kynurenate was found to be selective for the CAP, lacking effect on the CM, SP and COCP, and having only a slight

effect on the EP. Therefore the dose-dependent suppression of

the CAP that we observe with GAMS and kynurenate could possibly have resulted from a blockade of the hair cell neurotransmitter at the afferent nerve endings. The effect of kynurenate on EP does suggest that we exercise caution in interpreting the action of the drugs as being solely at this site. Nevertheless. these results are con- sistent with the hypothesis that an excitatory amino acid, such as L-glutamate, is the hair cell neuro- transmitter. Future studies will examine various agonists against each antagonist at the single neu- ron level to determine the site of action more specifically.

Acknowledgements

Thanks to Dr. L. Hughes, Dr. J.K. Cullen, Jr., C. Wiesendanger and A. Florez for designing and building the computer and writing the software which was used to collect the CAP, CM and SP data, and to John Carr, editorial consultant at

81

LSU School of Dentistry. Supported by NSF re- search grant BNS8419241 and the Louisiana Lions Eye Foundation.

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