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Japan. J. Pharmacol. 30, 29-36 (1980) 29 THE ACTION OF CHLORPHENESIN CARBAMATE ON THE FROG SPINAL CORD Hironaka AIHARA, Michio KURACHI, Sadao NAKANE, Michitada SASAJIMA and Masahiro OHZEKI Research Laboratories, Taisho Pharmaceutical Co., Ltd., Yoshino-cho1-403, Omiya, Saitama 330, Japan Accepted September 10, 1979 Abstract-Studies were carried out to elucidate the mechanism of action of chlor- phenesin carbamate (CPC) and to compare the effect of the drug with that of mephenesin on the isolated bullfrog spinal cord. Ventral and dorsal root potentials were recorded by means of the sucrose-gap method. CPC caused marked hyperpolarizations and depressed spontaneous activities in both of the primary afferent terminals (PAT) and motoneurons (MN). These hyperpolarizations were observed even in high-Mg" and Ca2+-freeRinger's solution, suggesting that CPC has direct actions on PAT and MN. Various reflex potentials (dorsal and ventral root potentials elicited by stimulating dorsal and ventral root, respectively) tended to be depressed by CPC as well as by mephenesin. Excitatory amino acids (L-aspartic acid and L-glutamic acid) caused marked depolarizations in PAT and MN, and increased the firing rate in MN. CPC did not modify the depolarization but abolished the motoneuron firing induced by these amino acids. However, mephenesin reduced both the depolarization and the motoneuron firing. The dorsal and ventral root potentials evoked by tetanic stimu- lation (40 Hz) of the dorsal root were depressed by the drugs. These results indicate that CPC has an apparent depressing action on the spinal neuron, and this action may be ascribed to the slight hyperpolarization and/or the prolongation of refractory period. The skeletal muscle relaxant actions of mephenesin have been attributed to its depressant action on interneurons of polysynaptic reflex arcs in the spinal cord and at supraspinal levels (1, 2). Chlorphenesin carbamate (CPC), a drug structurally related to mephenesin (Fig. 1), has been reported to be a selective blocker of polysynaptic pathways at the spinal and supraspinal level (3). However, there are reports indicating that mephenesin and CPC depress monosynaptic as well as polysynaptic reflex (4, 5). Furthermore, these drugs were reported to have no effect on the arousal response evoked through polysynaptic pathways (6). Thus, it is unlikely that these drugs are selective blockers of polysynaptic pathways. As there is little direct evidence for specific actions of these drugs on spinal interneurons, FIG. 1. Chemical structures of CPC and mephenesin. 30 H. AIHARA ET AL. we studied the effect of CPC on the frog spinal cord in parallel with that of mephenesin. The mechanism of muscle relaxant activities of these drugs is also discussed. MATERIALS AND METHODS Bullfrog (Rana catesbeiana) was cooled in ice to an anaesthetic state, and the spinal cord with 9th or 10th ventral and dorsal roots was carefully isolated. As soon as possible, a glass cannula was inserted into the anterior spinal artery and the spinal cord was continuously perfused with oxygenated Ringer's solution (pH 7.3 +0.1, 16-18•Ž, perfusion velocity; 0.3 ml/min) consisted of NaCl120 mM, KCl2.5 mM, CaCl2 1.8 mM, Tris (hydroxy- methyl) aminomethane 1 mM and glucose 5.6 mM. In some experiments, 12 mM MgCl2 was added to and CaCl2 was deleted from Ringer's solution. The potential changes occurring at the dorsal root nerve terminals and at the motoneurons in the spinal cord were recorded by means of the sucrose-gap method, the procedure being much the same as that reported by Kudo et al. (7). A schematic drawing of the experimental arrangement is shown in Fig. 2. Potential differences between the spinal cord and the peripheral stumps of ventral root or dorsal root were detected by calomel electrodes and then amplified by a DC-amplifiers (San-ei Sokki 6L5), and the outputs were connected with a two pen DC-recorder (Tohshin). In some experiments, the rate of discharges from the ventral root was recorded by a meter of our own design. Stimuli were delivered to the appropriate root (Nihonkohden MSE-3R) via bipolar platinum wires. All drugs were dissolved in Ringer's solution, and applied by means of exchanging the perfusate for the drug containing Ringer's solution or the injection of the drug containing Ringer's solution into the polyethylene tube (Fig. 2). Drugs used were L-glutamate monosodium salt (Wako), strychnine HNO3 (Sanko), picrotoxin (Tokyo Kasei), mephenesin (Sigma) and chlorphenesin carbamate (Taisho Pharm.). The following abbreviations were used; DR-DRP, the dorsal root potential induced by the stimulation of the dorsal root; VR-DRP, the dorsal root potential induced by the stimulation of the ventral root; DR-VRP, the ventral root potential induced by the stimu- FIG. 2. Diagram of sucrose-gap method for recording the potential changes occurring at the dorsal root nerve terminals and motoneurons in the isolated perfused spinal cord of the bullfrog. DR: dorsal root, VR: ventral root, S: stimulater, c.e.: calomel electrode. CHLORPHENESIN CARBAMATE AND SPINAL CORD 31 lation of the dorsal root. RESULTS Effects on potential changes of the dorsal and ventral roots: When 0.3 ml of CPC (10-3 M) was applied to the spinal cord through a polyethylene tube inserted into the cannula (injection velocity; 30 sec/0.3 ml), a hyperpolarization was produced in the dorsal and the ventral roots (Fig. 3A). The slow spontaneous depolarizations in the dorsal and the ventral roots were diminished and the spontaneous discharges (not illustrated) in the ventral root were abolished by the injection of CPC. The rates of discharge in the ventral root caused by the stimulation of dorsal root were reduced by the application of CPC. Reflex potentials (DR-VRP, DR-DRP and VR-DRP) were depressed slightly by a one shot application of CPC (10-3 M, 0.3 ml, Fig. 3B) and were reduced markedly by the continuous application of CPC (not illustrated). A similar effect was seen with mephenesin (Fig. 3C). These effects were reversible. Effects of CPC in the high-Mg2+ and Ca2+-free Ringer's solution: Exposure of the spinal cord to a drug solution could affect the dorsal and the ventral roots indirectly by activating pathways which synapse onto those neurons. To determine the indirect synaptic effects, the responses obtained in a high-Mg2+ and Ca2+-free Ringer's solution were compared A B C FIG. 3. Effects of CPC and mephenesin on the DRPs and VRPs. A: spontaneous occurring potentials. B and C: the potentials elicited by stimulating dorsal or ventral root, and indicate the effects of CPC (10-3 M) and mephenesin (10-3 M), respectively. Filled circles indicate the injection of drugs. 32 H. AIHARA ET AL. with responses in normal Ringer's solution. Firstly, it was confirmed that the addition of MgCl2 to and the deletion of CaCl2 from Ringer's solution resulted in the disappearance of spontaneous activity and electrically evoked potentials. The hyperpolarization of dorsal and ventral roots by CPC occurred consistently in the high-Mg2+ and Cas+-free Ringer's solution (Fig. 4), suggesting a direct effect of this drug on dorsal and ventral roots. Effects of strychnine and picrotoxin on the CPC hyperpolarization: Mimicking the FIG. 4. Effect of high-Mg2+ and Ca2+-free Ringer's solution on the CPC response. Left and right traces represent CPC responses in Ringer's solution and high-Mg2+ and Ca2+-free Ringer's solution, respectively. A B FIG. 5. Effects of strychnine and picrotoxin on CPC hyperpolarization. A: CPC hyperpolarization was unaffected by strychnine (10-4 M). B: CPC hyperpolari- zation was augmented by the application of picrotoxin (10-4 M). CHLORPHENESIN CARBAMA TE AND SPINAL CORD 33 action of inhibitory amino acids could produce hyperpolarization in the dorsal and ventral roots. Thus, we attempted to determine whether CPC hyperpolarization was blocked by strychnine or picrotoxin, antagonists of inhibitory amino acids (8-12). The addition of strychnine (10-4 M) to the Ringer's solution had no effect on the CPC hyperpolarization (Fig. 5A), while picrotoxin (10-4 M) augmented CPC hyperpolarization (Fig. 5B). Accordingly, it was concluded that CPC did not mimick the action of inhibitory amino acids. Effects of CPC on L-glutamate responses: Acidic amino acids, L-glutamate and L- aspartate are considered to be candidates for excitatory neurotransmitters in the vertebrate central nervous system. Therefore, the application of drugs antagonizing these amino acids might result in the depression at the central nervous system. When L-glutamate (10-s M, 0.3 ml) or L-aspartate (10-3 M, 0.3 ml, not illustrated) was applied to the spinal cord, depolarizing potentials were recorded in the dorsal and the ventral roots, and the burst of discharges in the ventral root was observed. It was thus confirmed that repetitive applications of these acidic amino acids resulted in responses of identical size and constant firing rates. As shown in Fig. 6A, continuous application (for 10 min) of CPC (10-s M) did not affect the depolarization induced by L-glutamate, while occasionally a longer application (for 40 min) of CPC did affect it. The bursts in ventral root evoked by amino acid were abolished, despite the same size in depolarization seen in the control. On the other hand, mephenesin (10-3 M) slightly depressed the depolarization caused by L-glutamate in the dorsal and the ventral roots, and completely blocked the burst in the ventral root by L-glutamate (Fig. 6B). Effects of CPC on the potentials evoked by multiple stimuli: As the centrally acting muscle relaxants may depress the repetitive activity in motoneurons arising in muscle tonus, we investigated the effects of CPC on the potentials evoked by multiple stimuli. When multiple stimuli (pulse duration; 0.05 msec, frequency; 40 Hz, intensity; 4V, for 5 sec) were A B FIG.

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