Equilibrium Res Suppl. 3, 1972 DISTRIBUTION OF CHOLINE ACETYLASE AND CHOLIN- ESTERASE AND THE ACTION OF CHOLINERGIC DRUGS ON THE CAT VESTIBULAR SYSTEM IZURU MATSUOKA INTRODUCTION It is well known that anticholinergic drugs are useful antimotion remedies. There- fore, it is pertinent to determine the role of cholinergic mechanisms in vestibular function. The known histochernical localization of acetylcholinesterase (AChE) in the cat nucleus vestibularis is complex. AChE is high in the superior and lateral nuclei, but low in the medial and inferior portions. In nucleus vestibularis lateralis (NVL) the cell bodies of Deiters' giant neurons contain almost all of the AChE. Shute and Lewis and Ross, have also shown AChE in the vestibular ganglion of Scarpa. Steiner and Weber and Yamamoto have reported that NVL neurons activated by vestibular stimulation were also excited by the iontophoretic application of acetylcholine (ACh). Further neurophar- macological investigations on the cholinergic mechanisms in the vestibular system are obviously needed. The present manuscript describes our studies on the gross distribution of choline ace- tylase (ChAc), AChE, cholinesterase (ChE), and total cholinesterase in the cat vestibular nerve and nucleus vestibularis lateralis (NVL). In addition, spontaneous unitary dis- charges were recorded in NVL neurons as well as field potentials during vestibular nerve stimulation before and after various cholinergic agonists and antagonists. METHODS Neurochemical analysis Eight adult cats of either sex were used. After 30 mg/kg i.v. of pentobarbital sodium, each animal was placed in a stereotaxic instrument. In order to locate NVL, a small elec- trode was inserted into the right side of the nucleus and fixed to the cranium with dental cement. The right vestibular nerve and brain stem were exposed under microscopic sur- gery using a Zeiss microscope (X6-X40). The vestibular nerve including Scarpa's ganglion and NVL was dissected out for chemical assay. ChAc was assayed radiochemically using a modification of the method of Schrier and Schuster29 and McCaman and Hunt". Total ChE, AChE and ChE were assayed radiochemically using the method of Siakatos et al.". Neuropharmacological studies Thirty-nine normal adult cats of either sex, weighing 2.5 to 4.0 kg, were used. All surgery was performed under diethyl ether-oxygen anesthesia. The trachea, femoral artery Department of Otolaryngology, Faculty of Medicine, University of Kyoto, Kyoto Equilibrium Res Suppl. 3 78 I. MATSUOKA and vein were cannulated. About 8 mm in diameter of the mucous membrane of the middle ear cavity was removed through an approach via the right tympanic bulla. A small concentric bipolar steel electrode was inserted through the round window according to the method of Fredrickson et al.. Another electrode was stereotaxically inserted through the cerebellum into the reticular formation (RF). The recording microelectrodes were gold tipped stainless steel and tungsten wires. Their electrical resistance was bet- ween 0.75 to 10.0 Mƒ¶. The recording electrode was inserted into the right vestibular nucleus, primarily NVL, with the aid of a stereotaxic brain atlas. The microelect- rode was connected through a cathode follower to a Grass P-5 amplifier and the potentials displ;iyed on a Tektronix dual beam oscilloscope. An amplitude discriminator was used to convert the action potentials into a pulse of constant amplitude and duration (TMC, Fig.1. Evoked potential responses of nucleus vestibularis lateralis to ipsilateral vestibular nerve stimulation. Evoked potentials at varying depths in NVL are illustrated. Note that following the stimulus artifact, prominent N1, N2, and N3 waves are re- corded at especially positions 3 through 7 in this nucleus. Note that the voked potential of N, wave has two peaks at position 6. Time base and voltage calibrations are as shown in this and subsequent figures. Equilibrium Res Suppl. 3 CHOLINERGIC MECHANISMS IN THE VESTIBULAR SYSTEM 79 Model 605, 606). This was fed into a CAT 400B computer and data printer (Model 500A) which yielded on-line poststimulus time histograms of single unit activity. In each recording thirty sweeps following stimulation or spontaneous unit activity were summed on the computer. All data displayed on the oscilloscope were photogra- phed on Kodak plus x film using a Grass recording camera. In some cases, the data from the computer were also displayed on a dual beam oscilloscope and recorded on Po- laroid film. After all operative procedures were completed, the animal was locally anesthetized at all wound edges with 0.5% lidocaine and then immobilized with decamethonium (1.0 mg/kg/hr i.v.). Artificial respiration was maintained immediately after injection of deca- methonium. Body temperature was kept at 36•‹ to 38•Ž by means of an automatic heat- ing pad (German Rupp Industry Inc., Model K-1-3). Under these conditions, the mean •} S.E. values of 5 animals' arterial blood of pH, pO, and pCO, were as follows: pH 7.37 •} 0.01, pO2=105.0•}5.7 mmHg, and pCO2=39.0•}6.0 mmHg. These values were almost the same as normal control animals. The arterial blood pressure was recorded by means of a strain gauge from the femoral artery. The right vestibular nerve and the RF were stimulated with a square wave pulse, 0.05 msec and 0.1 msec in width, delivered from a Grass S-8 stimulator. The intensity of stimuli ranged from 1.0 to 5.0 V. Single shock stimuli were usually used, but in some cases pulse trains (50, 150, and 250 Hz, 40 msec train duration and 0.1 msec pulse width) were applied. At the end of each experiment, a 22.5 V dc was applied to the microelectrode so that the site of recording could be confirmed histologically (see Fig.1). RESULTS Cholinergic neurochemistry of the vestibular system As shown in Table 1, the content of ChAc, AChE, ChE and total ChE was higher in NVL than in the vestibular nerve including Scarpa's ganglion. Field potentials in NVL to electrical stimulation of the ipsilateral vestibular nerve Field potentials evoked to vestibular nerve stimulation were classified as N1, N2 and Table 1. Cholinergic neurochemistry of the cat vestibular axon and necleus vestibularis lateralis ChAc is expressed as mean uMol ACh/g/hr•}S.E. AChE was massured using 14C-labelled methacholine, ChE using 14C-butyrylcholine and total ChE using "C-ACh as substrates and expressed as mean uMol/g/hr. Protein is expressed as mg/ml of homogenate. Equilibrium Res Suppl. 3 80 I. MATSUOKA N3 waves. These potentials were almost the same as the P, N, and N2 waves of Shimazu and Precht25.The N,-wave was a small negative and sometimes positive potential. The N2-wave was a large sharp negative potential and the N3-wave was a wide negative po- tential. The means•}S.E. of the latency on these three waves for 7 animals were N1= 0.50•}0.03, N2=1.09•}0.03, and N3=2.43•}0.21 msec. These values were almost the same as Shimazu's. When the microelectrode was inserted into NVL, a characteristic field potential was evoked by single shocks to the vestibular nerve. The typical response was always elicited in the middle portion of NVL. The N2-wave sometimes had two or three peaks. This indicates that there are different conducting axons in the peripheral vestibular nerve. The alterations in the N,- and N2-waves during different placements of the microelectrode from a dorsal to a ventral trajectory are shown in Fig. 1. The po- tentials are helpful for locating single neuronal responses to specific portions of NVL. The mean amplitudes•}S.E. of these waves in seven animals are as follows: N,=96.7•} 10.7, N2=176.3•}14.5, and N3=123.4•}17.5ƒÊV, as illustrated in the bar graph in Fig. 2. Effects of cholinergic agonists The mean amplitude of the N1-wave was reduced slightly to 73.5 •} 10.5ƒÊV 2 min. after administration of nicotine (25ƒÊg/kg i.v.), but this was not statistically significant. The mean amplitude of the N2-wave was reduced to 101.2•}36.1 pV (P<0.01) while the am- plitude of the N3-wave did not change. Five minutes after administration of physostig- Fig.2. Effects of various cholinergic drugs on the amplitude of evoked nucleus vestibularis lateralis potentials elicited with single shocks to the ipsilateral vestibular nerve. Each bar height represents the mean amplitude of a specific wave as designated. Note the dramatic effects of physostigmine in facilitating the N-2wave as well as scopolamine in reducing it. This wave was reduced following nicotine. All responses were measured 5 minutes after drug administration except after nicotine, which was measured 2 minutes after injection.*= P<.05, **=P<.01, ***=P<.001. Equilibrium Res Suppl. 3 CHOLINERGIC MECHANISMS IN THE VESTIBULAR SYSTEM 81 mine (25ƒÊg/kg i.v.), the mean amplitude of the N2-wave increased to 315.0±29.8 pV (P< 0.001). However, the N, and N3-waves did not change (Fig. 2). The accumulative dose response curve of physostigmine is shown in Fig. 3. The N,-wave slightly increased with doses of 25 and 50 ug/kg i.v., but this was not statistically significant. The N2-wave was markably enhanced even in small doses (5ƒÊg/kg i.v.). The N3 wave did not change at all. The optimal dose of physostigmine which facilitated the N2-wave was 25 pg/kg i.v. Larger doses caused a slight reduction. Effects of cholinergic antagonists As described above, the amplitude of the N2-wave was enhanced by physostigmine in doses of 25,ag/kg i.v. This enhancement was reduced by scopolamine in doses of 0.5 mg/kg i.v. In this dose scopolamine did not reduce the N, and N,-waves.