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J. Phyeiol. (1970), 210, pp. 751-760 751 With 5 text-figurew Printed in Great Britain CENTRIFUGAL INHIBITORY PROCESSES AFFECTING NEURONES IN THE CAT COCHLEAR NUCLEUS BY S. D.

1 J. Phyeiol. (1970), 210, pp With 5 text-figurew Printed in Great Britain CENTRIFUGAL INHIBITORY PROCESSES AFFECTING NEURONES IN THE CAT COCHLEAR NUCLEUS BY S. D. COMIS From the Neurocommunications Research Unit, University of Birmingham, Birmingham 15 (Received 13 April 1970) SUMMARY 1. Stimulation of the lateral part of the olivary S-segment in the cat inhibited neurones in the ipsilateral cochlear nucleus. A smaller number of neurones located in the ventral division of the cochlear nucleus were excited. 2. It is suggested that inhibition in the ipsilateral cochlear nucleus may be mediated directly by fibres making synaptic connexions on the cochlear nucleus neurones, or indirectly by inhibitory fibres acting at the cochlea. 3. The direct inhibitory process at the cochlear nucleus is unaffected by strychnine, whereas the inhibitory process at the cochlea is abolished by strychnine. 4. A cochlear nucleus neurone can be influenced simultaneously by excitatory and inhibitory processes. INTRODUCTION It is well established that direct connexions exist between the olivary S-segment and the ipsilateral cochlear nucleus (Rasmussen, 1960, 1964, 1967), and that certain olivo-cochlear nucleus fibres stain positively for acetylcholinesterase by the Koelle technique (Rasmussen, 1964, 1967). In a previous study (Comis & Whitfield, 1968), it was reported that direct current stimulation of cell bodies within the superior olivary S- segment of the cat enhances the activity of some neurones in the ipsilateral ventral cochlear nucleus. Neurones which could be thus activated by olivary stimulation were found to respond comparably to locally applied acetylcholine by means of the microtap (Comis, Evans & Whitfield, 1964). Furthermore, the effects of olivary stimulation could be attenuated by applying drugs with known anti-acetylcholine actions at the cochlear nucleus neurone. It was pointed out by Comis & Whitfield (1968) that while only excitatory effects had been observed among all neurones that

752 S. D. COMIS responded to olivary stimulation, the lateral part of the S-segment had not been extensively studied due to anatomical restriction.

2 752 S. D. COMIS responded to olivary stimulation, the lateral part of the S-segment had not been extensively studied due to anatomical restriction. In the present series of experiments an attempt has been made to investigate the effect of stimulation of the region hitherto unexplored. METHODS The experiments were performed on cats anaesthetized with Nembutal (30 mg/kg). The general method was essentially that used by Comis & Whitfield (1968). In order to approach the most lateral portion of the S-segment, sufficient bone was removed from the medial aspect of the bulla by means of a high speed dental bur. The middle ear was fully exposed by opening the bulla and when required the middle ear muscles were cut, or visually examined through a Zeiss otoscope during stimulation. The middle ear muscles were cut by means of a suitably sharpened hook. In some experiments cochlear potentials were recorded from the round window by means of Ag/AgCl electrodes. The monopolar stimulating electrode consisted of a stainless-steel needle, the tip of which was etched to 10 pu, and the shaft insulated with Damarda lacquet- The stimulating electrode was inserted into the superior olive through a hole drilled in the basisphenoid bone with a No. 10 round bur (Claudius Ash & Sons Co. Ltd.), some 8 mm from the ventral edge of the foramen magnum. Direct current stimulation was effected from a high impedance source (1 MQ) and short pulses of alternate polarity were used. The cochlear nucleus was approached according to the method described by Stopp & Whitfield (1963) and recordings were taken via the microtap. Acoustic stimulation was supplied through an Ionofane loudspeaker coupled to a hollow earbar. All data was amplified and recorded on magnetic tape. Neuronal spike activity was also passed through a pulse height discriminator and rate meter to a pen recorder. Electrode placements in the animal were histologically verified after the experiment. RESULTS In the majority of these experiments, the results were obtained in response to stimulation of the lateral half of the superior olivary S- segment with direct current of either negative or positive polarity. The merits of direct current stimulation have been discussed elsewhere (Comis & Whitfield, 1968; Comis & Davies, 1969b). Neuronal activity was recorded from the dorsal and ventral divisions of the cochlear nucleus. From experiments in which olivary stimulation proved effective, a total of sixty neurones were studied. Of these, twenty-six could be inhibited and eleven were excited by the stimulation. Histological examination after the experiment showed that the tip of the stimulating electrode was located in the lateral part of the S-segment, or just dorsal to it. Fig. 1 shows a typical section. In the ipsilateral cochlear nucleus, neurones that could be inhibited by olivary stimulation were located at all electrode depths, that is in both the dorsal and ventral divisions. However, the neurones that exhibited

3 CENTRIFUGAL INHIBITORY PROCESSES 753 excitation were predominantly to be found in the ventral division. These latter neurones were identical in behaviour to the ones extensively studied by Comis & Whitfield (1968). It is not possible at the present time to give a very precise picture regarding the functional distribution of centrifugal neurones in or around the S-segment which innervate the ipsilateral cochlear nucleus, other than to say that stimulation of the medial aspect of the S-segment produces predominantly excitatory effects in the ventral cochlear nucleus, whereas stimulation of neurones within, or dorsal to, the lateral part of the S-segment produces inhibition in the majority of cases all over the cochlear nucleus. Fig. 1. Transverse section through the cat olivary complex (Kiuver stain). The arrow indicates the direction of the electrode track and points to the position of the electrode tip. The magnitude of the current required to produce an effect in the cochlear nucleus was of the order of 50,uA, only one polarity being effective, positive or negative, in any one case as in previous experiments (Comis & Whitfield, 1968). It was suggested by these authors that the differing effective polarity was due to different configurations between the neurone stimulated, and the stimulating electrode tip. Inhibition and excitation are effective on the spontaneous as well as on tone induced activity.

4 754 S. D. COMIS Effect of middle ear muwde8 In some experiments it was noticed that if the stimulating electrode tip was placed at a depth of 4 mm (measured from the ventral surface of the brain) the majority of neurones with characteristic frequencies below about 2 khz could be inhibited. Subsequent histological examination revealed that the electrode tip was located in, or near, the motor nucleus of the trigeminal nerve. It was confirmed during experiments that stimulation of this site was accompanied by contractions of the tensor tympani muscle. When the muscle was cut, the above inhibitory effect was abolished. Neurones with high characteristic frequencies appear to be relatively unaffected by contractions of the tensor tympani. Stimulation of the motor nucleus of the trigeminal nerve was therefore avoided and in subsequent experiments the middle ear muscles were cut as a double precaution. Pharmacology The pharmacology of the excitatory effect has already been described and much of the evidence presented is in favour of acetylcholine as being the transmitter (Whitfield, 1968; Comis & Whitfield, 1968; Comis & Davies 1969a, b). Neurones which could be inhibited by olivary stimulation in the present study could also be inhibited by locally applied noradrenaline and excited by acetylcholine. Attempts to block olivo-cochlear nucleus inhibition by strychnine have so far failed. The question arose of whether partly or wholly, this inhibition is mediated directly on to the cochlear nucleus neurone, or whether all that was being seen was a reduction in the cochlear nucleus input as a result of an inhibitory process taking place at the cochlea. Such an inhibitory effect could arise by activating the neurones from which the uncrossed fibres of the olivo-cochlear bundle originate (Rasmussen, 1960, 1967). It has been firmly established that stimulation of the olivo-cochlear bundle reduces the activity of single auditory nerve fibres (Fex, 1962). Stimulation of the crossed and uncrossed olivo-cochlear fibres reduces the size of the cochlear N, responses (Galambos, 1956; Desmedt & Monaco, 196 1; Desmedt & LaGrutta, 1963; Fex, 1967). Therefore, any inhibitory effects occurring at the cochlea must be accounted for. This problem was approached in the following way. A neurone in the cochlear nucleus, which was found to be inhibited by olivary stimulation, was excited by the local application of glutamate or acetylcholine. The inhibitory stimulus was applied during the period of increased firing. If the firing rate of the neurone could be reduced to zero or at least reduced by an amount greater than the resting activity, it is very likely that in such a case the inhibitory effect is acting directly on the cochlear nucleus neurone. Fig. 2 illustrates such a case.

CENTRIFUGAL INHIBITORY PROCESSES Caseswere encountered, however, where increased neural activity induced as stated above could not be adequately inhibited via the olivocochlear nucleus pathway.

5 CENTRIFUGAL INHIBITORY PROCESSES Caseswere encountered, however, where increased neural activity induced as stated above could not be adequately inhibited via the olivocochlear nucleus pathway. Application of strychnine 1: 400 on the round window (Desmedt & LaGrutta, 1963) in some of these cases abolished the inhibitory effect on the activity of the cochlear nucleus neurone (Fig. 3). It was also observed that in some of these cases the cochlear N1 response to a 100 pssec click could be reduced by ipsilateral olivary stimulation (Fig. 4) as also described by Desmedt & LaGrutta (1963) and by Fex (1967). It would appear, therefore, that two independent inhibitory pathways can be activated following olivary stimulation; one set offibres proceeds towards the cochlear nucleus of the same side, and another set of fibres proceeds towards the cochlea. The fact that inhibition in the cochlear nucleus resulting from olivary stimulation is strychnine resistant supports the hypothesis that we are dealing with two chemically and perhaps even functionally distinct inhibitory pathways. 1s1ll l Iu 1M I 1111 fi IItI 1 I I I 11 I1t IIIltiS b~l & 1 II 1 1 II 11d1 I-11 fllitll. II, II1 755 Ilidilisfillt I cosinletttal "i. 1 sec 2 mv Fig. 2. The top trace shows the inhibitory effect of ipsilateral olivary stimulation on a spontaneously firing cochlear nucleus neurone. The bottom trace shows that the inhibitory stimulus is still effective when the spontaneous firing of the neurone is increased following the local application of acetylcholine. The black bar indicates the period of olivary stimulation. Comics & Davies (1969 a, b) showed that hemicholinium (H.C. 3) can block the excitatory effect of olivary stimulation on cochlear nucleus neurones, following pulsatile stimulation of the superior olive. It was suggested by these authors that this was due to the depletion of acetylcholine in the centrifugal cholinergic nerve endings. In one experiment hemicholinium was applied to a cochlear nucleus neurone in an effort to block excitation of the neurone caused by stimulation of the ipsilateral olivary S-segment following the method of Comis & Davies (1969 a, b). Fig. 5 shows that having abolished the excitatory

6 756 S. D. COMIS j2 mv I sec Fig. 3. The top trace shows the normal inhibitory effect on a cochlear nucleus neurone, of ipsilateral olivary stimulation. The bottom trace shows the abolition of the inhibitory effect following the application of strychnine (1: 400) on the round window. The black bar indicates the period of olivary stimulation. p p mv 10 msec Fig. 4. The effect of ipsilateral olivary stimulation on the N1 response to a 100 sse click. The middle record shows the N1 response during olivary stimulation. The top and bottom records show the N1 response before and after olivary stimulation respectively.

7 CENTRIFUGAL INHIBITORY PROCESSES 757 effect of olivary stimulation, subsequent olivary stimulation had an inhibitory effect on the same neurone and with the stimulating electrode position unchanged. A possible interpretation of this result is that in this case a dominant excitatory action was abolished thus revealing an underlying simultaneous inhibitory effect. On the basis of this observation it would appear that in some neurones in the cochlear nucleus, there is convergence of excitatory and inhibitory centrifugal actions having a common anatomical origin. -ml nil[ nil dal I 1111iffludill" IdIIIIII A th-i I III flik T A- I III F I III I IIIPI [I'll- Twu 41 a " III [III I I I III 11 IF I I I -Ik iiiiuiip qi~ D II i airnj u'a'ijff i tipi IsII I ij flqmqjw'" I fhai'i I 5l& s' 1r mym 1 sec Fig. 5. The top record shows the response of a cochlear nucleus neurone to a tone T (3 4 khz, 5 db above threshold). The middle trace shows the excitatory effect of ipsilateral olivary stimulation. Note the short initial silent period. The bottom trace shows the inhibitory effect of the same olivary stimulus following the local application of hemicholinium and pulsatile olivary stimulation. The black bar indicates the period of olivary stimulation. In Fig. 5 it can also be seen that before the application of hemicholinium, olivary stimulation resulted in an initial short silent period. This could be due to an initial inhibitory barrage occurring before the excitatory process invaded the neurone. Such interactions between inhibitory and excitatory processes could explain, partly at least, the long latencies which are associated with centrifugal excitation of cochlear nucleus neurones (Comis & Whitfield, 1968). DISCUSSION The results indicate that two separate groups of fibres leave the region of the olivary S-segment for the ipsilateral cochlear nucleus. One group originating mainly from the medial portion of the S-segment is excitatory in function while the second group which originates from neurones lying

8 758 S. D. COMIS within, or dorsal to, the lateral part of the S-segment is inhibitory. Cochlear nucleus neurones have also been shown to respond by excitation or inhibition following stimulation of the crossed component of the olivocochlear bundle (Starr & Wernick, 1968), and of the contralateral nuclei of the lateral lemniscus (Comis & Whitfield, 1968). The olivo-cochlear bundle and the nuclei of the lateral lemniscus are known to make direct connexion with the cochlear nucleus (Rasmussen, 1960, 1964, 1967). We have no data at, present regarding the pharmacological identity of neurones which give rise to inhibitory fibres terminating within the cochlear nucleus. There is increasing evidence that the olivo-cochlear inhibitory effect may be mediated by acetylcholine (Schunecht, Churchill & Doran, 1959; Gisselsson, 1960; Amaro, Guth & Wanderlinder, 1966; Daigneault & Brown, 1966; Fex, 1968; Guth & Amaro, 1969) although some evidence does not support this hypothesis (Sohmer & Feinmesser, 1963; Desmedt & LaGrutta, 1963; Katsuki, Tanaka & Miyoshi, 1965; Tanaka & Katsuki, 1966). It is known that inhibitory effects in the cochlea resulting from stimulation of the crossed and the uncrossed olivo-cochlear bundle can be blocked by strychnine (Desmedt & Monaco, 1961; Desmedt & LaGrutta, 1963). Strychnine is not a strong anticholinergic substance. A hypothesis which has been proposed to explain the action of strychnine on the cochlea suggests that this compound may be acting by preventing the release of acetylcholine from nerve endings of the olivo-cochlear bundle within the cochlea (McKinstry & Koelle, 1967). The fact that strychnine abolishes the inhibitory process at the cochlea but not in the cochlear nucleus suggests that there are two pharmacologically distinct inhibitory pathways arising from the superior olive. No compound can be proposed at present as a possible inhibitory transmitter responsible for the olivo-cochlear nucleus inhibitory process. Among compounds with an inhibitory action on cochlear nucleus are y-aminobutyric acid (GABA) (Whitfield & Comis, 1966), glycine (S. D. Comis, unpublished observations) and noradrenaline (Comis & Whitfield, 1968). It has been shown that noradrenaline-containing nerve terminals exist within the cochlear nucleus of the cat (Comis & Whitfield, 1968), but the origin of these terminals remains obscure. Attempts to trace the fibres giving rise to these terminals by means of the fluorescence method (Falck & Owman, 1965) after L-Dopa administration have not yet been fruitful. In the rat it appears that noradrenergic fibres terminating in the dorsal cochlear nucleus may originate in the region of the medulla and pons (Anden, Fuxe & Larsson, 1966). Clearly, much more work is required before any of the above compounds which have inhibitory actions in the cochlear nucleus can with any degree of certainty be connected with the olivocochlear nucleus inhibitory process.

9 CENTRIFUGAL INHIBITORY PROCESSES 759 Capps & Ades (1968) sectioned the olivo-cochlear bundle between the facial genua in the squirrel monkey. They were able to demonstrate that the animal's ability to perform frequency discriminations was markedly impaired following such operations. Dewson (1968) also demonstrated with a different training technique, that similar section of the olivocochlear bundle rendered squirrel monkeys less able to pick out a stimulus in the presence ofwhite noise. It has been pointed out by Rasmussen (1967) that in the cat, section of the olivo-cochlear bundle between the facial genua also involves the degeneration of collaterals of the olivo-cochlear bundle which enter the cochlear nucleus. It is possible therefore that many other centrifugal pathways share such functions as have been assigned to the olivo-cochlear bundle above. However, more experiments are necessary before we can be sure about the function of the olivo-cochlear nucleus centrifugal pathways. I wish to thank Dr I. C. Whitfield for his advice and valuable criticism. I am grateful to Mr T. L. Hayward for his highly skilled technical assistance. The financial support of the S.R.C. is gratefully acknowledged. REFERENCES AMARO, J., GumH, P. S. & WANDENFlDER, L. (1966). Inhibition of auditory nerve action potentials by acetylcholine and physostigmine. Br. J. Pharmac. Chemother. 28, AND1N, N.-E., FuEm, K. & LAnsSON, K. (1966). Effect of large mesencephalicdiencephalic lesions on the noradrenaline, dopamine and 5-hydroxytryptamine neurons of the central nervous system. Experientia 22, CAPPs, M. J. & ADES, H. W. (1968). Auditory frequency discrimination after transection of the olivo-cochlear bundle in squirrel monkeys. Expi Neurol. 21, CoMrs, S. D. & DAvIEs, W. E. (1969a). The effect of hemicholinium on a centrifugal auditory pathway. J. Physiol. 200, P. Cows, S. D. & DAvIEs, W. E. (1969b). Acetylcholine as a transmitter in the cat auditory system. J. Neurochem. 16, Comrs, S. D., EvAns, E. F. & Wmlruis, I. C. (1964). A microtap for controlling the application of drugs to single neurones. J. Phyaiol. 173, 4-6P. Corns, S. D. & WEITFELD, I. C. (1968). Influence of centrifugal pathways on unit activity in the cochlear nucleus. J. Neurophysiol. 31, DAiGNEAuLT, E. A. & BROWN, R. D. (1966). Acetylcholine suppression of the N, component of round window recorded cochlear potentials. Arch8 int. Pharmacodyn. Thdr. 162, DESMEDT, J. E. & LAGRuTTA, V. (1963). Function of the uncrossed efferent olivocochlear fibres in the cat. Nature, Lond. 200, DESMEDT, J. E. & MoNAco, P. (1961). Mode of action of the efferent olivo-cochlear bundle on the inner ear. Nature, Lond. 192, DEwsoN, J. H. (1968). Efferent olivo-cochlear bundle: some relationships to stimulus discrimination in noise. J. Neurophysiol. 31,

760 S. D. COMIS FALCK, B. & OwMAN, C. (1965). A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines. Acta Univ. lund. sect. ii, 5-23.

10 760 S. D. COMIS FALCK, B. & OwMAN, C. (1965). A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic monoamines. Acta Univ. lund. sect. ii, FEx, J. (1962). Auditory activity in centrifugal and centripetal fibres in cat. Acta physiol. scand. 55, suppl. 189, FEX, J. (1967). Efferent inhibition in the cochlea related to hair-cell dc-activity: Study of postsynaptic activity of the crossed olivo-cochlear fibres in the cat. J. acoust. Soc. Am. 41, FEX, J. (1968). Efferent inhibition in the cochlea by the olivo-cochlear bundle. In Hearing Mechanisms in Vertebrates, ed. DE REUCK, A. V. S. & KNIGHT, J., pp London: Churchill. GALAMMos, R. (1956). Suppression of auditory nerve activity by stimulation of efferent fibres to cochlea. J. Neurophysiol. 19, GIsSELSsoN, L. (1960). Effect on microphonics of acetylcholine injected into the endolymphatic space. Acta oto-lar. 51, GuTn, P. S. & AMARo, J. (1969). A possible cholinergic link in olivo-cochlear inhibition. Int. J. Neuropharmac. 8, KATSUKI, Y., TANAKA, Y. & Miyosm, T. (1965). Action of acetylcholine on cochlear responses. Nature, Lond. 207, MCKINSTRY, D. N. & KOELLE, G. B. (1967). Inhibition of release of acetylcholine by strychnine and its implications regarding transmission by the ohvo-cochlear bundle. Nature, Lond. 213, RASMUSSEN, G. L. (1960). Efferent fibers of the cochlear nerve and cochlear nucleus. In Neural Mechanism of the Auditory and Vestibular Systems, ed. RASMUSSEN, G. L. & WnwDLE, W. F., pp Springfield: Thomas. RASMUSSEN, G. L. (1964). Anatomic relationships of the ascending and descending auditory systems. In Neurological Aspects of Auditory and Vestibular Disorders, ed. FIELDs, W. S. & ALFORD, R. R., pp Springfield: Thomas. RASMUSSEN, G. L. (1967). Efferent connections of the cochlear nucleus. In Sensorineural Hearing Processes and Disorders, ed. GRAHAM, A. B., pp Boston: Little Brown. Scnu1EcHT, H. F., CHURCH*TI, J. A. & DORAN, R. (1959). The localization of acetylcholinesterase in the cochlea. Arche Otolar. 69, SOHMER, H. & FEINNESSER, M. (1963). Studies on the influence of acetylcholine, eserine and atropine on cochlear potentials in the guinea pig and the cat. Archs int. Pharmacodyn. Ther. 144, STARR, A. & WERNICK, J. S. (1968). Olivo-cochlear bundle stimulation: effects on spontaneous and tone-evoked activities of single units in cat cochlear nucleus. J. Neurophysiol. 31, STOPP, P. E. & W=.=xLD, I. C. (1963). The influence of microelectrodes on neuronal discharge patterns in the auditory system. J. Physiol. 167, TANAKA, Y. & KATSI, Y. (1966). Pharmacological investigations of cochlear responses and of olivo-cochlear inhibition. J. Neurophysiol. 29, WxrriELD, I. C. (1968). Centrifugal control mechanisms of the auditory pathway. In Hearing Mechanisms in Vertebrates, ed. DE REUCK, A. V. S. & KNIGHT, J., pp London: Churchill. WHxFrLD, I. C. & Comis, S. D. (1966). The interaction of centrifugal and centripetal stimulation on neurones of the cochlear nucleus. Final Report, Part II, AF EOAR, pp

INHIBITION OF AUDITORY NERVE ACTION POTENTIALS BY ACETYLCHOLINE AND PHYSOSTIGMINE

Br. J. Pharmac. Chemother. (1966), 28, 207-211. INHIBITION OF AUDITORY NERVE ACTION POTENTIALS BY ACETYLCHOLINE AND PHYSOSTIGMINE BY J. AMARO, P. S. GUTH AND L. WANDERLINDER From the Department of Pharmacology,