440
Brain Research, 159 (1978) 440-444 © Elsevier/North-Holland Biomedical Press
a-Bungarotoxin blocks reversibly cholinergic inhibition in the cochlea
JORGEN FEX and JOE C. ADAMS
Laboratory of Neuro-otolarjmgology, National Institute of Neurological ahd Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) (Accepted August 31st, 1978)
a-Bungarotoxin (aBTX), a neurotoxin obtained from the venom of the snake
Bungarus multicinctus, irreversibly blocks neuromuscular transmissiona,Z4, 26 and is widely used as a ligand of nicotinic cholinergic receptors. There is evidence that the postsynaptic receptors of the crossed olivocochlear efferent nerve fibers in the cochlea are cholinergiO ,2,1z-14,17,27; it has not been determined whether they may be nicotinic or muscarinic. When repetitively stimulated the crossed efferents inhibit auditory nerve activity 1°,16, increase cochlear microphonics6, 9 and evoke a slow potential 11,13. We report here that aBTX blocks these three effects of the crossed cochlear efferents and that most, if not all, of the block is reversible. We used 44 cats which were anesthetized with pentobarbital sodium, paralyzed with gallamine triethiodide, and artificially respirated. The crossed chochlear efferents were stimulated electrically in the floor of the fourth ventricle, cochlear potentials evokes by such stimulation, and by sound presented to the ear, were recorded from the rim of the round window with a Pt-Ir electrode, 0.1 mm in diameter. Most of the membrane of the round window was removed, by cauterization to avoid bleeding. The head of the animal was tilted to avoid spontaneous refill of fluid in the tympanic scala. As a control that conditions were stable before aBTX was applied, a series of cochlear potentials were recorded between repeated substitutions of perilymph in the basal turn of the tympanic scala with artificial perilymph (mM: NaC1, 140; NaHCOa, 12.5; KC1, 3.5; CaC12, 1.3; MgCI2, 1.14 and glucose, 3.4; bubbled with 9 5 ~ 02 q- 5 ~ Co2 at 38-40 °C, transferred to ice and reheated to 38-40 °C immediately before use). aBTX was added to the artificial perilymph to concentrations of 1.0-10.0/~M (8-80/~g/ml). When aBTX was used, bovine serum albumen was added to a concentration of 0.2 ~ . Two batches of aBTX were used, giving the same results. One batch was perified in our laboratory from the venom of Bungarus multicinctus through a method similar to that of Mebs et al.25 (Robert J. Wenthold, personal communication). The venom was obtained from the Miami Serpentarium Laboratories as was the other batch of purified aBTX. No change in the potency of the aBTX purified here, as judged by the experimental results, was found during the period of 1-7 months after purification. We found that aBTX applied repeatedly to the cochlea at a concentration of 1.0 /~M partially blocked inhibition of the response of the auditory nerve to sound stimuli
441
o 1
1 taM
Wash
10 taM
Wash
t
l
i
t
.
0
1
~
"o
®
•- >
(40 dB)
0.s ~ ! '-~t
J G
~
0
~-
/
~, I
_ _,~_ . 120 dB)
1
2
3
4
Time (h) Fig. 1. Effect of aBTX on efferent inhibition of sound-evoked activity of the auditory nerve and on the efferently evoked slow cochlear potential; cat; results from one experiment. The upper two traces (40 dB) represent the peak-to-peak amplitude of the sound (40 dB above threshold)-evoked action potential of the auditory nerve plotted against time, with 100 msec of repetitive stimulation of crossed cochlear efferents preceding the sound by 4 msec (open circles), and with no such stimulation (filled circles). Similarly, the lowertwo traces (20 dB) represent such amplitudes generated with sound at 20 dB above threshold. The vertical distance between filled and open circle traces represents efferent inhibition at the 40 dB sound level and at the 20 dB level, respectively. The dashed line with open squares (eft) represents t he slow potential evoked by repetitive stimulation of the crossed cochlearefferents, amplitude plotted against time. At the first arrow (1 #M), 1/~M aBTX in artificial perilymph was applied to the basilar turn of the tympanic scala. At the second arrow (Wash), artificial perilymph was applied to wash out aBTX; at the third (10/~M), 10 FM aBTX was applied; at the fourth (Wash), washout of aBTX. For application of fluid, the basal turn of the tympanic scala was emptied by suction and refilled with artificial perilymph (about 8/d) 3 times within 2 min and at intervals of 15 min, except for washout of aBTX. For washout, fluid was exchanged 7, 5 and 3 times, during 5, 3, and 2 rain, respectively, with intervals of 5-7 min between substitutions, after which the application schedule was followed. Sound (clicks) was produced by a 0.1 msec square wave fed into a 1 in. condenser microphone.
at 40 dB above threshold (Fig. 1; 40 dB; between first two arrows). At this concentration of aBTX, but with sound stimuli only 20 dB above threshold, the inhibition was only slightly blocked (Fig. 1 ; 20 dB; between first two arrows) or not blocked at all. Most or all of the efferently evoked slow potential was blocked (Fig. 1 ; eft; between first two arrows). With 10/~M aBTX and with sound stimuli at 40 dB above threshold, most (Fig. 1 ; 40 dB; between last two arrows) or all of the inhibition was blocked. At this concentration, but with sound stimuli only 20 dB above threshold (Fig. 1 ; 20 dB; between last two arrows), results were spread between no block or total block of inhibition, aBTX at 10/~M always totally blocked the efferently evoked slow potential (Fig. 1 ; eft; between last two arrows). Fig. 1 illustrates typical observations on the time course of the blocks and reversals. The block of inhibition was not completely reversed with one hour washout of the toxin but such reversal as is illustrated (Fig. 1 ; 40 dB; between second and third arrows) or more was seen several times. The block of the efferently evokes slow potential was completely reversed in many experiments, even after 1 h of total block with 10 # N aBTX. The effect of aBTX on the efferently caused increase of the cochlear microphonics was studied in some detail in only 4 experiments, using tone bursts of 1.0-1.1
442 kHz as sounds stimuli. The increase of the cochlear microphonics was blocked later than was the efferently evoked slow potential and restored earlier. It was shown in a recent study 7, using experimental exchange of fluids in the cat chochlea, that artificial perilymph may be diluted by cerebrospinal fluid seeping through the cochlear aqueduct into the basal turn of the cochlea. In the present study such seeping caused outflow of fluid through the round window of the chochlea during preparations for the experiment. When tilting the head of the cat had stopped the outflow we assumed that dilution of artificial perilymph was negligible during the experiment. It also may be assumed that the accessibility of aBTX to synapses of the crossed efferents in the organ of Corti was good in the present study since block induced by aBTX often developed fast and could be washed out quickly. Therefore, the high concentration of aBTX necessary in our experiments to induce a functional block probably cannot be explained in terms of unintentional dilution of aBTX solution or lack of penetration of the toxin. Conclusive evidence shows that in the cat the crossed cochlear efferents terminate with large endings on outer hair cells z2,32. The evidence that in the cat crossed efferents form synapses elsewhere in the cochlea is not compelling 31-33. The possibility that inhibition in the cat cochlea through the crossed efferents can be explained in terms of efferent synapses only with outer hair cells was recently discussed in detail 7,1a. Suggestive but inconclusive evidence for more than one kind of synapse of crossed cochlear efferents were previous finding~ 7,1a that changes of the efferently evoked slow potential and of the inhibition did not run in parallel. Similarly suggestive but inconclusive are the present results that the efferently evoked slow potential often was blocked by aBTX with no block of inhibition (at the sound level of 20 dB above treshold). Available evidence indicates that the effects of repetitively stimulated crossed cochlear efferents in evoking a slow potential and increasing the cochlear microphonics are mediated through the large efferent synapses on outer hair cells7,13. Therefore, the present findings indicate that aBTX blocks receptors at efferent synapses with outer hair cells, leaving the possibility open that there are receptor sites for binding of aBTX elsewhere in the organ of Corti. Non-specific binding of aBTX has been reported (see ref. 28) and it has been shown that aBTX binds to the axonal cholinergic binding macromolecule of lobster ~ and horseshoe crab is leg nerve membrane. However, no extrasynaptic effect of aBTX has been reported that can explain a functional block of neuronally induced activity in vertebrates. Several unsuccessful attempts to cause functional block with aBTX at neuronal nicotinic receptors to which aBTX had access have been reported a,s,29. aBTX was applied electrophoretically and by pressure ejection close to Renshaw cells of the cat without significantly blocking cholinergic excitation of these cells s. In two recent studies of sympathetic neurons that bind aBTX, aBTX did not functionally block the cholinergic receptors of these cells and in both studies a distinction between the ACh receptor and the aBTX receptor was demonstrated a,~9. On the other hand, Freeman ~5 has reported that aBTX causes an irreversible block at nicotinic synapses in the toad tectum. Also of interest here is a study of Survey et al. a°. They reported a reversible reaction with aBTX in a small fraction, about 1 ~ , of the total population at
443 the normal neuromuscular endplate region of skeletal muscle of the mouse; a small e n d p l a t e p o t e n t i a l b u t n o t a twitch r e t u r n e d after w a s h o u t o f a B T X for several hours. In A p l y s i a , three types o f A C h receptors o f central neurons are k n o w n 2°. One t y p e mediates i n h i b i t i o n t h r o u g h a n increase o f chloride p e r m e a b i l i t y 19 as the evidence indicates t h a t p o s t s y n a p t i c receptors o f the crossed cochlear efferents d o 7. This t y p e o f A p l y s i a A C h r e c e p t o r only, o u t o f the three, was f o u n d to be functionally blocked with a B T X in a study by K e h o e et al. 21. A c o n c e n t r a t i o n o f 10 ~ M o f a B T X was needed for the b l o c k to a p p e a r ; the b l o c k was reversible 21. These findings have recently been confirmed ( D a v i d O. C a r p e n t e r , p e r s o n a l c o m m u n i c a t i o n ) . O u r present findings t h a t a B T X functionally blocks crossed cochlear efferents s u p p o r t previous evidence t h a t these efferents are cholinergic, T h e reversibility o f the b l o c k indicates t h a t these receptors are different f r o m A C h receptors at o t h e r v e r t e b r a t e synapses.
We are grateful to Dr. Robert J. Wenthold for critical review of the manuscript, to William D. Livingston for assembling and programming the computer, to Michael J. Frye for technical assistance and to Judy F. Virts for typing. 1 Bobbin, R. P. and Konishi, T., Acetylcholine mimics crossed olivocochlear bundle stimulation, Nature New Biok, 231 (1971) 222-223. 2 Bobbin, R. P. and Konishi, T., Action of cholinergic and antichohnergic drugs at the crossed olivocochlear bundle-hair cell junction, Acta oto-laryng. (Stockh.), 77 (1974) 56-65. 3 Carbonetto, S.T.,Fambrough,D. M. and Muller, K. J.,Nonequivalenceofbungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 75 0978) 1016-1020. 4 Chang, C. C. and Lee, C. Y., Isolation ofneurotoxins from the venom ofGungarus multicinctus and their modes of neuromuscular blocking action, Arch. int. Pharmacodyn., 144 (1963) 241-257. 5 Denburg, J. L., Eldefrawi, M. E. and O'Brien, R. D., Maeromolecules from lobster axon membranes that bind cholinergic ligands and local anesthetics, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 177-181. 6 Desmedt, J. E. and Monaco, P., Mode of action of the efferent olivo-cochlear bundle on the inner ear, Nature (Land.), 193 0961) 1263-1265. 7 Desmedt, J. E. and Robertson, D., Ionic mechanism of the efferent olivo-cochlearinhibition studied by cochlear perfusion in the cat, J. Physiol. (Lond.), 247 (1975) 407-428. 8 Duggan, A. W., Hall, J. G. and Lee, C. Y., Alpha-bungarotoxin, cobra neurotoxin and excitation of Renshaw cells by acetylcholine, Brain Research, 107 (1976) 166-170. 9 Fex, J., Augmentation of the cochlear microphonics by stimulation of efferent fibres to cochlea, Acta oto-laryng. ( Stockh.) , 50 (1959) 540-541. 10 Fex, J., Auditory activity in centrifugal and centripetal cochlear fibres in cat. A study of a feedback system, Actaphysiol. scand., 55, Suppl. 189 (1962) 1-68. 11 Fex, J., Calcium action at an inhibitory synapse, Nature (Lot,d.), 213 (1967) 1233-1234. 12 Fex, J., Efferent inhibition in the cochlea by the olivo-cochlear bundle. In A. V. S. De Reuck and J. Knight (Eds.), Hearing Mechanisms in Vertebrates, Churchill, London, 1968, pp. 169-181. 13 Fex, J.,Neuropharmacologyandpotentialsoftheinnerear. InA. R. Moller(Ed.),BasicMechanisms in Hearing, Academic Press, New York, 1973, pp. 377-421. 14 Fex, J. and Wenthold, R. J., Choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the cochlea and cochlear nucleus of the guinea pig, Brain Research, 109 (1976) 575585. 15 Freeman, J. A., Possible regulatory function of acetylcholine receptor in maintenance of rectinotectal synapses, Nature (Lond.), 269 (1977) 218-222. 16 Galambos, R., Suppression of auditory nerve activity by stimulation of efferent fibers to cochlea, J. Neurophysiol., 19 (1956) 424-437. 17 Jasser, A. and Guth, P. S., The synthesis of acetylcholine by the olivo-cochlear bundle, J. Neurochem., 20 (1973) 45-53.
444 18 Jones, S. W., Galasso, R. T. and O'Brien, R. D., Nicotine and a-bungarotoxin binding to axonal and non-neural tissues, J. Neurochem., 29 (1977) 803-809. 19 Kehoe, J., Ionic mechanisms of a two-component cholinergic inhibition in Aplysia neurones, J. Physiol. (Lond.), 225 (1972) 85-114. 20 Kehoe, J., Three acetylcholine receptors in Aplysia neurones, J. Physiol. (Lond.), 225 (1972) 115146. 21 Kehoe, J., Sealock, R. and Bon, C., Effects of a-toxins from Bungarus multicinctus and Bungarus caeruleus on cholinergic responses in Aplysia neurones, Brain Research, 107 (1976) 527-540. 22 Kimura, R. and Wers~ll, J., Termination of the olivo-cochlear bundle in relation to the outer hair cells of the organ of Corti in guinea pig, Acta oto-laryng. (Stockh.), 55 (1962) 11-32. 23 Konishi, T. and Slepian, J., Summating potential with electrical stimulation ofcrossed olivocochlear bundles, Science, 172 (1971) 483-484. 24 Lee, C. Y., Tseng, L. F. and Chiu, T. H., Influence of denervation on localization of neurotoxins from Clapid venoms in rat diaphragm, Nature (Lond.), 215 (1967) 1177-1178. 25 Mebs, D., Narita, K., Iwanaga, S., Samejima, Y. and Lee, C.-Y., Purificat ion, properties and amino acid sequence of a-bungarotoxin from the venom of Bungarus multicinctus, Hoppe-Seyler's Z. Physiol. Chem., 353 (1972) 243-262. 26 Miledi, R. and Potter, L. T., Acetylcholine receptors in muscle fibres, Nature (Lond.), 233 (1971) 599-603. 27 Norris, C. H. and Guth, P. S., The release of acetylcholine (ACh) by the crossed olivo-cochlear bundle (COCB), Acta oto-laryng. (Stockh.), 77 (1974) 318-326. 28 O'Brien, R. D., Thompson, W. R. and Gibson, R. E., A comparison of acetylcholine and a-bungarotoxin binding to soluble Torpedo receptor. In E. de Robertis and J. Schacht (Eds.), Neurochemistry of Cholinergic Receptors, Raven Press, New York, 1974, pp. 49-62. 29 Patrick, J. and Stallcup, W. B., Immunological distinction between acetylcholine receptor and the a-bungarotoxin-binding component on sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4689-4692. 30 Sarvey, J. M., Albuquerque, E. X., Eldefrawi, A. T. and Eldefrawi, M., Effects of a-bungarotoxin and reversible cholinergic ligands on normal and denervated mammalian skeletal muscle, Membrane Biochem., 1 (1978) 131-157. 31 Spoendlin, H., Innervation patterns in the organ of Corti of the cat, Acta oto-laryng. (Stockh.), 67 (1969) 239-254. 32 Spoendlin, H., Structural basis of peripheral frequency analysis. In R. Plomp and F. G. Smoorenburg (Eds.), Frequency Analysis andPeriodicity Detection in Hearing, Sij thoff, Leiden, 1970, pp. 2-40. 33 Spoendlin, H., Neuroanatomical basis of cochlear coding mechanisms, Audiology, 14 (1975) 383407.
Brain Research, 159 (1978) 440-444 © Elsevier/North-Holland Biomedical Press
a-Bungarotoxin blocks reversibly cholinergic inhibition in the cochlea
JORGEN FEX and JOE C. ADAMS
Laboratory of Neuro-otolarjmgology, National Institute of Neurological ahd Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Md. 20014 (U.S.A.) (Accepted August 31st, 1978)
a-Bungarotoxin (aBTX), a neurotoxin obtained from the venom of the snake
Bungarus multicinctus, irreversibly blocks neuromuscular transmissiona,Z4, 26 and is widely used as a ligand of nicotinic cholinergic receptors. There is evidence that the postsynaptic receptors of the crossed olivocochlear efferent nerve fibers in the cochlea are cholinergiO ,2,1z-14,17,27; it has not been determined whether they may be nicotinic or muscarinic. When repetitively stimulated the crossed efferents inhibit auditory nerve activity 1°,16, increase cochlear microphonics6, 9 and evoke a slow potential 11,13. We report here that aBTX blocks these three effects of the crossed cochlear efferents and that most, if not all, of the block is reversible. We used 44 cats which were anesthetized with pentobarbital sodium, paralyzed with gallamine triethiodide, and artificially respirated. The crossed chochlear efferents were stimulated electrically in the floor of the fourth ventricle, cochlear potentials evokes by such stimulation, and by sound presented to the ear, were recorded from the rim of the round window with a Pt-Ir electrode, 0.1 mm in diameter. Most of the membrane of the round window was removed, by cauterization to avoid bleeding. The head of the animal was tilted to avoid spontaneous refill of fluid in the tympanic scala. As a control that conditions were stable before aBTX was applied, a series of cochlear potentials were recorded between repeated substitutions of perilymph in the basal turn of the tympanic scala with artificial perilymph (mM: NaC1, 140; NaHCOa, 12.5; KC1, 3.5; CaC12, 1.3; MgCI2, 1.14 and glucose, 3.4; bubbled with 9 5 ~ 02 q- 5 ~ Co2 at 38-40 °C, transferred to ice and reheated to 38-40 °C immediately before use). aBTX was added to the artificial perilymph to concentrations of 1.0-10.0/~M (8-80/~g/ml). When aBTX was used, bovine serum albumen was added to a concentration of 0.2 ~ . Two batches of aBTX were used, giving the same results. One batch was perified in our laboratory from the venom of Bungarus multicinctus through a method similar to that of Mebs et al.25 (Robert J. Wenthold, personal communication). The venom was obtained from the Miami Serpentarium Laboratories as was the other batch of purified aBTX. No change in the potency of the aBTX purified here, as judged by the experimental results, was found during the period of 1-7 months after purification. We found that aBTX applied repeatedly to the cochlea at a concentration of 1.0 /~M partially blocked inhibition of the response of the auditory nerve to sound stimuli
441
o 1
1 taM
Wash
10 taM
Wash
t
l
i
t
.
0
1
~
"o
®
•- >
(40 dB)
0.s ~ ! '-~t
J G
~
0
~-
/
~, I
_ _,~_ . 120 dB)
1
2
3
4
Time (h) Fig. 1. Effect of aBTX on efferent inhibition of sound-evoked activity of the auditory nerve and on the efferently evoked slow cochlear potential; cat; results from one experiment. The upper two traces (40 dB) represent the peak-to-peak amplitude of the sound (40 dB above threshold)-evoked action potential of the auditory nerve plotted against time, with 100 msec of repetitive stimulation of crossed cochlear efferents preceding the sound by 4 msec (open circles), and with no such stimulation (filled circles). Similarly, the lowertwo traces (20 dB) represent such amplitudes generated with sound at 20 dB above threshold. The vertical distance between filled and open circle traces represents efferent inhibition at the 40 dB sound level and at the 20 dB level, respectively. The dashed line with open squares (eft) represents t he slow potential evoked by repetitive stimulation of the crossed cochlearefferents, amplitude plotted against time. At the first arrow (1 #M), 1/~M aBTX in artificial perilymph was applied to the basilar turn of the tympanic scala. At the second arrow (Wash), artificial perilymph was applied to wash out aBTX; at the third (10/~M), 10 FM aBTX was applied; at the fourth (Wash), washout of aBTX. For application of fluid, the basal turn of the tympanic scala was emptied by suction and refilled with artificial perilymph (about 8/d) 3 times within 2 min and at intervals of 15 min, except for washout of aBTX. For washout, fluid was exchanged 7, 5 and 3 times, during 5, 3, and 2 rain, respectively, with intervals of 5-7 min between substitutions, after which the application schedule was followed. Sound (clicks) was produced by a 0.1 msec square wave fed into a 1 in. condenser microphone.
at 40 dB above threshold (Fig. 1; 40 dB; between first two arrows). At this concentration of aBTX, but with sound stimuli only 20 dB above threshold, the inhibition was only slightly blocked (Fig. 1 ; 20 dB; between first two arrows) or not blocked at all. Most or all of the efferently evoked slow potential was blocked (Fig. 1 ; eft; between first two arrows). With 10/~M aBTX and with sound stimuli at 40 dB above threshold, most (Fig. 1 ; 40 dB; between last two arrows) or all of the inhibition was blocked. At this concentration, but with sound stimuli only 20 dB above threshold (Fig. 1 ; 20 dB; between last two arrows), results were spread between no block or total block of inhibition, aBTX at 10/~M always totally blocked the efferently evoked slow potential (Fig. 1 ; eft; between last two arrows). Fig. 1 illustrates typical observations on the time course of the blocks and reversals. The block of inhibition was not completely reversed with one hour washout of the toxin but such reversal as is illustrated (Fig. 1 ; 40 dB; between second and third arrows) or more was seen several times. The block of the efferently evokes slow potential was completely reversed in many experiments, even after 1 h of total block with 10 # N aBTX. The effect of aBTX on the efferently caused increase of the cochlear microphonics was studied in some detail in only 4 experiments, using tone bursts of 1.0-1.1
442 kHz as sounds stimuli. The increase of the cochlear microphonics was blocked later than was the efferently evoked slow potential and restored earlier. It was shown in a recent study 7, using experimental exchange of fluids in the cat chochlea, that artificial perilymph may be diluted by cerebrospinal fluid seeping through the cochlear aqueduct into the basal turn of the cochlea. In the present study such seeping caused outflow of fluid through the round window of the chochlea during preparations for the experiment. When tilting the head of the cat had stopped the outflow we assumed that dilution of artificial perilymph was negligible during the experiment. It also may be assumed that the accessibility of aBTX to synapses of the crossed efferents in the organ of Corti was good in the present study since block induced by aBTX often developed fast and could be washed out quickly. Therefore, the high concentration of aBTX necessary in our experiments to induce a functional block probably cannot be explained in terms of unintentional dilution of aBTX solution or lack of penetration of the toxin. Conclusive evidence shows that in the cat the crossed cochlear efferents terminate with large endings on outer hair cells z2,32. The evidence that in the cat crossed efferents form synapses elsewhere in the cochlea is not compelling 31-33. The possibility that inhibition in the cat cochlea through the crossed efferents can be explained in terms of efferent synapses only with outer hair cells was recently discussed in detail 7,1a. Suggestive but inconclusive evidence for more than one kind of synapse of crossed cochlear efferents were previous finding~ 7,1a that changes of the efferently evoked slow potential and of the inhibition did not run in parallel. Similarly suggestive but inconclusive are the present results that the efferently evoked slow potential often was blocked by aBTX with no block of inhibition (at the sound level of 20 dB above treshold). Available evidence indicates that the effects of repetitively stimulated crossed cochlear efferents in evoking a slow potential and increasing the cochlear microphonics are mediated through the large efferent synapses on outer hair cells7,13. Therefore, the present findings indicate that aBTX blocks receptors at efferent synapses with outer hair cells, leaving the possibility open that there are receptor sites for binding of aBTX elsewhere in the organ of Corti. Non-specific binding of aBTX has been reported (see ref. 28) and it has been shown that aBTX binds to the axonal cholinergic binding macromolecule of lobster ~ and horseshoe crab is leg nerve membrane. However, no extrasynaptic effect of aBTX has been reported that can explain a functional block of neuronally induced activity in vertebrates. Several unsuccessful attempts to cause functional block with aBTX at neuronal nicotinic receptors to which aBTX had access have been reported a,s,29. aBTX was applied electrophoretically and by pressure ejection close to Renshaw cells of the cat without significantly blocking cholinergic excitation of these cells s. In two recent studies of sympathetic neurons that bind aBTX, aBTX did not functionally block the cholinergic receptors of these cells and in both studies a distinction between the ACh receptor and the aBTX receptor was demonstrated a,~9. On the other hand, Freeman ~5 has reported that aBTX causes an irreversible block at nicotinic synapses in the toad tectum. Also of interest here is a study of Survey et al. a°. They reported a reversible reaction with aBTX in a small fraction, about 1 ~ , of the total population at
443 the normal neuromuscular endplate region of skeletal muscle of the mouse; a small e n d p l a t e p o t e n t i a l b u t n o t a twitch r e t u r n e d after w a s h o u t o f a B T X for several hours. In A p l y s i a , three types o f A C h receptors o f central neurons are k n o w n 2°. One t y p e mediates i n h i b i t i o n t h r o u g h a n increase o f chloride p e r m e a b i l i t y 19 as the evidence indicates t h a t p o s t s y n a p t i c receptors o f the crossed cochlear efferents d o 7. This t y p e o f A p l y s i a A C h r e c e p t o r only, o u t o f the three, was f o u n d to be functionally blocked with a B T X in a study by K e h o e et al. 21. A c o n c e n t r a t i o n o f 10 ~ M o f a B T X was needed for the b l o c k to a p p e a r ; the b l o c k was reversible 21. These findings have recently been confirmed ( D a v i d O. C a r p e n t e r , p e r s o n a l c o m m u n i c a t i o n ) . O u r present findings t h a t a B T X functionally blocks crossed cochlear efferents s u p p o r t previous evidence t h a t these efferents are cholinergic, T h e reversibility o f the b l o c k indicates t h a t these receptors are different f r o m A C h receptors at o t h e r v e r t e b r a t e synapses.
We are grateful to Dr. Robert J. Wenthold for critical review of the manuscript, to William D. Livingston for assembling and programming the computer, to Michael J. Frye for technical assistance and to Judy F. Virts for typing. 1 Bobbin, R. P. and Konishi, T., Acetylcholine mimics crossed olivocochlear bundle stimulation, Nature New Biok, 231 (1971) 222-223. 2 Bobbin, R. P. and Konishi, T., Action of cholinergic and antichohnergic drugs at the crossed olivocochlear bundle-hair cell junction, Acta oto-laryng. (Stockh.), 77 (1974) 56-65. 3 Carbonetto, S.T.,Fambrough,D. M. and Muller, K. J.,Nonequivalenceofbungarotoxin receptors and acetylcholine receptors in chick sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 75 0978) 1016-1020. 4 Chang, C. C. and Lee, C. Y., Isolation ofneurotoxins from the venom ofGungarus multicinctus and their modes of neuromuscular blocking action, Arch. int. Pharmacodyn., 144 (1963) 241-257. 5 Denburg, J. L., Eldefrawi, M. E. and O'Brien, R. D., Maeromolecules from lobster axon membranes that bind cholinergic ligands and local anesthetics, Proc. nat. Acad. Sci. (Wash.), 69 (1972) 177-181. 6 Desmedt, J. E. and Monaco, P., Mode of action of the efferent olivo-cochlear bundle on the inner ear, Nature (Land.), 193 0961) 1263-1265. 7 Desmedt, J. E. and Robertson, D., Ionic mechanism of the efferent olivo-cochlearinhibition studied by cochlear perfusion in the cat, J. Physiol. (Lond.), 247 (1975) 407-428. 8 Duggan, A. W., Hall, J. G. and Lee, C. Y., Alpha-bungarotoxin, cobra neurotoxin and excitation of Renshaw cells by acetylcholine, Brain Research, 107 (1976) 166-170. 9 Fex, J., Augmentation of the cochlear microphonics by stimulation of efferent fibres to cochlea, Acta oto-laryng. ( Stockh.) , 50 (1959) 540-541. 10 Fex, J., Auditory activity in centrifugal and centripetal cochlear fibres in cat. A study of a feedback system, Actaphysiol. scand., 55, Suppl. 189 (1962) 1-68. 11 Fex, J., Calcium action at an inhibitory synapse, Nature (Lot,d.), 213 (1967) 1233-1234. 12 Fex, J., Efferent inhibition in the cochlea by the olivo-cochlear bundle. In A. V. S. De Reuck and J. Knight (Eds.), Hearing Mechanisms in Vertebrates, Churchill, London, 1968, pp. 169-181. 13 Fex, J.,Neuropharmacologyandpotentialsoftheinnerear. InA. R. Moller(Ed.),BasicMechanisms in Hearing, Academic Press, New York, 1973, pp. 377-421. 14 Fex, J. and Wenthold, R. J., Choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the cochlea and cochlear nucleus of the guinea pig, Brain Research, 109 (1976) 575585. 15 Freeman, J. A., Possible regulatory function of acetylcholine receptor in maintenance of rectinotectal synapses, Nature (Lond.), 269 (1977) 218-222. 16 Galambos, R., Suppression of auditory nerve activity by stimulation of efferent fibers to cochlea, J. Neurophysiol., 19 (1956) 424-437. 17 Jasser, A. and Guth, P. S., The synthesis of acetylcholine by the olivo-cochlear bundle, J. Neurochem., 20 (1973) 45-53.
444 18 Jones, S. W., Galasso, R. T. and O'Brien, R. D., Nicotine and a-bungarotoxin binding to axonal and non-neural tissues, J. Neurochem., 29 (1977) 803-809. 19 Kehoe, J., Ionic mechanisms of a two-component cholinergic inhibition in Aplysia neurones, J. Physiol. (Lond.), 225 (1972) 85-114. 20 Kehoe, J., Three acetylcholine receptors in Aplysia neurones, J. Physiol. (Lond.), 225 (1972) 115146. 21 Kehoe, J., Sealock, R. and Bon, C., Effects of a-toxins from Bungarus multicinctus and Bungarus caeruleus on cholinergic responses in Aplysia neurones, Brain Research, 107 (1976) 527-540. 22 Kimura, R. and Wers~ll, J., Termination of the olivo-cochlear bundle in relation to the outer hair cells of the organ of Corti in guinea pig, Acta oto-laryng. (Stockh.), 55 (1962) 11-32. 23 Konishi, T. and Slepian, J., Summating potential with electrical stimulation ofcrossed olivocochlear bundles, Science, 172 (1971) 483-484. 24 Lee, C. Y., Tseng, L. F. and Chiu, T. H., Influence of denervation on localization of neurotoxins from Clapid venoms in rat diaphragm, Nature (Lond.), 215 (1967) 1177-1178. 25 Mebs, D., Narita, K., Iwanaga, S., Samejima, Y. and Lee, C.-Y., Purificat ion, properties and amino acid sequence of a-bungarotoxin from the venom of Bungarus multicinctus, Hoppe-Seyler's Z. Physiol. Chem., 353 (1972) 243-262. 26 Miledi, R. and Potter, L. T., Acetylcholine receptors in muscle fibres, Nature (Lond.), 233 (1971) 599-603. 27 Norris, C. H. and Guth, P. S., The release of acetylcholine (ACh) by the crossed olivo-cochlear bundle (COCB), Acta oto-laryng. (Stockh.), 77 (1974) 318-326. 28 O'Brien, R. D., Thompson, W. R. and Gibson, R. E., A comparison of acetylcholine and a-bungarotoxin binding to soluble Torpedo receptor. In E. de Robertis and J. Schacht (Eds.), Neurochemistry of Cholinergic Receptors, Raven Press, New York, 1974, pp. 49-62. 29 Patrick, J. and Stallcup, W. B., Immunological distinction between acetylcholine receptor and the a-bungarotoxin-binding component on sympathetic neurons, Proc. nat. Acad. Sci. (Wash.), 74 (1977) 4689-4692. 30 Sarvey, J. M., Albuquerque, E. X., Eldefrawi, A. T. and Eldefrawi, M., Effects of a-bungarotoxin and reversible cholinergic ligands on normal and denervated mammalian skeletal muscle, Membrane Biochem., 1 (1978) 131-157. 31 Spoendlin, H., Innervation patterns in the organ of Corti of the cat, Acta oto-laryng. (Stockh.), 67 (1969) 239-254. 32 Spoendlin, H., Structural basis of peripheral frequency analysis. In R. Plomp and F. G. Smoorenburg (Eds.), Frequency Analysis andPeriodicity Detection in Hearing, Sij thoff, Leiden, 1970, pp. 2-40. 33 Spoendlin, H., Neuroanatomical basis of cochlear coding mechanisms, Audiology, 14 (1975) 383407.
