- Email: [email protected]
Cholinergic nicotenic receptors in the vestibular epithelia
174
Brain Research, 561 (1991) 174-176 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391
BRES 24863
Cholinergic nicotenic receptors in the vestibular epithelia R . A . Thornhill School of Biological Sciences, The University of Birmingham, Birmingham (U.K.) (Accepted 2 July 1991) Key words: Sensory cell; Vestibular epithelium; Cholinergic receptor
Receptor binding studies specific for nicotinic cholinergic receptors have been carried out on isolated vestibular epithelia of the frogs Rana catesbiana and Rana temporaria. Evidence is presented for the presence of nicotinic-like cholinergic receptors specificallyassociated with the sensory areas. The sensory cells of the vestibular epithelia are innervated by two main types of nerve endings. Sensory neurones make contact with the sensory hair cells via afferent endings, and transmit primary sensory information from the sensory cell to the central nervous system. Efferent nerve endings, which are inhibitory, make contact either with the sensory cell to give rise to presynaptic inhibition, or with afferent nerve endings to give rise to postsynaptic inhibition. Both afferent and efferent nerve endings are believed to be chemically mediated. The nature of the transmitter at the afferent ending is still unclear, but at the efferent synapse, acetylcholine (ACh) is the strongly favoured candidate 1'7"12'13A6A9. There are a number of criteria that need to be satisfied before a substance can be considered a putative neurotransmitter. In the case of the vestibular efferent system, ACh has been shown to satisfy some of these criteria. Isolated sensory areas have been shown to be able to synthesize A C h 5'7"1°'19 and acetylcholinesterase and choline acetyltransferase have been found to be closely associated with the vestibular efferent system H' 19. The effects of efferent stimulation are blocked by nicotinic antagonist 4'6'18, and perfusion of the scala media of the cochlea with ACh reduced tone evoked single fibre responses particularly in the presence of acetylcholinesterase inhibitors such as eserine 2'8. In the frog labyrinth, transection of the vestibular nerve is followed by a gradual reduction in the level of choline acetyltransferase activity ~3. There is, therefore, a broad body of evidence to support the contention that ACh acts as an efferent transmitter in vertebrate vestibular epithelia. An important criteria which must also be satisfied when designating putative neurotransmitters is that spe-
cific receptors for the transmitter, in this case cholinergic receptors, should be present. Nicotinic receptors (nAChRs) are implicated 1'6, but some evidence TM tends to support muscarinic-like receptors, or more than one type 3. Even if the precise nature of the receptors is not easy to classify using conventional criteria, the presence of any specific cholinergic receptors would provide further support for ACh as an efferent transmitter in this system. The binding of a-bungarotoxin (BTx) to nAChRs is highly specific and essentially irreversible under non-denaturing conditions 17. It has been used as a probe for nAChRs in a variety of tissues. The availability of radiolabelled BTx of high specific activity makes it feasible to assay nAChRs in the small quantities of tissue obtainable from vestibular sense organs. Significantly, Fex and Adams 4 have shown that BTx blocks the inhibitory effects of crossed olivo-cochlea bundle stimulation in the cochlea. For each of these experiments, the labyrinths of 10-20 Rana temporaria or Rana catesbiana were rapidly dissected out from freshly pithed frogs and stored in ice cold frog saline (in mM: 115 NaC1, 3.2 KCI, 1.8 CaC12, 2.4 NaHCO3; pH 7.4). The saccular sensory area, 3 ampullae, and an equivalent quantity of non-sensory canals were dissected from each labyrinth and each tissue type pooled. Each sample was then homogenized in 30/~1 of 10 mM NaH2PO 4, 50 mM NaCI, 0.02% NAN3, 10 -4 M PMSF and 1% Triton or Lubrol at pH 7.4 in a miniature homogenizer. The homogenizer was rinsed twice with homogenizing buffer and made up to 100/~1. Then 10 #1 was taken for protein determination 9 and the remainder divided equally into experimental and control samples.
Correspondence: R.A. Thornhill, School of Biological Sciences, The University of Birmingham, P.O. Box 363, Birmingham B15 2TI', U.K.
175
The control sample was pre-incubated for 2 h with either 10-5 M MTx (Sigma) or 10-3 M gallamine triethiode, before incubation with 10 -8 M 125I-BTx (Amersham International, spec. act. 700 Ci/mmol). After incubation, toxin binding to nAChRs in the homogenates was assayed using a D E A E disc assay 17. Aliquots of homogenate were applied to numbered D E A E (Whatman DE81, 2.3 cm diameter) discs and allowed to dry. The discs were then washed in phosphate buffer with regular changes and counted in a Nuclear Enterprise SR7 gamma counter at an efficiency of 68%. In some preliminary experiments, the binding of iodinated toxin to isolated sensory areas or to pelleted homogenates of sensory tissue were investigated using a protocol similar to that described above. In this case, the unbound toxin was rinsed out with repeated change of phosphate buffer. All binding assays were carried out at 4 °C. It was found that the iodinated toxin bound to the tissue. The proportion of this binding that was removed by pre-treatment with a 1000-fold excess (10 -5 M) of nonradioactive BTx was taken as 'specific' binding. Defined in this way, some specific binding occurred in non-sensory canal preparations. This, however, was normally about 5-10% of the specific binding associated with the sensory areas. Specific binding to the saccular sensory area homogenate was found to be 1.95 -+ 0.5 fmol/sensory area, which is not significantly different (P > 0.1) from the value of 2.4 --- 0.9 fmol/sensory area for nonhomogenized samples (Table I). Pre-incubation with 10 -3 M gallamine, a nicotinic cholinergic antagonist, inhibited
about 90% of the specific binding observed in the saccular sensory tissue. This is consistent with the physiological response to gallamine, which was found to suppress efferent inhibition 6. When solubilized nAChRs in sensory area homogenates were assayed using the D E A E disc assay, apparent specific binding to saccular receptors was 3.8 fmol/ sensory area 1 h after incubation, falling to 2.1 fmol/ sensory area at 25 h. The fall in the amount of 125I-toxin/ receptor complex bound to D E A E discs with time was investigated in more detail using solubilized saccular receptors. The dissocation of the toxin/receptor complex was studied after the addition of a 1000-fold excess of cold BTx following equilibration with a saturating dose of 10-8 M 125I-BTx. Controls were incubated with a 1000-fold excess of cold BTx to block specific binding sites prior to 10-8 M 125I-BTx treatment. Aliquots were taken at various times and assayed for receptor/toxin complex. Analysis of the binding kinetics of these data 14 reveals that the dissocation of the toxin/receptor complex is biphasic. The initial, more rapidly dissociating component constitutes 56% of the total binding, has a tl/2 of 15.2 h and a first order dissociation constant of 1.27.10-5/s. The second component (44%) has a tl/2 of 22.6 h and a first order dissociation constant of 8.53.10-7[s. This is similar to the solubilized rat brain receptor/toxin complex which is reported to have a halflife of 15.6 (ref. 15). The dissociation of BTx from nAChRs from toad and goldfish brain is also reported to be biphasic, although the time course was shorter than those reported here. In the goldfish, the complex has a fast dissociating component with a half-life 17.6 min, and a slowly dissociating component having a half-life of 44.7 h (refs. 14, 15). The level of toxin binding in saccular sensory areas corresponds to about 20 frnol/mg wet tissue, which may be compared with 10 fmol/mg wet tissue found in whole frog brain assayed under very similar conditions 14. These data suggest that the isolated vestibular sensory epithelia of the frog possess nicotinic-like cholinergic receptors with characteristics similar to those reported in the CNS and retina of other vertebrates. This finding strengthens the contention that the vestibular sensory system possesses a 'nicotinic-like' cholinergic innervation, and is consistent with much of the physiological and pharmacological data mentioned above.
1 Bernard, C., Cochran, S.L. and Precht, W., Presynaptic actions of cholinergic agents upon the hair cell-afferent synapse in the vestibular labyrinth of the frog, Brain Research, 338 (1985) 225236. 2 Cornis, S.D. and Leng, G., Action of putative neurotrans-
mitters in the guinea pig cochlea, Exp. Brain Res., 36 (1979) 119-128. 3 Eybalin, M., Choline acetyltransferase (CHAT)immunoelectron microscopy distinguishes at least three types of efferent synapses in the organ of Corti, Exp. Brain Res., 65 (1987) 261-
TABLE I Binding of 1251-BTx Sacculus
Whole sensory areas Range (n = 8) Mean -+ S.E.M. Homogenates DEAE disc assay 1h 25 h
0.3-3.5 2.4-+0.9
Ampulla Canal (fmol/sensory area) 0.9-3.4 2.7-+1.2
0.1-0.15 0.2-+0.15
1.95---0.5
2.01-+0.3
0.15-+0.1
3.8-+0.3 2.1-+0.1
4.5-+0.9 2.3-+0.2
0.2-+0.1 0.2+-0.1
176 270. 4 Fex, J. and Adams, J.C., Alpha-bungarotoxin blocks reversibly cholinergie inhibition in the cochlea, Brain Research, 159 (1978) 440--444. 5 Fex, J. and Wenthold, R.J., Choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the cochlea and cochlea nucleus of the guinea pig, Brain Research, 109 (1976) 575-585. 6 Flock, A. and Russell, I.J., The post-synaptic action of efferent fibres in the lateral line organ of the burbot Lota iota, J. PhysIOL, 235 (1973) 591-605. 7 Flock, A. and Lam, D.M.K., Neurotransmitter synthesis in the inner ear and lateral line sense organs, Nature, 249 (1974) 142144. 8 Galley, N., Klinke, R., Oertel, W., Pause, M. and Storch, W.H., The effect of intracochlearly administered acetylcholineblocking agents on the efferent synapses of the cochlea, Brain Research, 64 (1974) 55-63. 9 Geiger, P.J., Bessman and S.P., Protein determination by Lowry's method in the presence of sulphydril reagents, Anal Biochem., 49 (1972) 447-473. 10 Godfrey, D.A., Krzanowski, J.J. and Matchinsky, EM., Activities of enzymes of the eholinergic system in the guinea pig cochlea, J. Histochem. Cytochem., 24 (1976) 468-472. 11 Iurato, S., Luciano, L., Pannese, E. and Reale, E., In Iocalisation of acetylcholinesterase in the auditory and vestibular sys-
12 13
14
15
16
17
18
19
tems, M. Santini (Ed.), Golgi Centennial Symposium Proceedings, Raven, New York, 1975, pp. 553-567. Klinke, R., Neurotransmission in the inner ear, Hear. Res., 22 (1986) 235-243. Lopez, I. and Meza, G., Neurochemieal evidence for afferent GABAergic and efferent cholinergic neurotransmission in the frog vestibule, Neuroscience, 25 (1988) 13-18. Oswald, R.E. and Freeman, J.A., Characterization of the nicotinic acetyicholine receptor isolated from goldfish brain, J. Biol. Chem., 254 (1979) 3419-3426. Oswald, R.E. and Freeman, J.A., Alpha-bungarotoxin binding and central nervous system nicotinic acetylcholine receptors, Neuroscience, 6 (1981) 1-1. Robertson, D. and Johnstone, B.M., Efferent transmitter substance in the mammalian cochlea: single neuron support for the acetylcholine, Hear. Res., 1 (1978) 31-37. Salvaterra, P.M. and Mahler, H.R., Nicotinic acetylcholine receptor from the rat brain: solubilization, partial purification and characterization, J. Biol. Chem., 251 (1976) 6327-6334. Shigemoto, T. and Ohmori, H., Muscarinic agonists and ATP increase the intracellular Ca 2+ concentration in the chick cochlear hair cells, J. PhysioL, 420 (1990) 127-148. Thornhill, R.A., Biochemical and histochemical studies on neurotransmission. In J. Schwarzkopf (Ed.), Mechanoreception, Vol. 53, Westdeutscher Verlag, 1974, pp. 209-223.
Brain Research, 561 (1991) 174-176 © 1991 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/91/$03.50 ADONIS 0006899391
BRES 24863
Cholinergic nicotenic receptors in the vestibular epithelia R . A . Thornhill School of Biological Sciences, The University of Birmingham, Birmingham (U.K.) (Accepted 2 July 1991) Key words: Sensory cell; Vestibular epithelium; Cholinergic receptor
Receptor binding studies specific for nicotinic cholinergic receptors have been carried out on isolated vestibular epithelia of the frogs Rana catesbiana and Rana temporaria. Evidence is presented for the presence of nicotinic-like cholinergic receptors specificallyassociated with the sensory areas. The sensory cells of the vestibular epithelia are innervated by two main types of nerve endings. Sensory neurones make contact with the sensory hair cells via afferent endings, and transmit primary sensory information from the sensory cell to the central nervous system. Efferent nerve endings, which are inhibitory, make contact either with the sensory cell to give rise to presynaptic inhibition, or with afferent nerve endings to give rise to postsynaptic inhibition. Both afferent and efferent nerve endings are believed to be chemically mediated. The nature of the transmitter at the afferent ending is still unclear, but at the efferent synapse, acetylcholine (ACh) is the strongly favoured candidate 1'7"12'13A6A9. There are a number of criteria that need to be satisfied before a substance can be considered a putative neurotransmitter. In the case of the vestibular efferent system, ACh has been shown to satisfy some of these criteria. Isolated sensory areas have been shown to be able to synthesize A C h 5'7"1°'19 and acetylcholinesterase and choline acetyltransferase have been found to be closely associated with the vestibular efferent system H' 19. The effects of efferent stimulation are blocked by nicotinic antagonist 4'6'18, and perfusion of the scala media of the cochlea with ACh reduced tone evoked single fibre responses particularly in the presence of acetylcholinesterase inhibitors such as eserine 2'8. In the frog labyrinth, transection of the vestibular nerve is followed by a gradual reduction in the level of choline acetyltransferase activity ~3. There is, therefore, a broad body of evidence to support the contention that ACh acts as an efferent transmitter in vertebrate vestibular epithelia. An important criteria which must also be satisfied when designating putative neurotransmitters is that spe-
cific receptors for the transmitter, in this case cholinergic receptors, should be present. Nicotinic receptors (nAChRs) are implicated 1'6, but some evidence TM tends to support muscarinic-like receptors, or more than one type 3. Even if the precise nature of the receptors is not easy to classify using conventional criteria, the presence of any specific cholinergic receptors would provide further support for ACh as an efferent transmitter in this system. The binding of a-bungarotoxin (BTx) to nAChRs is highly specific and essentially irreversible under non-denaturing conditions 17. It has been used as a probe for nAChRs in a variety of tissues. The availability of radiolabelled BTx of high specific activity makes it feasible to assay nAChRs in the small quantities of tissue obtainable from vestibular sense organs. Significantly, Fex and Adams 4 have shown that BTx blocks the inhibitory effects of crossed olivo-cochlea bundle stimulation in the cochlea. For each of these experiments, the labyrinths of 10-20 Rana temporaria or Rana catesbiana were rapidly dissected out from freshly pithed frogs and stored in ice cold frog saline (in mM: 115 NaC1, 3.2 KCI, 1.8 CaC12, 2.4 NaHCO3; pH 7.4). The saccular sensory area, 3 ampullae, and an equivalent quantity of non-sensory canals were dissected from each labyrinth and each tissue type pooled. Each sample was then homogenized in 30/~1 of 10 mM NaH2PO 4, 50 mM NaCI, 0.02% NAN3, 10 -4 M PMSF and 1% Triton or Lubrol at pH 7.4 in a miniature homogenizer. The homogenizer was rinsed twice with homogenizing buffer and made up to 100/~1. Then 10 #1 was taken for protein determination 9 and the remainder divided equally into experimental and control samples.
Correspondence: R.A. Thornhill, School of Biological Sciences, The University of Birmingham, P.O. Box 363, Birmingham B15 2TI', U.K.
175
The control sample was pre-incubated for 2 h with either 10-5 M MTx (Sigma) or 10-3 M gallamine triethiode, before incubation with 10 -8 M 125I-BTx (Amersham International, spec. act. 700 Ci/mmol). After incubation, toxin binding to nAChRs in the homogenates was assayed using a D E A E disc assay 17. Aliquots of homogenate were applied to numbered D E A E (Whatman DE81, 2.3 cm diameter) discs and allowed to dry. The discs were then washed in phosphate buffer with regular changes and counted in a Nuclear Enterprise SR7 gamma counter at an efficiency of 68%. In some preliminary experiments, the binding of iodinated toxin to isolated sensory areas or to pelleted homogenates of sensory tissue were investigated using a protocol similar to that described above. In this case, the unbound toxin was rinsed out with repeated change of phosphate buffer. All binding assays were carried out at 4 °C. It was found that the iodinated toxin bound to the tissue. The proportion of this binding that was removed by pre-treatment with a 1000-fold excess (10 -5 M) of nonradioactive BTx was taken as 'specific' binding. Defined in this way, some specific binding occurred in non-sensory canal preparations. This, however, was normally about 5-10% of the specific binding associated with the sensory areas. Specific binding to the saccular sensory area homogenate was found to be 1.95 -+ 0.5 fmol/sensory area, which is not significantly different (P > 0.1) from the value of 2.4 --- 0.9 fmol/sensory area for nonhomogenized samples (Table I). Pre-incubation with 10 -3 M gallamine, a nicotinic cholinergic antagonist, inhibited
about 90% of the specific binding observed in the saccular sensory tissue. This is consistent with the physiological response to gallamine, which was found to suppress efferent inhibition 6. When solubilized nAChRs in sensory area homogenates were assayed using the D E A E disc assay, apparent specific binding to saccular receptors was 3.8 fmol/ sensory area 1 h after incubation, falling to 2.1 fmol/ sensory area at 25 h. The fall in the amount of 125I-toxin/ receptor complex bound to D E A E discs with time was investigated in more detail using solubilized saccular receptors. The dissocation of the toxin/receptor complex was studied after the addition of a 1000-fold excess of cold BTx following equilibration with a saturating dose of 10-8 M 125I-BTx. Controls were incubated with a 1000-fold excess of cold BTx to block specific binding sites prior to 10-8 M 125I-BTx treatment. Aliquots were taken at various times and assayed for receptor/toxin complex. Analysis of the binding kinetics of these data 14 reveals that the dissocation of the toxin/receptor complex is biphasic. The initial, more rapidly dissociating component constitutes 56% of the total binding, has a tl/2 of 15.2 h and a first order dissociation constant of 1.27.10-5/s. The second component (44%) has a tl/2 of 22.6 h and a first order dissociation constant of 8.53.10-7[s. This is similar to the solubilized rat brain receptor/toxin complex which is reported to have a halflife of 15.6 (ref. 15). The dissociation of BTx from nAChRs from toad and goldfish brain is also reported to be biphasic, although the time course was shorter than those reported here. In the goldfish, the complex has a fast dissociating component with a half-life 17.6 min, and a slowly dissociating component having a half-life of 44.7 h (refs. 14, 15). The level of toxin binding in saccular sensory areas corresponds to about 20 frnol/mg wet tissue, which may be compared with 10 fmol/mg wet tissue found in whole frog brain assayed under very similar conditions 14. These data suggest that the isolated vestibular sensory epithelia of the frog possess nicotinic-like cholinergic receptors with characteristics similar to those reported in the CNS and retina of other vertebrates. This finding strengthens the contention that the vestibular sensory system possesses a 'nicotinic-like' cholinergic innervation, and is consistent with much of the physiological and pharmacological data mentioned above.
1 Bernard, C., Cochran, S.L. and Precht, W., Presynaptic actions of cholinergic agents upon the hair cell-afferent synapse in the vestibular labyrinth of the frog, Brain Research, 338 (1985) 225236. 2 Cornis, S.D. and Leng, G., Action of putative neurotrans-
mitters in the guinea pig cochlea, Exp. Brain Res., 36 (1979) 119-128. 3 Eybalin, M., Choline acetyltransferase (CHAT)immunoelectron microscopy distinguishes at least three types of efferent synapses in the organ of Corti, Exp. Brain Res., 65 (1987) 261-
TABLE I Binding of 1251-BTx Sacculus
Whole sensory areas Range (n = 8) Mean -+ S.E.M. Homogenates DEAE disc assay 1h 25 h
0.3-3.5 2.4-+0.9
Ampulla Canal (fmol/sensory area) 0.9-3.4 2.7-+1.2
0.1-0.15 0.2-+0.15
1.95---0.5
2.01-+0.3
0.15-+0.1
3.8-+0.3 2.1-+0.1
4.5-+0.9 2.3-+0.2
0.2-+0.1 0.2+-0.1
176 270. 4 Fex, J. and Adams, J.C., Alpha-bungarotoxin blocks reversibly cholinergie inhibition in the cochlea, Brain Research, 159 (1978) 440--444. 5 Fex, J. and Wenthold, R.J., Choline acetyltransferase, glutamate decarboxylase and tyrosine hydroxylase in the cochlea and cochlea nucleus of the guinea pig, Brain Research, 109 (1976) 575-585. 6 Flock, A. and Russell, I.J., The post-synaptic action of efferent fibres in the lateral line organ of the burbot Lota iota, J. PhysIOL, 235 (1973) 591-605. 7 Flock, A. and Lam, D.M.K., Neurotransmitter synthesis in the inner ear and lateral line sense organs, Nature, 249 (1974) 142144. 8 Galley, N., Klinke, R., Oertel, W., Pause, M. and Storch, W.H., The effect of intracochlearly administered acetylcholineblocking agents on the efferent synapses of the cochlea, Brain Research, 64 (1974) 55-63. 9 Geiger, P.J., Bessman and S.P., Protein determination by Lowry's method in the presence of sulphydril reagents, Anal Biochem., 49 (1972) 447-473. 10 Godfrey, D.A., Krzanowski, J.J. and Matchinsky, EM., Activities of enzymes of the eholinergic system in the guinea pig cochlea, J. Histochem. Cytochem., 24 (1976) 468-472. 11 Iurato, S., Luciano, L., Pannese, E. and Reale, E., In Iocalisation of acetylcholinesterase in the auditory and vestibular sys-
12 13
14
15
16
17
18
19
tems, M. Santini (Ed.), Golgi Centennial Symposium Proceedings, Raven, New York, 1975, pp. 553-567. Klinke, R., Neurotransmission in the inner ear, Hear. Res., 22 (1986) 235-243. Lopez, I. and Meza, G., Neurochemieal evidence for afferent GABAergic and efferent cholinergic neurotransmission in the frog vestibule, Neuroscience, 25 (1988) 13-18. Oswald, R.E. and Freeman, J.A., Characterization of the nicotinic acetyicholine receptor isolated from goldfish brain, J. Biol. Chem., 254 (1979) 3419-3426. Oswald, R.E. and Freeman, J.A., Alpha-bungarotoxin binding and central nervous system nicotinic acetylcholine receptors, Neuroscience, 6 (1981) 1-1. Robertson, D. and Johnstone, B.M., Efferent transmitter substance in the mammalian cochlea: single neuron support for the acetylcholine, Hear. Res., 1 (1978) 31-37. Salvaterra, P.M. and Mahler, H.R., Nicotinic acetylcholine receptor from the rat brain: solubilization, partial purification and characterization, J. Biol. Chem., 251 (1976) 6327-6334. Shigemoto, T. and Ohmori, H., Muscarinic agonists and ATP increase the intracellular Ca 2+ concentration in the chick cochlear hair cells, J. PhysioL, 420 (1990) 127-148. Thornhill, R.A., Biochemical and histochemical studies on neurotransmission. In J. Schwarzkopf (Ed.), Mechanoreception, Vol. 53, Westdeutscher Verlag, 1974, pp. 209-223.