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Structural changes in insect neuromuscular junctions in lanthanum solution
Camp. Biothem. Phrsiol. Vol. 79A, No. 4, pp. 591-595, Printed in Great B&in
0300-9629/84 $3.00 + 0.00 cc 1984 Pergamon Press Ltd
1984
STRUCTURAL CHANGES IN INSECT JUNCTIONS IN LANTHANUM HIROSHI WASHIO
Laboratory
of Neurophysiology.
and
Mitsubishi-Kasei Telephone:
NEUROMUSCULAR SOLUTION
ITSUKO NIHONMATSU
Institute of Life Sciences, Machida, (0427) 26-1211
Tokyo
194, Japan.
(Received 26 March 1984) Abstract-l. An electron microscopic study was made of the effect of lanthanum ions on the neuromuscular junction of the cockroach Periplunrru americana leg muscles. Experimental muscles were treated with 5 mM La-saline for 2, 4 and I7 hr at 4 ‘C or 2 hr at room temperature (2&22”C). 2. After exposure to lanthanum for 2 hr at 4 C synaptic vesicles appeared to be slightly reduced in number, but the terminal mitochondria appeared unchanged. After the exposure for 4 hr at 4°C the majority of the terminals were fairly depleted of vesicles. No synaptic contact was recognized at this time. By I7 hr all terminals were almost devoid of synaptic vesicles. 3. The effect of lanthanum ions on the structural changes was markedly affected by temperature, 2 hr of treatment with La-saline being enough to deplete the synaptic vesicles at room temperature. 4. From the results obtained and literature cited the ultrastructural changes may correlate with the functional changes in the insect neuromuscular junction.
INTRODUCTION
glutalaldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.05) at 4’ C overnight and post-fixed with I?: 0~0, at 4-C for I hr. The coxal cuticle was removed after the first 2 hr of prefixation and the muscle continued being pre-fixed. In later experiments cacodylate buffer (0.1 M. pH 7.05) was substituted for the phosphate buffer and was compared with the result obtained, because of possible insoluble salt in the extracellular space (Heuser and Miledi, 1971). The standard bathing solution had the following composition (mM): NaCl 158; KCI 10.8; CaClz 5; HEPES 5; adjusted to pH 7.0 with NaOH. The lanthanum saline was prepared by substituting LaCl, for NaCI, keeping the osmolarity constant.
In our previous work on the insect neuromuscular junction (Washio and Miyamoto. 1983), lanthanum ions have been shown to block neuromuscular transmission. On the other hand, spontaneous release of transmitter markedly increased after lanthanum was added in the bathing medium (Blioch et d., 1968; Heuser and Miledi, 1971; DeBassio et al., 1972; Washio and Miyamoto, 1983). The high frequency of miniature excitatory postsynaptic potentials (MEPSPs) caused by 5 mM lanthanum lasted for less than I hr, resulting in cessation of the MEPSPs in the cockroach muscle (Washio and Miyamoto, 1983). Thus it appears that treatment with lanthanum induces a functional change in the myoneural junction in insect muscles associated with structural disintegration (cf. Heuser and Miledi, 1971). The present work was undertaken to discover any structural changes induced by treatment with lanthanum in the insect nerve terminal. Isolated cockroach leg muscles were exposed to 5 mM La-saline for different periods and were prepared for electronmicroscopic study.
RESULTS
Control muscles A typical junction of a coxal depressor muscle of the cockroach Periplaneta americana is shown in Fig. I. The normal junction of the insect muscle fibre has been characterized by accumulation of spherical synaptic vesicles (45-55 nm in diameter) at the presynaptic membrane, by the presence of mitochondria within the nerve terminal and by the appearance of thickening of the post-synaptic membrane (cf. Atwood et al., 1969; Faeder and Salpeter, 1970; Rees and Usherwood, 1972; Osborne, 1975). The presence of neurotubules and vacuoles was also observed. Occasionally coated vesicles were found in the terminal. As pointed out by Usherwood et a!. (1968), we also confirmed the structural undifferentiation of post-synaptic membrane of the cockroach muscle. The pre- and post-synaptic membrane were separated by a synaptic cleft, about 30-35 nm wide. Synaptic contacts could be clearly observed in control muscle fibers. Control terminals maintained for 2 hr at 4% in normal saline showed no signs of any deterioration (Fig. 2, Al, and Fig. 3, Al). In the 4-hr control preparation at 4°C the electron micrograph showed
.MATERIALS AND METHODS
The coxal depressor muscles I78 and 136 (Carbonell, 1947). innervated by only one excitatory motor axon (Pearson and Iles. 1971). were isolated from the meta- and mesothoracic legs. respectively, of the cockroach Periplaneta crm~vkmrr for electron microscopy. In all experiments control and experimental muscles were isolated from legs of each side of the same animal. To keep the approximate resting length of the muscle, these coxal muscles were isolated together with the ventromedial coxal cuticle. Experimental muscles were treated for various period. with La-salme at 4’C or at room temperature (2G22 C), while control muscles were bathed in normal saline at the same temperature as the experimental ones for the equivalent time of treatment with La”. Muscles were pre-fixed with 2”; 591
592
HIROSHI WASHLO and
Fig.
I. Cross-section
of a normal cockroach nerveterminal. The left micrograph is an enlargement showing details of the synaptic contact. Scale bar: 0.5 pm.
an increase in the number of vacuoles but a normal number and density of synaptic vesicles. The terminal mitochondria appeared to be unchanged (Fig. 2, A2). Efect
ITSUKO NIHONMATSU
of lunthunurn
Experimental muscles were treated with 5 mM La-saline for 2, 4 and I7 hr at 4’ C or 2 hr at room temperature. After exposure to lanthanum for 2 hr at 4 C synaptic vesicles appeared to be slightly reduced in number but the terminal mitochondria appeared to be unchanged. Synaptic contacts were less clear but still observed (Fig. 2, Bl and Fig. 3, Bl). In some terminals membrane-bound structures were distinguished (Fig. 3, BI). No clear difference was found between the micrographs obtained in the phosphateand cacodylate-buffered fixatives for the 2-hr exposure to La. After exposure for 4 hr at 4’C (Fig. 2, B2) the majority of terminals were fairly depleted of the vesicles. No synaptic contact was recognized at this time. By 17 hr all terminals were almost devoid of synaptic vesicles (Fig. 3, B3). Mitochondria did not appear to be much affected in some terminals, but other terminal mitochondria showed signs of swelling and internal membrane disruption. When muscles were treated with the La-saline at room temperature the effect of La was much more effective than at 4°C. Electron microscopy (Fig. 3, B3) of one muscle showed accumulation of high-density material consisting of synaptic vesicles, granules and cytoplasmic materials in the terminal treated with La for 2 hr at room temperature, which was roughly comparable with the treatment for I6 hr at 4 C (Fig. 3, B2). This type of electron-dense aggregation has also been found in the denervated nerve terminals in both vertebrate (Birks et ul., 1960; Miledi and Slater, 1970) and invertebrate (Frank, 1974; Wood and Usherwood, 1979) muscles.
A granular lanthanum precipitate occurred in the synaptic cleft and even in the terminal when the muscle was fixed with the phosphate buffer after exposure to 5 mM La for more than IO hr at 4 C (Fig. 2, B3). When cacodylate buffer was substituted for the phosphate the intracellular precipitate was absent after the exposure for I6 hr at 4 C (Fig. 3, B2). This result agrees with the finding of increased retention of lanthanum by the phosphate-buffered fixative (Heuser and Miledi, 1971). DISCUSSION
Electron microscopic examination of nerve terminals in the coxal depressor muscles of the cockroach Peripluneta americana indicated that the treatment 01 the junction with La induced ultrastructural changes associated with the functional disorganization. From previous electrophysiological works on the insect muscle (Washio and Miyamoto, 1983) neuromuscular transmission in Tenehrio muscles treated with 0.5 mM La was blocked irreversibly within about IO min. The acceleratory effect of 5 mM La on transmitter release lasted for less than I hr in Periplunetu muscles at room temperature. Therefore, after exposure to 5 mM La for 2 hr at room temperature we may expect that neuromuscular transmission to all fibres would be blocked and the MEPSP frequency would already be markedly reduced, and possibly completely stopped. The present work on electron microscopy of the terminal under the same condition (Fig. 3, B3) clearly demonstrated degeneration of the ultrastructural organization in the nerve terminal. Thus the ultrastructural changes may correlate with the blockage of neuromuscular transmission and the failure of MEPSPs. Correlated ultrastructural and electrophysiological
Lanthanum
on insect nerve terminals
593
Fig. 2. Nerve terminals exposed to 5 mM lanthanum at 4 C for 2 hr (Bl), 4 hr (B2) and 17 hr (B3) and of the control muscle exposed to normal Ringer at 4 C for 2 hr (Al), 4 hr (A2) and I7 hr (A3). The preparation was prefixed with glutalaldehyde and paraformaldehyde in phosphate buffer. Scale bar:
0.5 /rm.
works were made on the cockroach muscle during the application of metabolic inhibitors by Rees (1974). The study has shown agglutination of synaptic vesicles and disruption of the mitochondria, suggesting that these changes are causally related to a metabolic dysfunction of the terminal mitochondria. Although the present work showed signs of swelling and internal membrane disruption of terminal mitocondria, further study is needed to correlate the degree of the distribution of the cyto-architecture of the mitochondria and functional disorganization in the presence of La. Also, application of black widow spider
venom to the locust neuromuscular junction has been found to cause correlated changes in the spontaneous release of transmitter with ultrastructural disorganization (Cull-candy et al., 1973). Recently Florey and Kriebel (1983) have reported complete loss of synaptic vesicles with 34 hr of La treatment at 23°C in frog neuromuscular junctions, but no depletion was noticed at 4°C. Comparing the results obtained in the present work to those of the frog, the insect neuromuscular junction may be impaired more delicately by lanthanum ions morphologically and physiologically.
HIK~SHI WASHIO and ITSUKO NIHONUATSU
594
Fig. 3. Nerve terminals exposed to 5 mM lanthanum at 4’C for 2 hr (91). 17 hr (92) and at 22 C for 2 hr (B3), and of the control muscle exposed to the normal Ringer at 4 C for 2 hr (Al). 17 hr (A2), and at 22-C for 2 hr (A3). The preparation was prefixed in cacodylate buffer. The bar in 93 indicates 0.5 ilm for all part of this figure.
wish to thank Prof. K. Fujino for his helpful comments on the manuscript and Dr T. Arima for his technical suggestion. Thanks are also due to Miss Junko Kaneko for her technical assistance.
Acknowledgements-We
REFERENCES
Atwood H. L., Smyth T. and Johnston H. S. (1969) Neuromuscular synapses in the cockroach extensor tibiae muscle. J. Insect Physiol. 15, 529-535. Birks R., Katz 9. and Miledi R. (1960) Physiological and structural changes at the amphibian myoneural junction in the course of nerve degeneratton. J. Physiol., Lond. 150, 145-166.
Bhoch Z. L., Glagoleva I. M.. Liberman E. A. and Nenashev V. A. (1968) A study of the mechanism of quanta1 transmitter release at a chemical synapse. J. Physiol.. Land. 199, I l-35. Carbonell C. S. (1947) The thoracic muscles of the cockroach, Periplaneta 107, l-23.
americutu
Smithoniun
Inst. misc. Coil.
DeBassio W. A., Schnitzler R. M. and Parson R. L. (1972) Influence of lanthanum on transmitter release at the neuromuscular junction. J. Ncurohiol. 2, 263-278. Faeder I. R. and Salpeter M. M. (1970) The tine structure of nerve branches and neuromuscular junctions in the coxal adductor of the cockroach. Gromphadorhina portentosa.
J. Morphol.
132, 225-234.
Lanthanum on insect nerve terminals Florey E. and Kriebel M. E. (1983) Changes in acetylcholine concentration, miniature end-plate potentials and synaptic vesicles in frog neuromuscular preparations during lanthanum treatment. Camp. Biochem. Physiol. 75C, 285-294.
Frank E. (1974) The sensitivity to glutamate of denervated muscles of the crayfish. J. Physiol., Lond. 242, 371-382. Heuser J. and Miledi R. (1971)Effect of lanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Sot. B. 179, 247-260. Miledi R. and Slater C. R. (I 970) On the degeneration at rat neuromuscular junctions after nerve section. J. Physiol., Land. 207, 507-528.
Osborne M. P. (1975) The ultrastructure of nerve-muscle synapses. In Insect Muscle (Edited by Usherwood P. N. R.). pp. 161-205. Academic Press, London. Pearson K. G. and Iles J. F. (1971) Innervation of coxal depressor muscles in the cockroach, Periplaneta americana. J. exp. Biol. 54, 215-232.
595
Rees D. (1974) The effect of metabolic inhibitors on the cockroach nerve-muscle synapse. J. exp. Biol. 61, 33 l-343.
Rees D. and Usherwood P. N. R. (1972) Fine structure of normal and degenerating motor axons and nerve-muscle synapses in the locust, Schistocerca gregaria. Comp. Biothem. Physiol. 43A, 83-101.
Usherwood P. N. R., Cochrane D. G. and Rees D. (1968) Changes in structural, physiological and pharmacological properties of insect excitatory nerve-muscle synapses after motor nerve section. Nature 218, 589-591. _ Washio H. and Miyamoto T. (1983) Effect of lanthanum ions on neuromuscular transmission in insects. J. exp. Biol. 107, 405-414.
Wood M. R. and Usherwood P. N. R. (1979) Uhrastructural changes in cockroach leg muscle following unilateral neurotomy-I. Degeneration. J. Ultrastruct. 68, 265-280.
0300-9629/84 $3.00 + 0.00 cc 1984 Pergamon Press Ltd
1984
STRUCTURAL CHANGES IN INSECT JUNCTIONS IN LANTHANUM HIROSHI WASHIO
Laboratory
of Neurophysiology.
and
Mitsubishi-Kasei Telephone:
NEUROMUSCULAR SOLUTION
ITSUKO NIHONMATSU
Institute of Life Sciences, Machida, (0427) 26-1211
Tokyo
194, Japan.
(Received 26 March 1984) Abstract-l. An electron microscopic study was made of the effect of lanthanum ions on the neuromuscular junction of the cockroach Periplunrru americana leg muscles. Experimental muscles were treated with 5 mM La-saline for 2, 4 and I7 hr at 4 ‘C or 2 hr at room temperature (2&22”C). 2. After exposure to lanthanum for 2 hr at 4 C synaptic vesicles appeared to be slightly reduced in number, but the terminal mitochondria appeared unchanged. After the exposure for 4 hr at 4°C the majority of the terminals were fairly depleted of vesicles. No synaptic contact was recognized at this time. By I7 hr all terminals were almost devoid of synaptic vesicles. 3. The effect of lanthanum ions on the structural changes was markedly affected by temperature, 2 hr of treatment with La-saline being enough to deplete the synaptic vesicles at room temperature. 4. From the results obtained and literature cited the ultrastructural changes may correlate with the functional changes in the insect neuromuscular junction.
INTRODUCTION
glutalaldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.05) at 4’ C overnight and post-fixed with I?: 0~0, at 4-C for I hr. The coxal cuticle was removed after the first 2 hr of prefixation and the muscle continued being pre-fixed. In later experiments cacodylate buffer (0.1 M. pH 7.05) was substituted for the phosphate buffer and was compared with the result obtained, because of possible insoluble salt in the extracellular space (Heuser and Miledi, 1971). The standard bathing solution had the following composition (mM): NaCl 158; KCI 10.8; CaClz 5; HEPES 5; adjusted to pH 7.0 with NaOH. The lanthanum saline was prepared by substituting LaCl, for NaCI, keeping the osmolarity constant.
In our previous work on the insect neuromuscular junction (Washio and Miyamoto. 1983), lanthanum ions have been shown to block neuromuscular transmission. On the other hand, spontaneous release of transmitter markedly increased after lanthanum was added in the bathing medium (Blioch et d., 1968; Heuser and Miledi, 1971; DeBassio et al., 1972; Washio and Miyamoto, 1983). The high frequency of miniature excitatory postsynaptic potentials (MEPSPs) caused by 5 mM lanthanum lasted for less than I hr, resulting in cessation of the MEPSPs in the cockroach muscle (Washio and Miyamoto, 1983). Thus it appears that treatment with lanthanum induces a functional change in the myoneural junction in insect muscles associated with structural disintegration (cf. Heuser and Miledi, 1971). The present work was undertaken to discover any structural changes induced by treatment with lanthanum in the insect nerve terminal. Isolated cockroach leg muscles were exposed to 5 mM La-saline for different periods and were prepared for electronmicroscopic study.
RESULTS
Control muscles A typical junction of a coxal depressor muscle of the cockroach Periplaneta americana is shown in Fig. I. The normal junction of the insect muscle fibre has been characterized by accumulation of spherical synaptic vesicles (45-55 nm in diameter) at the presynaptic membrane, by the presence of mitochondria within the nerve terminal and by the appearance of thickening of the post-synaptic membrane (cf. Atwood et al., 1969; Faeder and Salpeter, 1970; Rees and Usherwood, 1972; Osborne, 1975). The presence of neurotubules and vacuoles was also observed. Occasionally coated vesicles were found in the terminal. As pointed out by Usherwood et a!. (1968), we also confirmed the structural undifferentiation of post-synaptic membrane of the cockroach muscle. The pre- and post-synaptic membrane were separated by a synaptic cleft, about 30-35 nm wide. Synaptic contacts could be clearly observed in control muscle fibers. Control terminals maintained for 2 hr at 4% in normal saline showed no signs of any deterioration (Fig. 2, Al, and Fig. 3, Al). In the 4-hr control preparation at 4°C the electron micrograph showed
.MATERIALS AND METHODS
The coxal depressor muscles I78 and 136 (Carbonell, 1947). innervated by only one excitatory motor axon (Pearson and Iles. 1971). were isolated from the meta- and mesothoracic legs. respectively, of the cockroach Periplaneta crm~vkmrr for electron microscopy. In all experiments control and experimental muscles were isolated from legs of each side of the same animal. To keep the approximate resting length of the muscle, these coxal muscles were isolated together with the ventromedial coxal cuticle. Experimental muscles were treated for various period. with La-salme at 4’C or at room temperature (2G22 C), while control muscles were bathed in normal saline at the same temperature as the experimental ones for the equivalent time of treatment with La”. Muscles were pre-fixed with 2”; 591
592
HIROSHI WASHLO and
Fig.
I. Cross-section
of a normal cockroach nerveterminal. The left micrograph is an enlargement showing details of the synaptic contact. Scale bar: 0.5 pm.
an increase in the number of vacuoles but a normal number and density of synaptic vesicles. The terminal mitochondria appeared to be unchanged (Fig. 2, A2). Efect
ITSUKO NIHONMATSU
of lunthunurn
Experimental muscles were treated with 5 mM La-saline for 2, 4 and I7 hr at 4’ C or 2 hr at room temperature. After exposure to lanthanum for 2 hr at 4 C synaptic vesicles appeared to be slightly reduced in number but the terminal mitochondria appeared to be unchanged. Synaptic contacts were less clear but still observed (Fig. 2, Bl and Fig. 3, Bl). In some terminals membrane-bound structures were distinguished (Fig. 3, BI). No clear difference was found between the micrographs obtained in the phosphateand cacodylate-buffered fixatives for the 2-hr exposure to La. After exposure for 4 hr at 4’C (Fig. 2, B2) the majority of terminals were fairly depleted of the vesicles. No synaptic contact was recognized at this time. By 17 hr all terminals were almost devoid of synaptic vesicles (Fig. 3, B3). Mitochondria did not appear to be much affected in some terminals, but other terminal mitochondria showed signs of swelling and internal membrane disruption. When muscles were treated with the La-saline at room temperature the effect of La was much more effective than at 4°C. Electron microscopy (Fig. 3, B3) of one muscle showed accumulation of high-density material consisting of synaptic vesicles, granules and cytoplasmic materials in the terminal treated with La for 2 hr at room temperature, which was roughly comparable with the treatment for I6 hr at 4 C (Fig. 3, B2). This type of electron-dense aggregation has also been found in the denervated nerve terminals in both vertebrate (Birks et ul., 1960; Miledi and Slater, 1970) and invertebrate (Frank, 1974; Wood and Usherwood, 1979) muscles.
A granular lanthanum precipitate occurred in the synaptic cleft and even in the terminal when the muscle was fixed with the phosphate buffer after exposure to 5 mM La for more than IO hr at 4 C (Fig. 2, B3). When cacodylate buffer was substituted for the phosphate the intracellular precipitate was absent after the exposure for I6 hr at 4 C (Fig. 3, B2). This result agrees with the finding of increased retention of lanthanum by the phosphate-buffered fixative (Heuser and Miledi, 1971). DISCUSSION
Electron microscopic examination of nerve terminals in the coxal depressor muscles of the cockroach Peripluneta americana indicated that the treatment 01 the junction with La induced ultrastructural changes associated with the functional disorganization. From previous electrophysiological works on the insect muscle (Washio and Miyamoto, 1983) neuromuscular transmission in Tenehrio muscles treated with 0.5 mM La was blocked irreversibly within about IO min. The acceleratory effect of 5 mM La on transmitter release lasted for less than I hr in Periplunetu muscles at room temperature. Therefore, after exposure to 5 mM La for 2 hr at room temperature we may expect that neuromuscular transmission to all fibres would be blocked and the MEPSP frequency would already be markedly reduced, and possibly completely stopped. The present work on electron microscopy of the terminal under the same condition (Fig. 3, B3) clearly demonstrated degeneration of the ultrastructural organization in the nerve terminal. Thus the ultrastructural changes may correlate with the blockage of neuromuscular transmission and the failure of MEPSPs. Correlated ultrastructural and electrophysiological
Lanthanum
on insect nerve terminals
593
Fig. 2. Nerve terminals exposed to 5 mM lanthanum at 4 C for 2 hr (Bl), 4 hr (B2) and 17 hr (B3) and of the control muscle exposed to normal Ringer at 4 C for 2 hr (Al), 4 hr (A2) and I7 hr (A3). The preparation was prefixed with glutalaldehyde and paraformaldehyde in phosphate buffer. Scale bar:
0.5 /rm.
works were made on the cockroach muscle during the application of metabolic inhibitors by Rees (1974). The study has shown agglutination of synaptic vesicles and disruption of the mitochondria, suggesting that these changes are causally related to a metabolic dysfunction of the terminal mitochondria. Although the present work showed signs of swelling and internal membrane disruption of terminal mitocondria, further study is needed to correlate the degree of the distribution of the cyto-architecture of the mitochondria and functional disorganization in the presence of La. Also, application of black widow spider
venom to the locust neuromuscular junction has been found to cause correlated changes in the spontaneous release of transmitter with ultrastructural disorganization (Cull-candy et al., 1973). Recently Florey and Kriebel (1983) have reported complete loss of synaptic vesicles with 34 hr of La treatment at 23°C in frog neuromuscular junctions, but no depletion was noticed at 4°C. Comparing the results obtained in the present work to those of the frog, the insect neuromuscular junction may be impaired more delicately by lanthanum ions morphologically and physiologically.
HIK~SHI WASHIO and ITSUKO NIHONUATSU
594
Fig. 3. Nerve terminals exposed to 5 mM lanthanum at 4’C for 2 hr (91). 17 hr (92) and at 22 C for 2 hr (B3), and of the control muscle exposed to the normal Ringer at 4 C for 2 hr (Al). 17 hr (A2), and at 22-C for 2 hr (A3). The preparation was prefixed in cacodylate buffer. The bar in 93 indicates 0.5 ilm for all part of this figure.
wish to thank Prof. K. Fujino for his helpful comments on the manuscript and Dr T. Arima for his technical suggestion. Thanks are also due to Miss Junko Kaneko for her technical assistance.
Acknowledgements-We
REFERENCES
Atwood H. L., Smyth T. and Johnston H. S. (1969) Neuromuscular synapses in the cockroach extensor tibiae muscle. J. Insect Physiol. 15, 529-535. Birks R., Katz 9. and Miledi R. (1960) Physiological and structural changes at the amphibian myoneural junction in the course of nerve degeneratton. J. Physiol., Lond. 150, 145-166.
Bhoch Z. L., Glagoleva I. M.. Liberman E. A. and Nenashev V. A. (1968) A study of the mechanism of quanta1 transmitter release at a chemical synapse. J. Physiol.. Land. 199, I l-35. Carbonell C. S. (1947) The thoracic muscles of the cockroach, Periplaneta 107, l-23.
americutu
Smithoniun
Inst. misc. Coil.
DeBassio W. A., Schnitzler R. M. and Parson R. L. (1972) Influence of lanthanum on transmitter release at the neuromuscular junction. J. Ncurohiol. 2, 263-278. Faeder I. R. and Salpeter M. M. (1970) The tine structure of nerve branches and neuromuscular junctions in the coxal adductor of the cockroach. Gromphadorhina portentosa.
J. Morphol.
132, 225-234.
Lanthanum on insect nerve terminals Florey E. and Kriebel M. E. (1983) Changes in acetylcholine concentration, miniature end-plate potentials and synaptic vesicles in frog neuromuscular preparations during lanthanum treatment. Camp. Biochem. Physiol. 75C, 285-294.
Frank E. (1974) The sensitivity to glutamate of denervated muscles of the crayfish. J. Physiol., Lond. 242, 371-382. Heuser J. and Miledi R. (1971)Effect of lanthanum ions on function and structure of frog neuromuscular junctions. Proc. R. Sot. B. 179, 247-260. Miledi R. and Slater C. R. (I 970) On the degeneration at rat neuromuscular junctions after nerve section. J. Physiol., Land. 207, 507-528.
Osborne M. P. (1975) The ultrastructure of nerve-muscle synapses. In Insect Muscle (Edited by Usherwood P. N. R.). pp. 161-205. Academic Press, London. Pearson K. G. and Iles J. F. (1971) Innervation of coxal depressor muscles in the cockroach, Periplaneta americana. J. exp. Biol. 54, 215-232.
595
Rees D. (1974) The effect of metabolic inhibitors on the cockroach nerve-muscle synapse. J. exp. Biol. 61, 33 l-343.
Rees D. and Usherwood P. N. R. (1972) Fine structure of normal and degenerating motor axons and nerve-muscle synapses in the locust, Schistocerca gregaria. Comp. Biothem. Physiol. 43A, 83-101.
Usherwood P. N. R., Cochrane D. G. and Rees D. (1968) Changes in structural, physiological and pharmacological properties of insect excitatory nerve-muscle synapses after motor nerve section. Nature 218, 589-591. _ Washio H. and Miyamoto T. (1983) Effect of lanthanum ions on neuromuscular transmission in insects. J. exp. Biol. 107, 405-414.
Wood M. R. and Usherwood P. N. R. (1979) Uhrastructural changes in cockroach leg muscle following unilateral neurotomy-I. Degeneration. J. Ultrastruct. 68, 265-280.