Brain Research, 160 (1979) 163-169 © Elsevier/North-HollandBiomedicalPress
163
Multiterminal innervation: non-uniform density along single lobster muscle fibers
D. E. MEISS and C. K. GOVIND Scarborough College, University of Toronto, West Hill, Ont. M I C 1A4 (Canada)
(Accepted September 7th, 1978)
The rules governing synaptic connectivity must account for the number and distribution (density) of synaptic terminals formed by a neuron on its target cell(s). The problem may be studied relatively easily in a lobster limb muscle, the distal accessory flexor muscle (DAFM) because it consists of a small number of fibers (22)lz, multiterminally innervated by an excitatory and an inhibitory axon 23. The single excitatory axon to the DAFM gives rise to a heterogenous population of synapses which differ in their physiological properties of transmitter release and facilitationis. Such synaptic diversity from a single motor axon has been shown in other crustacean muscles2,3,8,2° including the proximal accessory flexor muscle (PAFM) in lobster 12. In the last named muscle, synapses along a single muscle fiber though arising from separate primary branches of the axon possess similar facilitation properties. This had led Frank 12 to suggest that the muscle fiber may govern the facilitation properties of the synapses, i.e. a myotypic hypothesis of synaptic differentiation. In contrast in the DAFM, we find that the quantal content of synaptic transmission is significantly higher at the tendon end of a muscle fiber than at its exoskeletal end. Correspondingly the tendon end has a higher density of terminals, synapses and presynaptic dense bodies than the exoskeletal end of the fiber. This nonuniform distribution of innervation along single fibers in the lobster DAFM appears to be related to the branching pattern of the motor axon. The distal accessory flexor muscle (DAFM) in the meropodite of the 1st and 2nd walking legs of adult lobsters (body weight approximately 500 g) was used. The preparations were bathed in lobster saline which was continuously aerated and maintained at 10-12 °C. The saline had the following composition: NaC1, 472 mM; KC1, 10 mM; CaCI2, 16 mM; MgCI2, 7 mM; glucose, 11 mM; Tris.maleate 10 mM; pH 7.4. Conventional methods of stimulation and electrical recording were used4,1°. Both extracellular records of potentials at single synaptic foci (ERSPs) and intracellular records of excitatory post-synaptic potentials (EPSPs) were made during low frequency (1Hz) stimulation of the single excitor axon. A single muscle fiber was prepared for electron microscopy by methods described previously14. Serial cross-sections of the fiber were examined with Zeiss 9S
164 i.] J. I~T-,
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B1 ~~.,~._~.~.,.....~
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Fig. 1. Simultaneous records of ERSPs (A1, B1) and EPSPs (A2, B2) from the tendon (A) and exoskeletal (B) ends of a single fiber of the lobster distal accessory flexor muscle. Traces on the left show a single response at fast sweep while traces on the right show a number of responses at a slower sweep. At the slower sweep speed in A1, BI, the deflection below the horizontal base line is the ERSP while the deflection above the line is the stimulus artefact. Note the tendon end (A1) has no transmission failures while the exoskeletal end (B1) has 3 failures indicated by arrows. Calibration : vertical 200 b~Vfor A1, B1 ; 40 mV for A2, B2. Horizontal 40 msec, (left); 2 sec (right).
and Siemens Elmiskop 102 electron microscopes and quantitative analysis of the n u m b e r and size of terminals, synapses and presynaptic dense bodies was made from micrographs magnified to 26,000 x and 70,800 × 14 Extracellular records of synaptic potentials (ERSPs) evoked by stimulation of the single excitor axon were made simultaneously from two synaptic foci, one near the t e n d o n end and the other near the exoskeletal end of the fiber (Fig. 1). This eliminated the possibility of differential temporal depression in transmitter release at the two synaptic loci which would have gone undetected if they had been monitored separately in time. Fibers from the proximal and distal edges o f the muscle were sampled as they represented fibers with widely divergent synapses. Neuromuscular synapses on the distal fibers are solely of the high quantal output type, with mean quantal content (m) > 1.0, while those on the proximal fibers are of the low output type (m
163
Multiterminal innervation: non-uniform density along single lobster muscle fibers
D. E. MEISS and C. K. GOVIND Scarborough College, University of Toronto, West Hill, Ont. M I C 1A4 (Canada)
(Accepted September 7th, 1978)
The rules governing synaptic connectivity must account for the number and distribution (density) of synaptic terminals formed by a neuron on its target cell(s). The problem may be studied relatively easily in a lobster limb muscle, the distal accessory flexor muscle (DAFM) because it consists of a small number of fibers (22)lz, multiterminally innervated by an excitatory and an inhibitory axon 23. The single excitatory axon to the DAFM gives rise to a heterogenous population of synapses which differ in their physiological properties of transmitter release and facilitationis. Such synaptic diversity from a single motor axon has been shown in other crustacean muscles2,3,8,2° including the proximal accessory flexor muscle (PAFM) in lobster 12. In the last named muscle, synapses along a single muscle fiber though arising from separate primary branches of the axon possess similar facilitation properties. This had led Frank 12 to suggest that the muscle fiber may govern the facilitation properties of the synapses, i.e. a myotypic hypothesis of synaptic differentiation. In contrast in the DAFM, we find that the quantal content of synaptic transmission is significantly higher at the tendon end of a muscle fiber than at its exoskeletal end. Correspondingly the tendon end has a higher density of terminals, synapses and presynaptic dense bodies than the exoskeletal end of the fiber. This nonuniform distribution of innervation along single fibers in the lobster DAFM appears to be related to the branching pattern of the motor axon. The distal accessory flexor muscle (DAFM) in the meropodite of the 1st and 2nd walking legs of adult lobsters (body weight approximately 500 g) was used. The preparations were bathed in lobster saline which was continuously aerated and maintained at 10-12 °C. The saline had the following composition: NaC1, 472 mM; KC1, 10 mM; CaCI2, 16 mM; MgCI2, 7 mM; glucose, 11 mM; Tris.maleate 10 mM; pH 7.4. Conventional methods of stimulation and electrical recording were used4,1°. Both extracellular records of potentials at single synaptic foci (ERSPs) and intracellular records of excitatory post-synaptic potentials (EPSPs) were made during low frequency (1Hz) stimulation of the single excitor axon. A single muscle fiber was prepared for electron microscopy by methods described previously14. Serial cross-sections of the fiber were examined with Zeiss 9S
164 i.] J. I~T-,
A2 _ . , f ~
I '
, I
I ~
! l 'f J lT
LLLU_LLLLLL
B1 ~~.,~._~.~.,.....~
J __t_l__l__i
B2 . _ _ ~ ~
L
J
_J
{L}_LLLL L(
Fig. 1. Simultaneous records of ERSPs (A1, B1) and EPSPs (A2, B2) from the tendon (A) and exoskeletal (B) ends of a single fiber of the lobster distal accessory flexor muscle. Traces on the left show a single response at fast sweep while traces on the right show a number of responses at a slower sweep. At the slower sweep speed in A1, BI, the deflection below the horizontal base line is the ERSP while the deflection above the line is the stimulus artefact. Note the tendon end (A1) has no transmission failures while the exoskeletal end (B1) has 3 failures indicated by arrows. Calibration : vertical 200 b~Vfor A1, B1 ; 40 mV for A2, B2. Horizontal 40 msec, (left); 2 sec (right).
and Siemens Elmiskop 102 electron microscopes and quantitative analysis of the n u m b e r and size of terminals, synapses and presynaptic dense bodies was made from micrographs magnified to 26,000 x and 70,800 × 14 Extracellular records of synaptic potentials (ERSPs) evoked by stimulation of the single excitor axon were made simultaneously from two synaptic foci, one near the t e n d o n end and the other near the exoskeletal end of the fiber (Fig. 1). This eliminated the possibility of differential temporal depression in transmitter release at the two synaptic loci which would have gone undetected if they had been monitored separately in time. Fibers from the proximal and distal edges o f the muscle were sampled as they represented fibers with widely divergent synapses. Neuromuscular synapses on the distal fibers are solely of the high quantal output type, with mean quantal content (m) > 1.0, while those on the proximal fibers are of the low output type (m
