Neuromuscular junctions in the buccal mass of Aplysia: Fine structure and electrophysiology of excitatory transmission

The lower extrinsic protractor muscle in the buccal mass of Aplysia consists of bundles of muscle fibers 4–12 m̈ in diameter, containing thick and thin filaments that are not arranged in a transversely striated pattern. Individual fibers come close to one another and form specialized junctional regions. Electrophysiological evidence indicates that the muscle fibers form an electrical syncytium. Muscle bundles are innervated by more than one excitatory axon at a number of points along their length. The presynaptic terminals contain spherical electron-lucent vesicles and a few larger electron-dense vesicles. There are no obvious structural postsynaptic specializations. Graded contraction can result from summation of excitatory junctional potentials in separate axons or from summation and facilitation of junctional potentials from a single axon. The buildup of facilitation during a train of stimuli results from the linear summation of facilitation remaining from preceding impulses.     

Fast-axon synapses of a crab leg muscle

Neuromuscular synapses of the "fast" excitatory axon supplying the main extensor muscle in the leg of the shore crab Pachygrapsus crassipes were studied with electrophysiological and electron-microscopic techniques. Electrical recording showed that many muscle fibers of the central region of the extensor muscle responded only to stimulation of the fast axon, and electron microscopy revealed many unitary subterminal axon branches. Maintained stimulation, even at a low frequency, resulted in depression of the excitatory junctional potentials (EJPs) set up by the fast axon but EJPs of different muscle fibers depressed at different rates, indicating some physiological heterogeneity among the fast-axon synapses. Focal recording at individual synaptic sites on the surfaces of the muscle fibers showed quantal contents ranging from 1.4 to 5.5 at different synapses; these values are relatively high in comparison with similar determinations made in the crayfish opener muscle. Synapse-bearing nerve terminals were generally relatively small in diameter and filiform, with many individual synaptic contact areas of uniform size averaging 0.6 micron2. All of the individual synapses had a presynaptic "dense body" at which synaptic vesicles clustered. If these structures represent release points for transmitter quanta, the initial high quantal content would have an ultrastructural basis. The mitochondial content of the nerve terminals, the synaptic vesicle population, and the specialized subsynaptic sarcoplasm were all much reduced in comparison with tonic axon synaptic regions in this and other crustaceans. The latter features may be correlated with the relatively infrequent use of this axon by the animal, and with rapid fatigue.     

Increased effectiveness of a motorneuron after partial denervation of its target muscle in the cricket Teleogryllus oceanicus

Fibers of the metathoracic extensor tibia muscle of the cricket Teleogryllus oceanicus are innervated by a slow excitatory axon (slow fibers), a fast excitatory axon (fast fibers), or by both slow and fast axons (dual fibers). Sectioning metathoracic nerve 5 removes the fast axon input to the muscle but not that of the slow axon. Following such partial denervation, the mechanical responses initiated by the slow axon increase progressively for at least 30 days; twitch tensions reach 5–10 times those of control muscles and tetanic tensions 10–30 times control values. After sectioning nerve 5, resting membrane potentials decrease in those fibers which originally received fast axon input and the input resistance of all fiber types increases, including that of slow fibers which are not innervated through nerve 5. Excitatory junctional potentials (EJPs) initiated by the slow axon become larger following partial denervation, accounting in part for the larger contraction amplitudes. The increased input resistance is adequate to account for the larger EJPs in slow fibers but not for the proportionally greater increase in EJP amplitude in fibers which were formerly dually innervated. The change in EJP amplitude is abrupt in slow fibers and gradual in formerly dual fibers.     

The Distribution of Pre- and Postsynaptic Inhibition at Crustacean Neuromuscular Junctions

The relative contribution of pre- and postsynaptic mechanisms to peripheral inhibition has been analyzed in the abdominal slow flexor muscles of crayfish and lobsters. The conductance of the muscle fiber membrane may be increased to five or more times its resting value by repetitive stimulation of the peripheral inhibitory axon, and this effect accounts for all of the attenuation exerted by the inhibitor against excitatory junctional potentials. No "critical interval" has been found at which an inhibitory nerve impulse produces anomalously large reduction of a following depolarizing junctional potential; electrotonic depolarizations and junctional potentials are identically affected under all phase conditions. The presynaptic inhibitory mechanism is, therefore, absent in this system. In the dactyl opener muscle, on the contrary, most of the attenuation of excitatory junctional potentials is achieved presynaptically, though equally large postjunctional conductance changes are also seen (Dudel and Kuffler, 1961). The difference is correlated with a difference in the reflex operation of the two muscles. Reflex inhibition in the abdominal slow flexors is primarily central, whereas in the dactyl opener, inhibition is brought about by an increase in inhibitory nerve discharge frequency without central suppression of the single excitatory axon. The function of peripheral inhibition in the abdominal flexors is presumably to terminate residual depolarization by reducing the long time-constant of the muscle fibers.     

Temperature effects on a slow-crustacean neuromuscular system

The membrane potential of the E2 axon and the bender muscle fibers increased with temperature. The input resistance of the axon, the spike amplitude and time course declined with temperature. Excitatory junctional potentials (ejps) exhibited maximum amplitudes and minimum facilitation at about the same temperature. Ejp time course and muscle membrane input resistance declined with temperature. Tension produced by the muscle also declined but then increased when additional spikes were generated in the periphery of the E2 axon.     

Peripheral generation and modulation of crustacean motor axon activity at high temperatures

Summary We have examined the effects of temperature changes on the stretcher muscle and its motor supply in a crab (Pachygrapsus crassipes). An increase in temperature caused a decrease in the amplitude of evoked excitatory junctional potentials (ejp's). Above a critical threshold a single action potential in the excitor (E) or specific inhibitor (SI) axon provoked multiple spikes in the appropriate axon and concomitant ejp's or inhibitory junctional potentials (ijp's) in the stretcher muscle fibers. The critical temperature for generation of peripheral spikes was dependent upon the crab's thermal history.In preparations in which a shock to the E axon evoked repetitive firing, stimulation of the SI axon at about the same time as the E axon abolished or curtailed the peripherally generated E axon responses. No reciprocal modulation of SI activity by the E axon was observed. GABA abolished the peripheral generation of E spikes and picrotoxin prevented SI modulation of E activity. We suggest that the site of SI modulation is at the axo-axonal synapses, possibly at the fine E axon branches and the bottlenecks along the E axon where inhibitory synapses have been observed.Abbreviations CI common inhibitor (axon) - E excitor (axon) - ejp excitatory junctional potential - ijp inhibitory junctional potential - SI specific inhibitor axon This work was supported by grants awarded to Dr. Atwood from the National Research Council of Canada and the Muscular Dystrophy Association of Canada.     

Penis-retractor muscle of Aplysia: excitatory motor neurons

Cobalt axonal iontophoresis and intracellular recordings were used to identify a cluster of several motor neurons innervating the penis-retractor muscle of Aplysia. Intracellularly recorded motor neuron action potentials elicited direct, one-for-one, constant latency excitatory junctional potentials (ejps) in individual muscle fibers. The axons of motor neurons could be recorded extracellularly in the penis-retractor nerve and stimulation of the nerve backfired the motor neurons. Perfusion of the ganglion, the muscle, or both with solutions of either increased Mg++/decreased Ca++ or increased Ca++ sea water indicated that the presumed motor neuron impaled was not a sensory cell and that interneurons were not intercalated in the pathway. Innervation of muscle fibers was found to be functionally polyneuronal and diffuse. The ejps were found to undergo marked facilitation with repetitive motor-neuron stimulation. The motor neurons were isolated in a distinct cluster in the right pedal ganglion. Their electrical activity was characterized by spontaneous irregular action potentials and a moderate input of postsynaptic potentials.     

Physiological properties of the penis retractor muscle of Aplysia

The properties of the penis retractor muscle of Aplysia have been studied using intracellular, sucrose gap and tension recording. The fibers are of the invertebrate smooth muscle type and exhibit slow contractions which occur spontaneously or in response to stretch in isolated preparations. Individual muscle fibers are innervated by excitatory and inhibitory axons. A variety of sizes of excitatory and inhibitory junctional potentials can be recorded from them. The innervation is probably diffuse and functionally polyneuronal. The fibers are electrically coupled, permeable to potassium and chloride at rest, and exhibit no overshooting active responses. The muscle shows graded responses of depolarization and contraction proportional to strength of nerve stimulation. Facilitation and depression of junctional potentials are seen with various frequencies of nerve stimulation. Post-tetanic potentiation occurs with nerve stimulation at frequencies from 2 to 50 Hz and is suppressed in the presence of increased extracellular calcium concentrations.     

Neuromuscular relationships in the abdomen of the Californian shore crab Pachygrapsus crassipes

There are two pairs of muscles in each abdominal segment of the crab; one pair of flexors and one pair of extensors. In the early larval stages the muscles have short sarcomeres--a property of fast fibers--and high thin to thick filament ratios--a property of slow fibers. In the adult the abdominal muscles are intermediate and slow, since they have fibers with intermediate and long sarcomeres, high thin to thick filament ratios, low myofibrillar ATPase activity, and high NADH diaphorase activity. The different fiber types are regionally distributed within the flexor muscle. Microelectrode recordings from single flexor muscle fibers in the adult showed that most fibers are supplied by three excitatory motor axons, although some are supplied by as many as five efferents. One axon supplies all of the flexor muscle fibers in its own hemisegment, and the evoked junctional potentials exhibit depression. This feature together with the innervation patterns of the fibers are similar to those reported for the deep flexor muscles of crayfish and lobsters. Therefore, in the adult crab, the abdominal flexor muscles have some features in common with the slow superficial flexors of crayfish and other features in common with the fast deep flexor muscles.     

Structure and contractile properties of the ostial muscle (musculus orbicularis ostii) in the heart of the American lobster

“Venous” blood enters the crustacean heart through bivalved ostia. Each ostium is a discrete anatomical unit that remains functional even when isolated from the heart. Muscle fibers produce overshooting action potentials that have a plateau of variable duration in response to nervous drive from the cardiac ganglion or during trains of electrical stimuli. Contractions show summation and facilitation when stimulated by trains of stimuli delivered at rates greater than 0.5 s⁻¹ and 0.2 s⁻¹, respectively. Contraction amplitude increases with stimulating impulse frequency and train duration. Maximum force occurs at 1.2 times the slack length. The morphology of ostial fibers resembles that of myocardial fibers. Interconnected bundles of myofilaments occur in both the ostial fibers and the myocardial fibers. In ostial and myocardial fibers, the myofilament bundles are invested by perforated sheets of sarcoplasmic reticulum, and these sheets interface with a network of sarcolemmal tubules to form dyadic interior couplings at the level of the sarcomeric H-bands. The contractile apparatus originates and terminates at intermediate junctions on the transverse cellular boundaries, and the lateral surfaces of the muscle fibers are linked by a modest number of communicating (gap) junctions. Accepted: 10 August 1999     

Crustacean Neuromuscular Mechanisms: Functional Morphology of Nerve Terminals and the Mechanism of Facilitation

Two aspects of crustacean neuromuscular physiology are discussed:(1) the ultrastructural identification of the excitatory andinhibitory nerve terminals, and (2) the characteristics of,and the possible mechanisms for, facilitation. The first problem was studied in crayfish opener muscle whichhas one excitatory and one inhibitory axon. One of the nerveswas stimulated in the presence of DNP until synaptic transmissionfailed; the preparations were then fixed for electron microscopy.Whenever the excitatory nerve was stimulated, the terminalswith round synaptic vesicles were depleted while nearby terminalswith smaller elongate vesicles were normal. When the inhibitorynerve was stimulated, the converse was true. The possible reasons for the diversity in crustacean neuromuscularproperties are discussed. Large EPSP's with a high quantal content(m), appear to be produced by terminals which are invaded bya propagated spike. Small EPSP's (small m) appear to be producedby terminals which don't spike and which are depolarized bya decrementally conducted potential. There is an inverse relationshipbetween m and the amount of facilitation. The physiologicalbasis for facilitation is discussed; previous hypotheses arefound wanting and a new one is proposed, that of slow depolarization.     

Fine structure of an octopaminergic neuron and its terminals

The large octopaminergic dorsal unpaired median neuron of the locust that innervates the extensor tibiae muscle, DUMETi, was examined electronmicroscopically. Its soma contains many Golgi complexes apparently making dense-core vesicles similar to those found in peripheral branches and terminals. There are also larger stores of the dense material in the soma, especially near the exit of the principal neurite, that are not in vesicular form. Since the neurons can be penetrated and stimulated by microelectrodes, they form favorable subjects for direct studies of the control of neurosecretion. Preterminal fine branches of the neuron were located in proximal outer bundles of muscle fibers into which they had been traced electrophysiologically. They contain numerous large dense-core vesicles arrayed in rows near microtubules. These fine branches have a thick layer of collagenous connective tissue between the axon and the muscle fiber. Final terminals have varicosities containing many vesicles, lying inside the outer layers of the sarcolemmal complex of muscle fibers. They do not form synaptic structures. Terminals of another DUM neuron, one that innervates the dorsal longitudinal flight muscles (DUMDL), were similar in detail to those of DUMETi. DUMETi swelled about 20-fold in cross-sectional area above a ligature, in a 12-hr period, indicating that there is an extensive centrifugal flow of material in it, and sprouted a branch.     

Neuromuscular Junctions in Flight and Tymbal Muscles of the Cicada

The tymbal muscle fiber in the cicada closely resembles the indirect flight muscle fiber in its structural detail. We agree with other authors that the tymbal muscle is a modified indirect flight muscle. The peripheral nerve branches to the tymbal and flight muscle fibers are similar to those in the wasp leg. The axon is loosely mantled by irregular turns of the mesaxon, enclosing cytoplasm. The nerve is therefore a tunicated nerve. The neuromuscular junction in the high frequency muscle fibers shows direct apposition of plasma membranes of axon and muscle fiber, large numbers of mitochondria and synaptic vesicles in the axon, and concentrations of mitochondria, aposynaptic granules, and endoplasmic reticulum in the postsynaptic area of the muscle fiber. Of special interest is the multitude of intracellular, opposing membranes in the postsynaptic area. They form laminated stacks and whorls, vesicles, cysternae, and tubules. They occasionally show continuity with the plasma membrane, the outer nuclear envelope, and the circumfibrillar endoplasmic reticulum. The membrane system in this area is designated "rete synapticum." It is believed to add to the electrical capacity of the neuromuscular junction, to serve in transmission of potentials, and possibly is the site of the oscillating mechanism in high-frequency muscle fibers.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : I. Cytological and Cytochemical Studies on the Neuromuscular Junction of Differentiating Muscle in the Regenerating Limb of the Newt Triturus

Development of the neuromuscular junction on differentiating muscle was investigated in the regenerating limb of the newt Triturus. Motor end-plate formation begins when vesicle-filled axon terminations approach differentiating muscle cells that have reached the stage of a multinucleate cell containing myofibrils. Slight ridges or elevations occur on the muscle surface, and there is an increase in density of the cytoplasm immediately beneath the plasma membrane of the elevation. The axon becomes more closely approximated to the muscle cell and comes to lie in a shallow depression or gutter on the surface of the muscle. The surface ridges increase in length and constrict at their bases to form junctional folds. In the axon terminal, focal accumulations of vesicles are found where the axon contour projects slightly opposite the secondary synaptic clefts. Cholinesterase activity in the developing junctions was demonstrated by the thiolacetic acid-lead nitrate method. Enzymatic activity is not found on intercellular nerve fibers or the muscle surface prior to close approximation of axon endings and muscle. Eserine- and DFP-sensitive activity appears concurrently with morphological differentiation. Activity occurs in membranous tubulovesicles in the sarcoplasm subjacent to the neuromuscular junction and in association with the sarcolemma. The largest reaction deposits occur at the tips of the emerging junctional folds. Smaller and less numerous localizations occur on the axon membrane and within the axoplasm. It is concluded from these studies that the nerve endings have an inductive effect on both the morphological and chemical specializations of the neuromuscular junction.     

The oculomotor system of Daphnia magna

Summary The highly mobile cyclopic compound eye of Daphnia magna is rotated by six muscles arranged as three bilateral pairs. The three muscles on each side of the head share a common origin on the carapace and insert dorsally, laterally and ventrally on the eye. The dorsal and ventral muscles are each composed of two muscle fibers and the lateral muscle is composed of from two to five fibers, with three the most common number. Individual muscle fibers are spindle-shaped mononucleated cells with organized bundles of myofilaments. Lateral eye-muscle fibers are thinner than those of the other muscles but are otherwise similar in ultrastructure. Two motor neurons innervate each dorsal and each ventral muscle and one motor neuron innervates each lateral muscle. The cell bodies of the motor neurons are situated dorsally in the supraesophageal ganglion (SEG) and are ipsilateral to the muscles they innervate. The dendritic fields of the dorsal-muscle motor neurons are ipsilateral to their cell bodies; those of the ventral-muscle motor neurons are bilateral though predominantly contralateral. The central projections of the lateral-muscle motor neurons are unknown. In the dorsal and ventral muscles one motor axon synapses principally with one muscle fiber; in each lateral muscle the single motor axon branches to, and forms synapses with, all the fibers. The neuromuscular junctions, characterized by pre- and postsynaptic densities and clear vesicles, are similar in all the eye muscles.     

Inhibitory innervation of a lobster muscle

Summary Inhibitory neuromuscular synapses formed by the common inhibitor (CI) neuron on the distal accessory flexor muscle (DAFM) in the lobster, Homarus americanus, were studied with electrophysiological and electron-microscopic (thin-section and freeze-fracture) techniques. Postsynaptic inhibition as indicated by inhibitory junctional potentials was several-fold stronger on distal compared to proximal muscle fibers. This difference correlated with the results of serial thin-section studies, which showed more inhibitory synapses on distal fibers than on their proximal counterparts. Effects of postsynaptic inhibition on excitatory junctional potentials via current shunting had a morphological correlate in the spatial relationship between inhibitory and excitatory synapses on the distal fibers. Inhibitory synapses were larger than their excitatory counterparts and had fewer glial processes. In freeze-fracture views, inhibitory synapses did not appear as raised plateaus in the P-face as do excitatory synapses, and their active zones were more widely scattered. The intramembrane particles in the inhibitory postsynaptic membrane-representing neurotransmitter receptors-are arranged in parallel rows in the sarcolemmal P-face and have complementary furrows in the sarcolemmal E-face. Altogether, our findings help to describe a population of inhibitory neuromuscular synapses formed by the CI neuron in lobster muscle.     

Excitatory neural control of posterograde heartbeat by the frontal ganglion in the last instar larva of a lepidopteran, Bombyx mori

The frontal ganglion of the silkworm (Bombyx mori) gives rise to a visceral nerve, branches of which include a pair of anterior cardiac nerves and a pair of the posterior cardiac nerves. Forward-fill of the visceral nerve with dextran labeled with tetramethyl rhodamine shows the anterior cardiac nerves innervate the anterior region of the dorsal vessel. Back-fill of the anterior cardiac nerves with Co²⁺ and Ni²⁺ ions and the fluorescent dye reveals that the cell bodies of two motor neurons are located in the frontal ganglion. Injection of 5, 6-carboxyfluorescein into the cell body of an identified motor neuron shows that the neuron gives rise to an axon running to the visceral nerve. Unitary excitatory junctional potentials (EJPs) were recorded from a myocardial cell at the anterior end of the heart. They responded in a one-to-one manner to electrical stimuli applied to the visceral nerve, or to impulses generated by a depolarizing current injected into the cell body. EJPs induced by stimuli at higher than 0.5 Hz showed facilitation while those induced at higher than 2 Hz showed summation. Individual EJPs without summation, or a train of EJPs with summation, caused acceleration in the phase of posterograde heartbeat and heart reversal from anterograde heartbeat to posterograde heartbeat. It is likely that the innervation of the anterior region of the dorsal vessel by the motor neurons, through the anterior cardiac nerves is responsible for the control of heartbeat in Lepidoptera, at least in part.     

Heterogeneity of excitatory synapses at the ends of single muscle fibers in lobster,Homarus americanus

Crustacean neuromuscular synapses arising from a single excitor axon are known to be well differentiated among different muscle fibers but little is known about their condition along single fibers. Focal recording techniques were used to examine the quantal transmitter release and facilitation properties of synapses in the single excitatory innervated distal accessory flexor muscle of the lobster, Homarus americanus. Synapses were reliably differentiated with respect to quantal output so that those located near the tendon end were 1.15–4.12 times greater than those at the opposite, exoskeletal end (p < 0.01, paired t-test). Regional differences were also seen in the amount of facilitation determined from twin pulse experiments. The fine structural basis for these differences was determined by serial section electron microscopy of 10-μm segments at each end to ensure that the area of focal recording was sampled. No quantitative differences were found in the terminals or synapses in the two regions. Instead, the physiological diversity was correlated with number and size of presynaptic dense bars. Thus, the tendon end had a greater number and larger mean surface area of dense bars compared to the exoskeletal end. This heterogeneity of excitatory multiterminal innervation is correlated with the axonal branching pattern. Thus, the main axon and the larger primary axon branches lie in close proximity to the tendon end of the muscle fibers, whereas the exoskeletal end is innervated by smaller secondary and tertiary axonal branches. This proximity to the large axonal branches of the higher quantal output synapses at the tendon end may be regulated by some neural influence including a timing of innervation and/or access to greater amounts of metabolites in the larger branches which may be conducive to forming high-output synapses.     

Immunogold electron microscopic demonstration of glutamate and GABA in normal and deafferented cerebellar cortex: correlation between transmitter content and synaptic vesicle size

Selective labeling of mossy fiber terminals and parallel fibers was obtained in rat cerebellar cortex by a glutamate antibody produced and characterized by Hepler et al. The high-resolution electron microscopic immunogold demonstration of this amino acid offered the possibility of determining the size and shape of synaptic vesicles in glutamate-positive mossy endings. Mossy terminals that stained with the glutamate antibody formed two distinct populations, one with spherical synaptic vesicles with an average diameter of 34.0 nm (more than 90% of all mossy fiber endings) and one with pleomorphic and smaller synaptic vesicles which had an average diameter of 28.5 nm. We present experimental evidence that the mossy terminals with large round vesicles are of extracerebellar origin, whereas those with small pleomorphic synaptic vesicles are endings of nucleocortical fibers. The presence of two distinct classes of gamma-aminobutyric acid (GABA)-containing axon terminals within cerebellar glomeruli has also been demonstrated; those originating from the cerebellar nuclei contain large (36.2 nm) synaptic vesicles, whereas the majority of GABA-stained axon terminals that are of local (cortical) origin contain small (29.1 nm) synaptic vesicles. It therefore appears that, at least in the case of glutamate and GABA, morphological characterization of the axon terminals based on the size and shape of synaptic vesicles is not a reliable indicator of their functional nature (i.e., whether they are excitatory or inhibitory); convincing evidence for the identity of the transmitter can be obtained only by electron microscopic immunostaining procedures. Our results also suggest the existence of both inhibitory and excitatory feedback from cerebellar nuclei to cerebellar cortex.     

DEVELOPMENT OF THE NEUROMUSCULAR JUNCTION : II. Cytological and Cytochemical Studies on the Neuromuscular Junction of Dedifferentiating Muscle in the Regenerating Limb of the Newt Triturus

Following amputation of the limb of the newt, Triturus viridescens, muscle fibers dedifferentiate giving rise to mesenchymal cells. The earliest changes detected in neuromuscular junctions of dedifferentiating muscle fibers are the appearance of a few vacuoles and decrease in density of the terminal axoplasm. Later, synaptic vesicles become tightly clustered in the axon termination, and their content appears denser than normal. Then, vesicles diminish in number until few are seen in the ending. While these changes are occurring, the area of contact of nerve with muscle becomes smaller. Junctional folds persist only where the nerve maintains contact with muscle, but these are shorter than normal and appear as slight ridges on the muscle surface. Subsequently, the nerve withdraws from the muscle cell and is completely invested by Schwann cell cytoplasm, and all traces of junctional folds are lost at the former region of contact. Cholinesterase activity was localized with the thiolacetic acid-lead nitrate method. Even before marked morphological changes occur in the junction, DFP- and physostigmine-sensitive activity in the cleft between nerve and muscle is decreased in intensity. Activity continues to decrease as the area of nerve-muscle contact diminishes and junctional folds disappear. When the nerve has withdrawn from the muscle surface, only a few small deposits of lead are left in the intervening region. These results show that as muscle becomes less specialized during dedifferentiation, the neuromuscular junction also loses the cytological and cytochemical specializations associated with synaptic function.