Structural definition of the neuromuscular system in the swimming-paddle opener muscle of blue crabs

Blue crabs are excellent swimmers, using their highly modified last pereiopods as sculling paddles. Hence, the hypertrophied paddle opener muscle was examined for adaptations of its motor innervation by an excitor and a specific inhibitor axon. The muscle has a uniform composition of slow fibers with long (6-12 microm) sarcomere lengths. Individual fibers are richly innervated with approximately two-thirds excitatory and one-third inhibitory innervation. The profuse excitatory innervation reflects the high activity levels of this motoneuron in swimming. Adaptation to sustained activity associated with swimming is also reflected in the motor nerve terminals by a high concentration of energy source, which is equally divided between glycogen granules and mitochondria, the former providing a more rapid source of energy. The excitor axon makes predominantly neuromuscular synapses, but also a few synapses onto the inhibitor axon. The location of these excitatory axoaxonal synapses suggests regional modulation of the inhibitor axon. The specific inhibitor axon makes less than two-thirds of its synapses with the muscle fiber, regulating contraction via postsynaptic inhibition. The remaining inhibitory synapses are onto the excitor axon, signaling very strong presynaptic inhibition. Such presynaptic inhibition will effectively decouple the opener muscle from the stretcher muscle even though both are innervated by a single excitor axon.     

The closer muscle is a second target for the stretcher inhibitor motoneuron of the crayfish's thoracic limbs

Summary The stretcher inhibitor motoneuron of each thoracic limb of a crayfish (Pacifastacus leniusculus) was consistently found to innervate parts of the closer muscle, in addition to the stretcher muscle; it is thus not a specific inhibitor as previously thought. The common inhibitory motoneuron also innervates parts of both muscles. Some individual closer muscle fibers are inhibited more strongly by one inhibitor, some by the other, and some fairly equally by both; no general rule governing the inhibitors' closer muscle outputs became evident. In the claw, the distal closer fibres with the longest membrane time constants are all strongly inhibited by the stretcher inhibitor, and some by the common inhibitor as well.No other thoracic limb muscles were found to receive the stretcher inhibitor. The opener inhibitor's effects could be detected only in the opener muscle. The common inhibitor inhibits all walking leg muscles effectively. In the cheliped, it consistently inhibits all except the opener muscle, where its output may be vestigial. Its axon emerges through the ganglion's first root, whereas the opener and stretcher inhibitors' axons pass through the second root. The fast and slow excitatory axons to the extensor muscle also exit separately through the first and second roots, as in locusts.Abbreviations CI common inhibitor - EJP excitatory junctional potential - IJP inhibitory junctional potential - OI opener inhibitor - SI stretcher inhibitor     

The distribution of the common inhibitory neuron in brachyuran limb musculature

Summary In the walking legs of two common crabs, antidromic stimulation of the common inhibitory axon (CI) from either opener or closer nerve produces inhibitory potentials in certain fibers of every muscle distal to the ischiopodite. In particular, CI inhibits the flexor and accessory flexor muscles of the meropodite and abolishes or reduces contractile force in the flexor. The specific opener inhibitor, OI, formerly believed to innervate the flexor, has no electrical or mechanical effect on this muscle. The brachyuran inhibitory limb innervation thus appears to be the same as that accepted for the anomurans, comprising one universally distributed common inhibitor and two truly specific inhibitors serving the opener and stretcher muscles.Abbreviations CI common inhibitor - OI opener inhibitor - SI stretcher inhibitor - FI flexor inhibitor - FE flexor excitor(s)     

Effects of stimulus timing on transmitter release and postsynaptic membrane potential at crayfish neuromuscular junctions

Summary Different synaptic terminals of the single excitor axon to the opener muscle of crayfish (Procambarus clarkii) often release transmitter in a very different manner when stimulated with the same equal-interval, doublet, or triplet patterns. Compared to synapses that show little facilitation (low F_e synapses), highly facilitating (high F_e) synapses show greater percentage increases in several measures of synaptic efficacy when stimulated with any of these patterns. Low F_e synapses usually show the greater absolute changes in these measures of synaptic efficacy. Changes in the span and pattern of doublets and triplets can independently affect both pre- and postsynaptic measures of synaptic efficacy at either low F_e or high F_e synapses.Abbreviations EJP excitatory junctional potential - MJP spontaneous miniature EJP - F _e ratio of EJP at 1 Hz to EJP amplitude at 10 Hz - F ₁ zero-time facilitation - A ₂,B ₂,C ₂ doubly corrected EJP amplitude of a particular pulse - average amplitude of doubly corrected EJPs in a train of equal-interval, doublets, and triplets, respectively - A_m, B_m, C_m maximum depolarization reached by a particular EJP - time constant of decay     

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.     

Presynaptic inhibition and the participation of GABAB receptors at neuromuscular junctions of the crab Eriphia spinifrons

Presynaptic inhibition exerted by the common inhibitor on the closer and opener muscles and by the specific inhibitor on the opener muscle was investigated in the crab Eriphia spinifrons. In the closer muscle, activation of GABA_B receptors by baclofen reduced the mean quantal content of excitatory junctional currents by about 25%. Blocking GABA_B receptors with CGP 55845 diminished presynaptic inhibition at a similar percentage. GABA_B receptor-mediated presynaptic inhibition is linked to G proteins. Application of pertussis toxin eliminated about 25% of the inhibition exerted by the common inhibitory neuron. GABA_B receptors participate in presynaptic inhibition at release boutons of the slow and the fast closer excitor at a similar percentage. In the opener muscle, presynaptic inhibition of transmitter release from the same endings of the opener excitor was about 15% stronger with the specific inhibitor than with the common inhibitor. About 10% of the presynaptic inhibition produced by either one of the two inhibitors could be abolished by blocking GABA_B receptors. The amplitudes of the excitatory junctional currents in the opener were reduced in the presence of baclofen by about 25%, suggesting that synaptic terminals of the opener excitor are endowed with a similar percentage of GABA_B receptors as terminals of the slow and the fast closer excitors. Baclofen had no effect on postsynaptic inhibition, indicating that GABA_B receptors are not involved in postsynaptic neuromuscular inhibition. Accepted: 8 January 2000     

Differences in synaptic output between excitatory and inhibitory motoneurons in a crayfish muscle

A pair of antagonistic motoneurons, one excitatory and one inhibitory, innervates the distal accessory flexor muscle in the walking limb of the crayfish Procambarus clarkii. The number and size of synapses formed by these two axons on the muscle fibers (neuromuscular synapses) and on each other (axo-axonal synapses) were estimated using thin-section electron microscopy. Although profiles of nerve terminals of the two axons occur in roughly equal proportions, the frequency of occurrence of neuromuscular synapses differed markedly: 73% were excitatory and 27% were inhibitory. However, inhibitory synapses were 4–5 times larger than excitatory ones, and consequently, the total contact areas devoted to neuromuscular synapses were similar for both axons. Axo-axonal synapses were predominantly from the inhibitory axon to the excitatory axon (86%), and a few were from the excitatory axon to the inhibitory axon (14%). The role of the inhibitory axo-axonal synapse is presynaptic inhibition, but that of the excitatory axo-axonal synapse is not known. The differences in size of neuromuscular synapses between the two axons may reflect intrinsic determinants of the neuron, while the similarity in total synaptic area may reflect retrograde influences from the muscle for regulating synapse number.     

Asymmetry of motor impulses and neuromuscular synapses produced in crayfish claws by unilateral immobilization

Summary The fast axon supplying the closer muscle in crayfish (Procambarus clarkii) normally fires few impulses and generates large excitatory postsynaptic potentials (EPSPs) that fatigue rapidly with repeated stimulation. When the dactyl of one claw is immobilized in the closed position, impulse production in the fast axon decreases on the immobilized side and increases on the contralateral side. On the immobilized side, EPSPs become larger but more readily depressed with repeated stimulation, while converse changes occur on the contralateral side.In order to establish whether the smaller number of impulses on the immobilized side was responsible for the changes in EPSPs, extra impulses were generated in the fast axon of immobilized claws by implanting electrodes in the claw. Raising the impulse production to equal or exceed that of the contralateral side did not prevent the changes in EPSPs produced by immobilization. Thus, it is probable that changes in the level of synaptic input to central parts of the fast closer excitor neuron are mainly responsible for altered physiological properties of peripheral synapses, rather than the fast axon's impulse traffic per se.     

Stimulation-induced changes at crayfish (Procambarus clarkii) neuromuscular terminals

Summary The fine structure of neuromuscular terminals of the single excitor axon was examined in the limb stretcher muscle of the crayfish Procambarus clarkii. A morphometric comparsion of the neuromuscular terminals of the left and right limbs of a control crayfish showed them to be similiar in qualitative as well as quantitative features. The excitor axon to the stretcher muscle of the right side was stimulated, by backfiring its branches in the adjacent opener muscle, at 20 Hz for 4–5 h per day over 4–5 days. The stretcher muscle on the left side was not stimulated and served as a control. Morphometric analysis of stimulated terminals revealed an increase in the number of dense bars and synaptic vesicles compared to their non-stimulated, contralateral counterparts. Since dense bars are regarded as active sites of transmitter release, changes in their number provide a morphological basis for synaptic plasticity.     

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.     

Innervation pattern of the deep extensor abdominal muscle in the crayfish species Astacus astacus

The deep extensor abdominal muscle consisting of one medial and two lateral muscle bundles together with the nerve innervating the muscles of crayfish species Astacus astacus, was prepared. Light microscopic investigations of methylene blue stained preparations showed that the nerve innervating the deep extensor abdominal muscle consists of five distinct axons. The five axons were stained separately with lucifer yellow and the innervation pattern of the axons was determined. To confirm the histological results the axons were also stimulated with a suction electrode to elicit excitatory postsynaptic currents on the muscle membrane which were detected using a macro patch electrode. The muscle is innervated by a common excitatory and a common inhibitory axon branching over all three muscle bundles and sending additionally a branch to the L1-bundle of the next posterior segment, and by two axons specific for the two lateral muscle bundles. The axon specific for the innervation of the L1-bundle sends also a branch to the L1-bundle of the next posterior segment. In addition there is one excitatory axon which directly innervates the medial muscle bundle of the next posterior segment branching in most of the cases also to the medial bundle of the segment where it originates.Abbreviations DEAM deep extensor abdominal muscle - EPSC excitatory postsynaptic current - IPSC inhibitory postsynaptic current - L lateral - M medial - GABA -aminobutyric acid     

The Cdk5 inhibitor Roscovitine increases LTP induction in corticostriatal synapses

In corticostriatal synapses, LTD (long-term depression) and LTP (long-term potentiation) are modulated by the activation of DA (dopamine) receptors, with LTD being the most common type of long-term plasticity induced using the standard stimulation protocols. In particular, activation of the D1 signaling pathway increases cAMP/PKA (protein kinase A) phosphorylation activity and promotes an increase in the amplitude of glutamatergic corticostriatal synapses. However, if the Cdk5 (cyclin-dependent kinase 5) phosphorylates the DARPP-32 (dopamine and cAMP-regulated phosphoprotein of 32 kDa) at Thr⁷⁵, DARPP-32 becomes a strong inhibitor of PKA activity. Roscovitine is a potent Cdk5 inhibitor; it has been previously shown that acute application of Roscovitine increases striatal transmission via Cdk5/DARPP-32. Since DARPP-32 controls long-term plasticity in the striatum, we wondered whether switching off CdK5 activity with Roscovitine contributes to the induction of LTP in corticostriatal synapses. For this purpose, excitatory population spikes and whole cell EPSC (excitatory postsynaptic currents) were recorded in striatal slices from C57/BL6 mice. Experiments were carried out in the presence of Roscovitine (20 μM) in the recording bath. Roscovitine increased the amplitude of excitatory population spikes and the percentage of population spikes that exhibited LTP after HFS (high-frequency stimulation; 100Hz). Results obtained showed that the mechanisms responsible for LTP induction after Cdk5 inhibition involved the PKA pathway, DA and NMDA (N-methyl-D-aspartate) receptor activation, L-type calcium channels activation and the presynaptic modulation of neurotransmitter release.     

The whole-body shortening reflex of the medicinal leech: motor pattern,sensory basis,and interneuronal pathways

The leech whole-body shortening reflex consists of a rapid contraction of the body elicited by a mechanical stimulus to the anterior of the animal. We used a variety of reduced preparations — semi-intact, body wall, and isolated nerve cord — to begin to elucidate the neural basis of this reflex in the medicinal leech Hirudo medicinalis. The motor pattern of the reflex involved an activation of excitatory motor neurons innervating dorsal and ventral longitudinal muscles (dorsal excitors and ventral excitors respectively), as well as the L cell, a motor neuron innervating both dorsal and ventral longitudinal muscles. The sensory input for the reflex was provided primarily by the T (touch) and P (pressure) types of identified mechanosensory neuron. The S cell network, a set of electrically-coupled interneurons which makes up a fast conducting pathway in the leech nerve cord, was active during shortening and accounted for the shortest-latency excitation of the L cells. Other, parallel, interneuronal pathways contributed to shortening as well. The whole-body shortening reflex was shown to be distinct from the previously described local shortening behavior of the leech in its sensory threshold, motor pattern, and (at least partially) in its interneuronal basis.Abbreviations conn connective - DE dorsal excitor motor neuron - DI dorsal inhibitor motor neuron - DP dorsal posterior nerve - DP:B1 dorsal posterior nerve branch 1 - DP:B2 dorsal posterior nerve branch 2 - MG midbody ganglion - VE ventral excitor motor neuron - VI ventral inhibitor motor neuron     

THREE-DIMENSIONAL ULTRASTRUCTURE OF THE CRAYFISH NEUROMUSCULAR APPARATUS

The synapse-bearing nerve terminals of the opener muscle of the crayfish Procambarus were reconstructed using electron micrographs of regions which had been serially sectioned. The branching patterns of the terminals of excitatory and inhibitory axons and the locations and sizes of neuromuscular and axo-axonal synapses were studied. Excitatory and inhibitory synapses could be distinguished not only on the basis of differences in synaptic vesicles, but also by a difference in density of pre- and postsynaptic membranes. Synapses of both axons usually had one or more sharply localized presynaptic "dense bodies" around which synaptic vesicles appeared to cluster. Some synapses did not have the dense bodies. These structures may be involved in the physiological activity of the synapse. Excitatory axon terminals had more synapses, and a larger percentage of terminal surface area devoted to synaptic contacts, than inhibitory axon terminals. However, the largest synapses of the inhibitory axon exceeded in surface area those of the excitatory axon. Both axons had many side branches coming from the main terminal; often, the side branches were joined to the main terminal by narrow necks. A greater percentage of surface area was devoted to synapses in side branches than in the main terminal. Only a small fraction of total surface area was devoted to axo-axonal synapses, but these were often located at narrow necks or constrictions of the excitatory axon. This arrangement would result in effective blockage of spike invasion of regions of the terminal distal to the synapse, and would allow relatively few synapses to exert a powerful effect on transmitter release from the excitatory axon. A hypothesis to account for the development of the neuromuscular apparatus is presented, in which it is suggested that production of new synapses is more important than enlargement of old ones as a mechanism for allowing the axon to adjust transmitter output to the functional needs of the muscle.     

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.     

Ultrastructure of the nerve-muscle junction in the pharynx of the earthworm,Lumbricus terrestris

Summary InLumbricus terrestris the wall of the pharynx is built up from obliquely striated longitudinal and circular muscle layers. The occurrence of perikarya and nerve bundles showing green fluorescence suggests the presence of aminergic innervation in the pharynx. A significant number of chemical synapses were detected in the neuropil among axon terminals. The junctional gap is generally 100–300 nm wide in type I junctions which resemble the cholinergic motor endplates of vertebrate skeletal muscle. A narrow junctional gap of about 25 nm is characteristic of the close contacts in the type II neuromuscular junction. Agranular spherical vesicles, together with small and large dense-cored granules, fill in these axon terminals.     

Intracellular activity of morphologically identified neurons of the grass frog's optic tectum in response to moving configurational visual stimuli

Summary In the grass frogRana temporaria, various classes of tectal neurons were identified by means of intracellular recording and iontophoretic staining using potassium-citrate/Co³⁺-lysine-filled micropipettes, which have been defined previously by extracellular recording methods. Class T5(1) neurons had receptive fields (RF) of 33°±5° diameter. In response to a moving 8°×8° square (S), a 2°×16° worm-like (W), or a 16°×2° antiworm-like (A) moving stripe, these cells showed excitatory postsynaptic potentials (EPSPs) and spikes which were interrupted occasionally by small inhibitory postsynaptic potentials (IPSPs). The excitatory responses (R) were strongest towards the square (R_S) and less to the worm (R_W). For the antiworm (R_A) the responses were smallest or equal to the worm stimulus yielding the relationship R_S>R_WR_A. Some of these cells were identified as pear-shaped or large ganglionic neurons, whose somata were located in the tectal cell layer 8. The somata of other large ganglionic neurons were found in layer 7 and the somata of other pear-shaped neurons at the top of layer 6, both displaying T5(1) properties. Class T5(2) neurons (RF=34°±3°) responded with large EPSPs and spikes, often interrupted by small IPSPs, when their RF was traversed by the square stimulus. The excitatory activity was somewhat less to the worm stimulus, whereas no activity at all, or only IPSPs, were recorded in response to the antiworm-stimulus; thus yielding the relationship for the excitatory activity R_S>R_W>R_A 0. Such a cell was identified as pyramidal neuron; the soma was located at the top of layer 6, with the long axon travelling into layer 7 to the medulla oblongata. Class T5(3) neurons (RF=29°±6°) showing EPSPs and spikes according to the relationship R_S>R_A>R_W have been identified as large ganglionic neurons. Their somata were located in layer 8. Class T5(4) neurons (RF=24±7°) responded only to the square stimulus with EPSPs and spikes, sometimes interrupted by IPSPs and yielding the relationship R_S>R_AR_W0. The somata of these large ganglionic or pear-shaped neurons were located in layer 8. Class T1(1) neurons (RF=30°–40°) were most responsive to stimuli moving at a relatively long distance in the binocular visual field, and have been identified as pear-shaped neurons. Their somata were located in layer 6.Further neurons are described and morphologically identified which have not yet been classified by extracellular recording methods. For example,IPSP neurons (RF=20°–30°) responded (R) with IPSPs only according to the relationship R_S>R_A R_W. The somata of these pear-shaped neurons were located in layer 6.The properties of tectal cells in response to electrical stimulation of the optic tract and to brisk changes of diffuse illumination suggest certain neuronal connectivity patterns. The results support the idea ofintegrative functional units (assemblies) of connected cells which are involved in various perceptual processes, such as configurational prey selection expressed by T5(2) prey-selective neurons.Abbreviations A antiworm-like 16°×2° stripe stimulus with long axis perpendicular to the direction of movement - W wormlike 2°×16° stripe stimulus with long axis oriented parallel to the direction of movement - S square 8°×8° moving stimulus - ERF excitatory receptive field - IRF inhibitory receptive field - RF receptive field - EPSP excitatory postsynaptic potential - IPSP inhibitory postsynaptic potential     

Electrical properties and transmitter output associated with growth and transformation in shrimp muscle fibers

Summary Comparisons were made of the passive electrical properties of closer muscle fibers in the dimorphic claws of snapping shrimp,Alpheus armillatus. During claw transformation the small fibers of pincer claws grow to become much larger snapper claw fibers. As muscle fibers grow, the relationship of fiber input resistance (R ₀) to fiber diameter (d) is predicted by the proportionality,R ₀d ^–3/2. Muscle fiber membrane resistance,R _m, is independent of fiber diameter, but membrane capacitance,C _m, grows with diameter. This results in a 40 to 50 fold reduction in fiber input impedance as fiber diameter enlarges during transformation. Reductions of muscle fiber impedance are partially compensated by 2–5 fold increases in quantal content at excitatory synapses on snapper muscle fibers. However, changes in quantal content during transformation apparently are independent of fiber diameter per se. Excitatory junction potentials in both pincer and snapper muscle fibers have equal amplitude. Because fiber input impedance decreases precipitously during transformation, and in view of the relatively small compensatory changes in quantal content at excitatory synapses, additional pre- or post-synaptic modifications must supplement increased quantal content to maintain synaptic efficacy in transformed muscle fibers.Abbreviations ejp excitatory junctional potential - epp endplate potential - mepp miniature endplate potential