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.

Changes in the sarcoplasmic reticulum and transverse tubular system of fast and slow skeletal muscles of the mouse during postnatal development.

The sarcoplasmic reticulum (SR) and transverse tubular system (TTS) of a fast-twitch muscle (extensor digitorum longus-EDL) and a slow-twitch muscle (soleus-SOL) of the mouse were examined during postnatal development. Muscles of animals newborn to 60 days old were fixed in glutaraldehyde and osmium tetroxide and examined with an electron microscope. At birth the few T tubules were often oriented longitudinally, but at the age of 10 days most of them had a transverse orientation. In the EDL, the estimated volume of the TTS increased from 0.08% at birth to 0.4% in the adult; corresponding values for the SOL were 0.04% at birth and 0.22% in the adult. A similar relative change was observed in surface area of the TTS during development. Calculated on the basis of a 30 microm diameter fiber, the surface area of the TTS in the EDL increased from 0.60 cm(2) TTS/cm(2) fiber surface in the newborn to 3.1 cm(2)/cm(2) in the adult, compared with 0.15 cm(2)/cm(2) at birth to 1.80 cm(2)/cm(2) in the adult for the SOL. The SR in the newborn muscles occurred as a loose network of tubules that developed rapidly within the subsequent 20 days, especially at the I band level. The volume of the SR increased in the EDL from 1.1% of fiber volume at birth to 5.5% in the adult. In the SOL the change was from 1.7% to 2.9%. The SOL approached the adult values more rapidly than the EDL, although the EDL had more SR and T tubules. Fibers of both EDL and SOL muscles showed variation in Z line thickness, mitochondrial content, and diameter, but over-all differences between the two muscles in amount of SR and TTS were significant. It is considered that the differing amounts of SR and TTS are closely related to the differing speeds of contraction that have been demonstrated for these two muscles.

Synaptic vesicles: selective depletion in crayfish excitatory and inhibitory axons.

Stimulation of the excitatory axon of the opener muscle of the crayfish in the presence of the metabolic inhibitor 2,4-dinitrophenol leads to depletion of synaptic vesicles in nerve terminals containing round vesicles. Stimulation of the inhibitory axon under these conditions produces depletion of vesicles in other nerve terminals containing more elongate synaptic vesicles. The experiments show that terminals with round synaptic vesicles are excitatory and that terminals with elongate synaptic vesicles are inhibitory. Replenishment of synaptic vesicles appears to require metabolic energy.

Synaptic facilitation: long-term neuromuscular facilitation in crustaceans.

Continuous stimulation at frequencies equal to or greater than 5 hertz for 20 to 30 minutes results in a two- to fivefold increase in the amplitudes of excitatory postsynaptic potential recorded from certain stretcher and opener muscles of decapod crustaceans. This long-term facilitation appears to result from an accumulation of sodium ions within the nerve terminals. It persists for at least 1 hour after stimulation has stopped.

Drosophila Hsc70-4 is critical for neurotransmitter exocytosis in vivo.

Previous in vitro studies of cysteine-string protein (CSP) imply a potential role for the clathrin-uncoating ATPase Hsc70 in exocytosis. We show that hypomorphic mutations in Drosophila Hsc70-4 (Hsc4) impair nerve-evoked neurotransmitter release, but not synaptic vesicle recycling in vivo. The loss of release can be restored by increasing external or internal Ca(2+) and is caused by a reduced Ca(2+) sensitivity of exocytosis downstream of Ca(2+) entry. Hsc4 and CSP are likely to act in common pathways, as indicated by their in vitro protein interaction, the similar loss of evoked release in individual and double mutants, and genetic interactions causing a loss of release in trans-heterozygous hsc4-csp double mutants. We suggest that Hsc4 and CSP cooperatively augment the probability of release by increasing the Ca(2+) sensitivity of vesicle fusion.

Neuroprotection at Drosophila synapses conferred by prior heat shock.

Synapses are critical sites of information transfer in the nervous system, and it is important that their functionality be maintained under stressful conditions to prevent communication breakdown. Here we show that synaptic transmission at the Drosophila larval neuromuscular junction is protected by prior exposure to heat shock that strongly induces expression of heat shock proteins, in particular hsp70. Using a macropatch electrode to record synaptic activity at individual, visualized boutons, we found that prior heat shock sustains synaptic performance at high test temperatures through pre- and postsynaptic alterations. After heat shock, nerve impulses release more quantal units at high temperatures and exhibit fewer failures of release (presynaptic modification), whereas the amplitude of quantal currents remains more constant than does that in nonheat-shocked controls (postsynaptic modification). The time course of these physiological changes is similar to that of elevated hsp70. Thus, stress-induced neuroprotective mechanisms maintain function at synapses by modifying their properties.

Role of cAMP cascade in synaptic stability and plasticity: ultrastructural and physiological analyses of individual synaptic boutons in Drosophila memory mutants.

Mutations of the genes rutabaga (rut) and dunce (dnc) affect the synthesis and degradation of cAMP, respectively, and disrupt learning in Drosophila. Combined ultrastructural analysis and focal electrophysiological recording in the larval neuromuscular junction revealed a loss of stability and fine tuning of synaptic structure and function in both mutants. Increased ratios of docked/undocked vesicles and poorly defined synaptic specializations characterized dnc synapses. In contrast, rut boutons possessed fewer, although larger, synapses with lower proportions of docked vesicles. At reduced Ca(2+) levels, decreased quantal content coupled with an increase in failure rate was seen in rut boutons and reduced pair-pulse facilitation were found in both rut and dnc mutants. At physiological Ca(2+) levels, strong enhancement, instead of depression, in evoked release was observed in some dnc and rut boutons during 10 Hz tetanus. Furthermore, increased variability of synaptic transmission, including fluctuation and asynchronicity of evoked release, paralleled an increase in synapse size variation in both dnc and rut boutons, which might impose problems for effective signal processing in the nervous system. Pharmacological and genetic studies indicated broader ranges of physiological alteration by dnc and rut mutations than either the acute effects of cAMP analogs or the available mutations that affect cAMP-dependent protein kinase (PKA) activity. This is consistent with previous reports of more severe learning defects in dnc and rut mutations than these PKA mutants and allows identification of the phenotypes involving long-term developmental regulation and those conferred by PKA.

Calcium entry related to active zones and differences in transmitter release at phasic and tonic synapses.

Synaptic functional differentiation of crayfish phasic and tonic motor neurons is large. For one impulse, quantal release of neurotransmitter is typically 100-1000 times higher for phasic synapses. We tested the hypothesis that differences in synaptic strength are determined by differences in synaptic calcium entry. Calcium signals were measured with the injected calcium indicator dyes Calcium Green-1 and fura-2. Estimated Ca(2+) entry increased almost linearly with frequency for both axons and was two to three times larger in phasic terminals. Tonic terminal Ca(2+) at 10 Hz exceeded phasic terminal Ca(2+) at 1 Hz, yet transmitter release was much higher for phasic terminals at these frequencies. Freeze-fracture images of synapses revealed on average similar numbers of prominent presynaptic active zone particles (putative ion channels) for both neurons and a two- to fourfold phasic/tonic ratio of active zones per terminal volume. This can account for the larger calcium signals seen in phasic terminals. Thus, differences in synaptic strength are less closely linked to differences in synaptic channel properties and calcium entry than to differences in calcium sensitivity of transmitter release.

Correlated electrophysiological and ultrastructural studies of a crustacean motor unit.

Structural and functional interrelationships between the pre- and postsynaptic elements of a singly motor innervated crab muscle (stretcher of Hyas araneus L.) were examined using electrophysiological and electron microscopic techniques. Excitatory postsynaptic potential (EPSP) amplitude at 1 Hz was found to be inversely related to the extent of facilitation, and directly related both to the amount of transmitter released at 1 Hz and the muscle fiber input resistance (R(in)). The extent of facilitation (F(e)), taken as the ratio of the EPSP amplitude at 10 Hz to that 1 Hz, was inversely related to muscle fiber R(in), tau(m), and sarcomere length. Sarcomere length was directly related to R(in) and tau(m). The excitatory nerve terminals of low F(e) muscle fibers had larger neuromuscular synapses than did those of high F(e) fibers. Inhibitory axo-axonal synapses were more often found in low F(e) muscle fibers. These structural features may account for the greater release of transmitter at low frequencies from the low F(e) nerve terminals as well as provide for a greater amount of presynaptic inhibition of low F(e) muscle fibers. The implications of these findings for the development and physiological performance of the crustacean motor unit are discussed. It is proposed that both nerve and muscle fiber properties may be determined by the developmental pattern of nerve growth.

Differential responses of crab neuromuscular synapses to cesium ion.

Excitatory postsynaptic potentials (EPSP's) generated in crab muscle fibers by a single motor axon, differ in amplitude and facilitation. Some EPSP's are large at low frequencies of stimulation and show little facilitation; others are smaller and show pronounced facilitation. When K(+) is replaced by Cs(+) in the physiological solution, all EPSP's increase in amplitude, but small EPSP's increase proportionately more than large ones. Quantal content of transmission, determined by external recording at single synaptic regions, undergoes a much larger increase at facilitating synapses. The increase in quantal content of transmission is attributable to prolongation of the nerve terminal action potential in Cs(+). After 1-2 h of Cs(+) treatment, defacilitation of synaptic potentials occurs at synapses which initially showed facilitation. This indicates that Cs(+) treatment drastically increases the fraction of the "immediately available" transmitter store released by each nerve impulse, especially at terminals with facilitating synapses. It is proposed that facilitating synapses normally release less of the "immediately available" store of transmitter than poorly facilitating synapses. Possible reasons for this difference in performance are discussed.

Turning behavior in Drosophila larvae: a role for the small scribbler transcript.

The Drosophila larva is extensively used for studies of neural development and function, yet the mechanisms underlying the appropriate development of its stereotypic motor behaviors remain largely unknown. We have previously shown that mutations in scribbler (sbb), a gene encoding two transcripts widely expressed in the nervous system, cause abnormally frequent episodes of turning in the third instar larva. Here we report that hypomorphic sbb mutant larvae display aberrant turning from the second instar stage onwards. We focus on the smaller of the two sbb transcripts and show that its pan-neural expression during early larval life, but not in later larval life, restores wild type turning behavior. To identify the classes of neurons in which this sbb transcript is involved, we carried out transgenic rescue experiments. Targeted expression of the small sbb transcript using the cha-GAL4 driver was sufficient to restore wild type turning behavior. In contrast, expression of this sbb transcript in motoneurons, sensory neurons or large numbers of unidentified interneurons was not sufficient. Our data suggest that the expression of the smaller sbb transcript may be needed in a subset of neurons for the maintenance of normal turning behavior in Drosophila larvae.

"Satellite cells" and nerve terminals in the crayfish opener muscle visualized with fluorescent dyes.

Nerve terminals and associated cells on the muscle's surface were visualized in the crayfish opener muscle with several fluorescent dyes in conjunction with confocal microscopy and conventional fluorescence microscopy. The nerve terminals of the excitatory and inhibitory axons were best seen with 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). This dye is selectively accumulated in mitochondria, which are numerous both in the axons and in synapse-bearing terminal varicosities. Muscle nuclei were also clearly visualized, because they excluded 4-Di-2-Asp but were stained by acridine orange (AO). A positive attraction between muscle nuclei and nerve terminals was evident by visual inspection and was confirmed by spatial statistics. Additional flat cells on the muscle's surface appeared as bright rings with elongated processes that were often close to or overlapped nearby nerve terminals. The structure of these cells was established by electron microscopy after labeling them with fluorescent polystyrene beads, which could be found over structures on the muscle surface in sections of embedded specimens. The flat surface cells were distinct from peripheral glial cells closely associated with axons and nerve terminals. Nevertheless, spatial statistics showed that the surface cells were grouped near nerve terminals. They occupied a small fraction of the muscle cell's surface. Their functional role has not been determined in crustacean muscles.

Axoaxonal synapse location and consequences for presynaptic inhibition in crustacean motor axon terminals.

Serial sections were made of several excitatory nerve terminals in the stretcher muscle of the spider crab, Hyas areneus , to document locations of inhibitory axoaxonal synapses responsible for physiologically powerful presynaptic inhibition. The excitatory terminals are varicose, often with small side branches joined to the main terminal by thin bottlenecks . Axoaxonal synapses occur predominantly on the varicosities, both primary and secondary, with a smaller number on bottlenecks . The distribution is often clustered at specific locations of the excitatory terminal. An electrical model was employed to ascertain the effectiveness of axoaxonal synapses at different locations on the terminal. The model plotted the potential distribution along the terminal with or without a synaptic conductance equivalent to one quantal unit of inhibitory transmitter action. It was assumed from recent work that terminal varicosities are not completely invaded by an action potential. The model predicts that large drops in potentials originating in the main axon occur in the terminals during inhibitory transmitter action, with the largest total drop produced by axoaxonal synapses on the terminal varicosities. The effectiveness of inhibitory action is critically dependent on the dimensions and internal resistance of the bottlenecks . Thus, the geometrical features of the excitatory terminal appear to play a key role in effectiveness of presynaptic inhibition.

Inhibitory axoaxonal and neuromuscular synapses in the crayfish opener muscle: membrane definition and ultrastructure.

The specific inhibitory motoneuron to the crayfish (Procambarus clarkii) opener muscle provides neuromuscular synapses to the muscle fibers and axoaxonal synapses to the excitatory motor nerve terminals. Freeze fracture of the membrane in both types of synapses show that the presynaptic active zone consists of clusters of large particles (putative calcium channels), which are often encircled by large depressions representing fused synaptic vesicles on the internal leaflet or P face of the presynaptic membrane. Corresponding pits and protrusions mark the external leaflet or E face of the presynaptic membrane. The postsynaptic receptor-bearing surface, characterized for neuromuscular synapses only, consists of rows of particles on both leaflets of the muscle membrane. The organization differs from that seen at excitatory synapses where particles occur only on the E-face leaflet. Serial thin sections of nerve terminals reveal that neuromuscular synapses are significantly larger in proximal fibers than in their central counterparts and support a greater number of presynaptic dense bars (active zones). Axoaxonal synapses also show regional differences; almost three times as many occur in the proximal region compared with the central region. Most synapses possess a single dense bar. The majority of synapses formed by the inhibitory axon are neuromuscular; a minority are axoaxonal. The latter occur in various locations along the excitatory nerve terminals as well as on branches of the axon itself. This preterminal or "off-shore" location could act to cut off entire populations of excitatory synapses or reduce the amplitude of the preterminal action potential.

Variation in terminal morphology and presynaptic inhibition at crustacean neuromuscular junctions.

Synaptic terminals of excitatory and inhibitory neurons supplying muscle fibers in leg muscles of crabs (Pachygrapsus crassipes and Hyas areneus) were investigated with light and electron microscopy. Terminals responsible for large excitatory postsynaptic potentials (EPSPs) at low frequencies of activation had a compact configuration with clusters of terminal boutons radiating from the main axon branch. Terminals responsible for small EPSPs had a more diffuse organization, with boutons often arranged in series along thin axon branches. Inhibitory neurons, when activated, produced both presynaptic and postsynaptic inhibitory effects, with the former being more potent at low frequencies of activation. Presynaptic inhibition was variable in magnitude but was generally strong in fibers with large EPSPs. Representative terminals from regions of strong and weak presynaptic inhibition were identified by activity-dependent uptake of horseradish peroxidase, serially sectioned, and reconstructed from electron micrographs. Both regions were found to contain axo-axonal synapses from inhibitory to excitatory terminals, with a larger number in the region of strong presynaptic inhibition. In addition, axo-axonal synapses were more uniformly distributed in the latter region. The number of inhibitory presynaptic dense bars (active zones) was somewhat higher in the region of weak inhibition, but larger individual dense bars occurred in the region of strong inhibition. Possible factors contributing to the differences in strength of inhibition include: (1) morphology and electrical properties of terminals; and (2) high probability of transmission at a relatively small number of inhibitory synapses during low frequency activation in the region of strong inhibition.

Structural features of crayfish phasic and tonic neuromuscular terminals.

We examined the fine structure of terminals of the phasic and tonic excitatory axon to the crayfish limb extensor muscle. The phasic terminals are known to release 50-100 times more transmitter for a small length of terminal for a single impulse. Phasic terminals labeled with horseradish peroxidase (HRP) were relatively thin and contained a single unbranched mitochondrion; tonic terminals were much thicker, and their varicosities contained several multibranched mitochondria. Tonic terminals devoted a larger proportion of their total volume to mitochondria. The percentage volume of clear synaptic vesicles was slightly higher in phasic axon terminals, but as the tonic axon terminals were fivefold larger in volume, the total synaptic volume is much greater in tonic than phasic terminals. The number of synapses per length of terminal, and the total number of active zones per length of terminal, were greater for tonic terminals, and individual synapses were, on average, slightly larger in surface contact area for tonic terminals. In contrast, individual active zones were, on average, longer in phasic synapses. A higher proportion (50%) of phasic synapses had multiple active zones than was the case for tonic synapses (16%), and pairs of closely spaced active zones were more frequently found on phasic synapses. These findings clearly rule out synapse and active zone number as a factor contributing to higher transmitter output, but suggest that active zone size and synaptic complexity, as evidenced by multiple closely spaced active zones in a single synapse, are likely to play a causal role in the greater transmitter release of the phasic terminal. Even synapse complexity would not be enough to account fully for the large difference in terminal transmitter output, and additional factors may include electrical and biochemical differences.

Quantal release at visualized terminals of a crayfish motor axon: intraterminal and regional differences.

Synaptic transmission was measured at visualized terminal varicosities of the motor axon providing the sole excitatory innervation of the "opener" muscle in walking legs of crayfish (Procambarus clarkii Girard). Two questions were addressed: 1) How uniform is quantal emission at different locations along terminals innervating a single muscle fiber, and 2) can differences in quantal emission account for the different excitatory postsynaptic potential (EPSP) amplitudes generated by terminals localized in defined regions of the muscle? Extracellular "macropatch" electrodes were placed over individual varicosities, viewed after brief exposure to a fluorescent dye, and synaptic currents were recorded to determine quantal content of transmission. Along terminals supplying a single muscle fiber, nonuniform release was found: Varicosities closer to the point of origin of the terminal branch released more transmitter than those located more distally. Quantal content was higher for varicosities of the muscle's proximal region (where large EPSPs occur) than for varicosities of the central region (where small EPSPs occur). The probability of transmitter release per synapse is estimated to be greater for the proximal varicosities. At low frequencies of stimulation, quantal content per muscle fiber is two to four times larger in the proximal region. Taken in conjunction with a twofold higher mean input resistance for the proximal muscle fibers, the difference in quantal content can account for a four- to eightfold difference in EPSP amplitude. The observed mean EPSP amplitude is at least eight times larger in the proximal region. We discuss factors contributing to differences in EPSP amplitudes.

Differential physiology and morphology of motor axons to ventral longitudinal muscles in larval Drosophila.

Morphological and physiological characteristics of the two major motor axons supplying the commonly studied ventral longitudinal muscle fibers (6 and 7) of third-instar Drosophila melanogaster larvae were investigated. The innervating terminals of the two motor axons differ in the size of their synapse-bearing varicosities. The terminal with the larger varicosities also fluoresces more brightly when stained with the vital fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-Asp) and occupies a larger total contact area on the muscle fiber. Through selective simultaneous recording of synaptic currents from identified boutons in living preparations during elicitation of synaptic potentials, it was shown that the axon with the smaller varicosities generates a large excitatory junction potential (EJP) in muscle 6 and that the axon with the larger varicosities generates a smaller EJP. Short-term facilitation is more pronounced for the smaller EJP. In preparations treated with 4-Di-2-Asp, the fluorescence of smaller varicosities increases with stimulation that elicits the large EJPs, indicating an activity-dependent entry of calcium that enhances mitochondrial fluorescence. The differences in morphology and physiology of the two axons are similar to, though less pronounced than, those observed in "phasic" and "tonic" motor axons of crustaceans.

Characterization and partial purification of different factors with contraction-potentiating activities from neurohaemal organs of the locust.

Phe-Met-Arg-Phe (FMRF-NH2) and structurally related peptides enhance neuromuscular transmission and contraction of the M. extensor tibiae preparations of the locusts Locusta migratoria and Schistocerca gregaria (Walther et al.: Neurosci. Lett. 45:99-104, '84). Similar effects could also be obtained with extracts of locust ganglia (Walther and Schiebe: Neurosci. Lett. 77:209-214, '87). By using two HPLC systems, we have partially purified extracts of the unpaired median nerves (including their neurohaemal organs) of different locust ganglia. The biological activity of the extracts served as an estimate for the degree of purification. Six different bioactive fractions were identified migrating at and close to retention times of known -RFamide peptides with similar bioactivity. No fraction coeluted with authentic FMRF-NH2 or FLRF-NH2. We demonstrate that extensor tibiae muscle contractions were potentiated by HPLC fractions from raw material with -RF-NH2 immunoreactivity, but also by HPLC fractions from raw material without such immunoreactivity.

Motor nerve terminals on abdominal muscles in larval flesh flies, Sarcophaga bullata: comparisons with Drosophila.

Motor nerve terminals on abdominal body-wall muscles 6A and 7A in larval flesh flies were investigated to establish their general structural features with confocal microscopy, transmission electron microscopy, and freeze-fracture procedures. As in Drosophila and other dipterans, two motor axons supply these muscles, and two morphologically different terminals were discerned with confocal microscopy: thin terminals with relatively small varicosities (Type Is), and thicker terminals with larger varicosities (Type Ib). In serial electron micrographs, Type Ib terminals were distinguished from Type Is terminals by their larger cross-sectional area, more extensive subsynaptic reticulum, more mitochondrial profiles, and more clear synaptic vesicles. Type Ib terminals possessed larger synapses and more synaptic contact area per unit terminal length. Although presynaptic dense bars of active zones were similar in mean length for the two terminal types, there were almost twice as many dense bars per synapse for Type Ib terminals. Freeze-fractures through the presynaptic membrane showed particle-free areas indicative of synapses on the P-face, within which were localized aggregations of large intramembranous particles indicative of active zones. These particles were similar in number to those found at active zones of several other arthropod neuromuscular junctions. In general, synaptic structural parameters strongly paralleled those of the anatomically homologous muscles in Drosophila melanogaster. In live preparations, simultaneous focal recording from identified varicosities and intracellular recording indicated that the two terminals produced excitatory junction potentials of similar amplitude in a physiological solution similar to that used for Drosophila.