Crustacean frequenins: Molecular cloning and differential localization at neuromuscular junctions

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium‐binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq‐like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin‐ and dynamin‐like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq‐like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium‐binding regions (EF hands) are conserved. The widespread occurrence of frq‐like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons. © 1999 John Wiley & Sons, Inc. J Neurobiol 41: 165–175, 1999     

Synaptic diversity and differentiation: Crustacean neuromuscular junctions

Crustacean motor neurons exhibit a wide range of synaptic responses. Tonically active neurons generally produce small excitatory postsynaptic potentials (EPSPs) at low impulse frequencies, and are able to release much more transmitter as the impulse frequency increases. Phasic neurons typically generate large EPSPs in their target cells, but have less capability for frequency facilitation, and undergo synaptic depression during maintained activity. These differences depend in part upon the neuron's ongoing levels of activity; phasic neurons acquire physiological and morphological features of tonic neurons when their activity level is altered. Molecules responsible for adaptation to activity can be sought in single identified phasic neurons with current techniques. The fact that both phasic and tonic neurons innervate the same target muscle fibers is evidence for presynaptic determination of synaptic properties, but there is also evidence for postsynaptic determination of specific properties of different endings of a single neuron. The occurrence of high- and low-output endings of the same tonic motor neurons on different muscle fibers suggests a target-specific influence on synaptic properties. Structural variation of synapses on individual terminal varicosities leads to the hypothesis that individual synapses have different probabilities for release of transmitter. We hypothesize that structurally complex synapses have a higher probability for release than the less complex synapses. This provides an explanation for the larger quantal contents of high-output terminals (where the proportion of complex synapses is higher), and also a mechanism for progressive recruitment of synapses during frequency facilitation.     

An Attempt to Account for the Diversity of Crustacean Muscles

Crustacean muscles are known to contain muscle fibers of variableproperties and to be innervated by phasic and/or tonic motoneuronswhich may possess synapses of diverse physiological properties.Frequently, phasic motor axons innervate short-sarcomere phasicmuscle fibers and tonic motor axons innervate long-sarcomeretonic muscle fibers, but some muscles receiving a single (tonic)motor axon contain both phasic and tonic muscle fibers. Althoughit is not known whether neural trophic influences are involvedin muscle differentiation, some neural trophic effects havebeen found in crustaceans, and it is reasonable to assume thatsuch influences may be involved in establishing the definitiveproperties of the muscle. Several other postulates must be made:(1) Phasic and tonic motor axons differ in their trophic effectiveness:(2) muscle fibers innervated relatively early in developmentby a tonic motor axon acquire the properties of tonic musclefibers, while those innervated later become intermediate orphasic muscle fibers; (3) the developmental stage of a growingor regenerating axon terminal plays a role in determinationof synaptic properties. Studies on regenerating limb buds supportthe hypothesis, which can account for the genesis of all observedtypes of crustacean neuromuscular system. Further experimentalwork is necessary to test the hypothesis.     

Crustacean frequenins: molecular cloning and differential localization at neuromuscular junctions.

Crustacean muscles are innervated by phasic and tonic motor neurons that display differential physiology and have morphologically distinct synaptic terminals. Phasic motor neurons release much more transmitter per impulse and have filiform terminals, whereas tonic motor neurons release less transmitter and have larger terminals with prominent varicosities. Using an antibody raised against Drosophila frequenin (frq), a calcium-binding protein that enhances transmitter release in Drosophila synaptic terminals, we found that frq-like immunoreactivity is prominent in many of the phasic, but not tonic nerve endings of crayfish motor neurons. In contrast, synapsin- and dynamin-like immunoreactivities are strongly expressed in both types of terminal. The immunocytochemical findings strongly suggested the presence of an frq-like molecule in crayfish, and its differential expression indicated a possible modulatory role in transmitter release. Therefore, we cloned the cDNA sequences for the crayfish and lobster homologues of Drosophila frq. Crustacean frequenins are very similar in sequence to their Drosophila counterpart, and calcium-binding regions (EF hands) are conserved. The widespread occurrence of frq-like molecules and their differential localization in crayfish motor neurons indicate a significant role in physiology or development of these neurons.     

Crustacean Neuromuscular Mechanisms

Properties of crustacean muscle fibers and neuromuscular synapsesare discussed, with particular reference to the problems offast and slow contraction, synaptic diversity, and peripheralinhibition. Electrical and mechanical responses of crustacean muscle fibersare variable, and govern to a large extent the muscle's performance.Fast and slow contractions are often mediated by distinct "phasic"and "tonic" muscle fibers, as in abdominal muscles, in whichsuch fibers are segregated into two parallel sets of muscles.In leg muscles the fibers are often heterogeneous in propertiesand innervation. In doubly-motor-innervated muscles of crabsthe axons producing fast and slow contractions preferentiallyinnervate rapidly and slowly contracting fibers, respectively. Crustacean neuromuscular synapses vary greatly in electricalbehavior and in ultrastructural characteristics. Some motoraxons possess both facilitating and nonfacilitating synapses.The proportion of the different types of synapse associatedwith a motor axon probably determines in large measure the propertiesof the postsynaptic potentials evoked by that axon. Pre-synaptic and post-synaptic inhibition both occur, sometimesin the same muscle. The latter type is more common. Pre-synapticinhibition is thought to be mediated by the action of an inhibitorytransmitter-substance on receptors of the motor nerve terminals.     

Excitatory Transmission in Insect Neuromuscular Systems

Many, but not all, visceral muscles in insects are innervatedby neurosecretory axons. The neurosecretory junctions with theheart muscle of the American cockroach, Periplaneta americana,show ultrastructural and electrophysiological evidence of chemicallytransmitting synapses, and cytochemical evidence for the presenceof monoamines. Electron microscopy of nerve terminals showsthat synaptic vesicles may be formed directly from electron-dense"neurosecretory" granules Neurotomy of motor axons to skeletal muscles in insects leadsto aggregation and clumping of synaptic vesicles after 48 hours.Treatment of in vitro nerve-muscle preparations with variousrespiratory poisons caused aggregation similar to that developedin neurotomized animals. This suggested that vesicle aggregationin both cases may have resulted from a decrease in availableadenosine triphosphate in the nerve terminal with subsequentalteration in the normal charge density which supports a repulsiveforce between the vesicles.     

Neuropeptide localization in varicosities of Aplysia sensory neurons is regulated by target and neuromodulators evoking long-term synaptic plasticity

The synapses between the sensory neuron (SN) and motor neuron of Aplysia undergo long-term functional and structural modulation with appropriate behavioral training or with applications of specific neuromodulators. Expression of molecules within the presynaptic terminals may be regulated in parallel with the changes evoked by the neuromodulators. We examined with immunocytochemical methods whether the level of sensorin, the SN-specific neuropeptide, is modulated in SN varicosities by the location of interaction with the target motor cell L7 and by applications of either 5-HT that evoke long-term facilitation or FMRFamide that evoke long-term depression of Aplysia sensorimotor connections in vitro. A significantly higher proportion of SN varicosities are sensorin positive when they are in contact with the proximal axons of L7 compared to varicosities of the same SNs in contact with distal L7 neurites. Both 5-HT and FMRFamide evoked changes in the efficacy and structure of sensorimotor connections that are accompanied by changes in the frequency of sensorin-positive varicosities contacting the axons of L7. More preexisting SN varicosities are stained after 5-HT, and fewer preexisting SN varicosities are stained after FMRFamide. These results suggest that the postsynaptic target and the neuromodulators not only regulate overall structure but also regulate the level of SN neuropeptide at synaptic sites. © 1996 John Wiley & Sons, Inc.     

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.Download video file.^{(61M, mov)}     

Sources of inputs to longitudinal muscle motor neurons and ascending interneurons in the guinea-pig small intestine

Light- and electron-microscopic studies were used to investigate connections between specific subgroups of neurons in the myenteric plexus of the guineapig small intestine. Inputs to two classes of calretinin-immunoreactive (IR) nerve cells, longitudinal muscle motor neurons and ascending interneurons, were examined. Inputs from calbindin-IR primary sensory neurons and from three classes of descending interneurons were studied. Electron-microscopic analysis showed that calbindin-IR axons formed two types of inputs, synapses and close contacts, on calretinin-IR neurons. About 40% of inputs to the longitudinal muscle motor neurons and 70% to ascending interneurons were calbindin-IR. Approximately 50% of longitudinal muscle motor neurons were surrounded by bombesin-IR dense pericellular baskets and 40% by closely apposed varicosities. At the electron-microscope level, the bombesin-IR varicosities were found to form synapses and close contacts with the motor neurons. Dense pericellular baskets with bombesin-IR surrounded 36% of all ascending interneurons, and a further 17% had closely apposed varicosities. Somatostatin-and 5-HT-IR descending interneurons provided no dense pericellular baskets to calretinin-IR nerve cells. Thus, calretinin-IR, longitudinal muscle motor neurons and ascending interneurons receive direct synaptic inputs from intrinsic primary sensory neurons and from non-cholinergic, bombesin-IR, descending interneurons.     

Structural plasticity at crustacean neuromuscular synapses

Crustacean motor axons innervate muscle fibers via a multiplicity of synaptic terminals which release small but variable amounts of transmitter. Differences in release performance appear to be correlated with the size of synaptic contacts and presynaptic dense bars (active zones). These structural parameters proliferate via sprouting from existing synaptic terminals and relocate to ever more distal sites during development and growth of an identified axon. Moreover, alterations in number of synaptic contacts and active zones occur in adults following stimulation or decentralization, demonstrating structural plasticity of crustacean neuromuscular synapses.     

Neural control of the pyloric region in the foregut of the shrimp Penaeus (Decapoda: Penaeidae)

Pyloric pattern-generating neurons that control the pyloric region of the foregut were identified in the stomatogastric ganglion of the most primitive decapod genus Penaeus. Five types of motor neurons and one interneuron are involved in generation of pyloric motor pattern. One cell type of motor neurons innervates muscles of both the gastric mill and the pylorus like the gastric motor neurons in Cancer, but unlike those in Panulirus. These identified neurons are connected to each other either by electrical or inhibitory chemical synapses to construct the neural circuit. This pyloric circuit is similar to the homologous circuit of other crustacean species though some differences are seen in synaptic connections, supporting the hypothesis that the basic design of the neural circuit has been conserved during evolution of the Malacostraca, and that differences have occurred in the synaptic connectivity as the foregut structure has become complex. The motor neurons use either acetylcholine or glutamate as a neurotransmitter like in reptantians. The foregut structure, the number of the pyloric cells, muscle innervation, neurotransmitters, and circuitry are compared among malacostracan crustaceans to provide insight into how the neural circuits change and evolve to produce the motor patterns mediating behaviour. Accepted: 18 April 1997     

Presynaptic mitochondria and the temporal pattern of neurotransmitter release

Mitochondria are critical for the function of nerve terminals as the cycling of synaptic vesicle membrane requires an efficient supply of ATP. In addition, the presynaptic mitochondria take part in functions such as Ca2+ buffering and neurotransmitter synthesis. To learn more about presynaptic mitochondria, we have examined their organization in two types of synapse in the lamprey, both of which are glutamatergic but are adapted to different temporal patterns of activity. The first is the giant lamprey reticulospinal synapse, which is specialized to transmit phasic signals (i.e. bursts of impulses). The second is the synapse established by sensory dorsal column axons, which is adapted to tonic activity. In both cases, the presynaptic axons were found to contain two distinct types of mitochondria; small 'synaptic' mitochondria, located near release sites, and larger mitochondria located in more central parts of the axon. The size of the synapse-associated mitochondria was similar in both types of synapse. However, their number differed considerably. Whereas the reticulospinal synapses contained only single mitochondria within 1 micron distance from the edge of the active zone (on average 1.2 per active zone, range of 1-3), the tonic dorsal column synapses were surrounded by clusters of mitochondria (4.5 per active zone, range of 3-6), with individual mitochondria sometimes apparently connected by intermitochondrial contacts. In conjunction with studies of crustacean neuromuscular junctions, these observations indicate that the temporal pattern of transmitter release is an important determinant of the organization of presynaptic mitochondria.     

Setogenesis and molting in planktonic crustaceans

The formation of new setae, termed setogenesis, is describedfor two taxa of planktonic crustaceans: euphausiids, Euphausiapacifica and Thysanoessa spinifera, and a calanoid copepod,Calanus marshallae. Characteristics of setal formation and eversionat ecdysis are described from two time-series of animals preservedat known intervals in their molt cycle. Results from these laboratoryreference series indicate that previous interpretations of setogenesisin the literature can by synthesized to describe a dynamic processof setal formation which is common to all crustacean taxa. The morphological characteristics of developing setae are usedto designate three specific phases in the molt cycle of planktoniccrustaceans (premolt, postmolt, and intermolt). This stagingtechnique may be used to study field-oriented problems relatedto molting in small planktonic crustaceans.     

GABA-immunoreactive neurons and terminals in the cat periaqueductal gray matter: A light and electron microscopic study

Immunocytochemical and electron microscopic methods were used to study the GABAergic innervation in adult cat periaqueductal gray matter (PAG). A mouse monoclonal antibody against γ -aminobutyric acid (GABA) was used to visualize the inhibitory neuronal system of PAG. At light microscopy, GABA-immunopositive (GABA_IP) neurons formed two longitudinally oriented columns in the dorsolateral and ventrolateral PAG that accounted for 36% of the neuronal population of both PAG columns; their perikaryal cross-sectional area was smaller than that of unlabeled (UNL) neurons found in the same PAG subdivisions. At electron microscopic level, patches of GABA immunoreactivity were readily detected in neuronal cell bodies, proximal and distal dendrites, axons and axon terminals. Approximately 35–36% of all terminals were GABA_IP; they established symmetric synapses with dendrites (84.72% of the sample in the dorsolateral PAG and 86.09% of the sample in the ventrolateral PAG) or with cell bodies (7–10% of the sample). Moreover, 49.15% of GABA_IP axon terminals in the dorsolateral and 52.16% in the ventrolateral PAG established symmetric synapses with GABA_IP dendrites. Immunopositive axon terminals and unlabeled terminals were also involved in the formation of a complex synaptic arrangment, i.e. clusters of synaptic terminals in close contact between them that were often observed in the PAG neuropil. Moreover, a fair number of axo-axonic synapses between GABA_IP and/or UNL axon terminals were present in both PAG subdivisions. Several dendro-dendritic synapses between labeled and unlabeled dendrites were also observed in both PAG subdivisions. These results suggest that in the cat PAG there exist at least two classes of GABArgic neurons. The first class could exert a tonic control on PAG projecting neurons, the second could act on those GABAergic neurons that in turn keep PAG projecting neurons under tonic inhibition. The functional implications of this type of GABAergic synapse organization are discussed in relation to the dishinibitory processes that take place in the PAG.     

Regenerated synaptic terminals on a crayfish slow muscle identify with transplanted phasic or tonic axons

Phasic or tonic nerves transplanted onto a denervated slow superficial flexor muscle in adult crayfish regenerated synaptic connections that displayed large or small excitatory postsynaptic potentials (EPSPs), respectively, suggesting that the neuron specifies the type of synapse that forms (Krause et al., J Neurophysiol 80:994-997, 1998). To test the hypothesis that such neuronal specification would extend to the synaptic structure as well, we examined the regenerated synaptic terminals with thin serial section electron microscopy. There are distinct differences in structure between regenerated phasic and tonic innervation. The phasic nerve provides more profuse innervation because innervation sites occurred more frequently and contained larger numbers of synaptic terminals than the tonic nerve. Preterminal axons of the phasic nerve also had many more sprouts than those of the tonic nerve. Phasic terminals were thinner and had a lower mitochondrial volume than their tonic counterparts. Phasic synapses were half the size of tonic ones, although their active zone-dense bars were similar in length. The density of active zones was higher in the phasic compared with the tonic innervation, based on estimates of the number of dense bars per synapse, per synaptic area, and per nerve terminal volume. Because these differences mirror those seen between phasic and tonic axons in crayfish muscle in situ, we conclude that the structure of the regenerated synaptic terminals identify with their transplanted axons rather than with their target muscle. Therefore, during neuromuscular regeneration in adult crayfish, the motoneuron appears to specify the identity of synaptic connections.     

Postsynaptic regulation of the development and long-term plasticity of Aplysia sensorimotor synapses in cell culture

The monosynaptic component of the neuronal circuit that mediates the withdrawal reflex of Aplysia californica can be reconstituted in dissociated cell culture. Study of these in vitro monosynaptic connections has yielded insights into the basic cellular mechanisms of synaptogenesis and long-term synaptic plasticity. One such insight has been that the development of the presynaptic sensory neurons is strongly regulated by the postsynaptic motor neuron. Sensory neurons which have been cocultured with a target motor neuron have more elaborate structures—characterized by neurites with more branches and varicosities—than do sensory neurons grown alone in culture or sensory neurons that have been cocultured with an inappropriate target cell. Another way in which the motor neuron regulates the development of sensory neurons is apparent when sensorimotor cocultures with two presynaptic cells are examined. In such cocultures the outgrowth from the different presynaptic cells is obviously segregated on the processes of the postsynaptic cell. By contrast, when two sensory neurons are placed into cell culture without a motor neuron, thier processes readily grow together. In addition to regulating the in vitro development of sensory neurons, the motor neuron also regulates learning-related changes in the structure of sensory neurons. Application of the endogenous facilitatory trasmitter serotonin (5-HT) causes long-term facilitation of in vitro sensorimotor synapses due in part to growth of new presynatpic varicosities. But 5-HT applied to sensory neurons alone in cultuer does not produce structural changes in these cells. More recently it has been found that sensorimotor synapses in cell culture can exhibit long-term potentiation (LTP). Like LTP of some hippocampal synapses, LTP of in vitro Aplysia syanpses is regulated by the voltage of the postsynaptic cell. Pairing high-frequency stimulation of sensory neurons with strong hyperpolarization of the motor neuron blocks the induction of LTP. Moreover, LTP of sensorimotor synapses can be induced in Hebbian fashion by pairing weak presynaptic stimulation with strong postsynaptic depolarization. These findings implicate a Habbian mechanism in classical conditioning in Aplysia. They also indicate that Hebbian LTP is a phylogenetically ancient form of synaptic plasticity. 1994 John Wiley & Sons, Inc.     

Regeneration from crayfish phasic and tonic motor axons in vitro

An explant culture system is described that allows examination of axonal growth from the tonically and phasically active motoneurons of the abdominal nerve cord of the crayfish. In this preparation, growth occurs from the cut end of the axon while the remainder of the motoneuron is undisturbed. In vitro growth from the branches of the third roots, which contain the axons from the tonic and phasic motoneurons of abdominal ganglia one through four, was verified as axonal by retrograde labeling of axons and neuronal somata within the nerve cord. Growth from the axons of phasic and tonic cells was observed as early as 24 h after plating and continued for an additional 7–10 days. The morphology and growth rates of the motor terminals differed between the tonic and phasic axons. The phasic axons grew significantly faster and branched more often than did the tonic motor axons. These differences in growth may be related to differences in motoneuron size or, may result from differences in electrical activity. Tonic motoneurons show spontaneous impulse activity for up to 6 days in culture, whereas phasic motoneurons show no spontaneous impulse activity. In addition, the differences in growth may be related to the morphological differences in tonic and phasic motor terminals observed in situ. © 1993 John Wiley & Sons, Inc.     

Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae

The innervation of ventral longitudinal abdominal muscles (muscles 6, 7, 12, and 13) of third-instar Drosophila larvae was investigated with Nomarski, confocal, and electron microscopy to define the ultrastructural features of synapse-bearing terminals. As shown by previous workers, muscles 6 and 7 receive in most abdominal segments “Type I” endings, which are restricted in distribution and possess relatively prominent periodic terminal enlargements (“boutons”); whereas muscles 12 and 13 have in addition “Type II” terminals, which are more widely distributed and have smaller “boutons.” Serial sectioning of the Type I innervation of muscles 6 and 7 showed that two axons with distinctive endings contribute to it. One axon (termed Axon 1) has somewhat larger boutons, containing numerous synapses and presynaptic dense bodies (putative active zones for transmitter release). This axon also has more numerous intraterminal mitochondria, and a profuse subsynaptic reticulum around or under the synaptic boutons. The second axon (Axon 2) provides somewhat smaller boutons, with fewer synapses and dense bodies per bouton, fewer intraterminal mitochondria, and less-developed subsynaptic reticulum. Both axons contain clear synaptic vesicles, with occasional large dense vesicles. Approximately 800 synapses are provided by Axon 1 to muscles 6 and 7, and approximately 250 synapses are provided by Axon 2. In muscles 12 and 13, endings with predominantly clear synaptic vesicles, generally similar to the Type I endings of muscles 6 and 7, were found, along with another type of ending containing predominantly dense-cored vesicles, with small clusters of clear synaptic vesicles. This second type of ending was found most frequently in muscle 12, and probably corresponds to a subset of the “Type II” endings seen in the light microscope. Type I endings are thought to generate the ?fast’? and ?slow’? junctional potentials seen in electrophysiological recordings, whereas the physiological actions of Type II endings are presently not known. © 1993 John Wiley & Sons, Inc.     

Structure of anterior dorsal ventricular ridge in snakes.

Anterior dorsal ventricular ridge (ADVR) is a major subcortical, telencephalic nucleus in snakes. Its structure was studied in Nissl, Golgi, and electron microscopic preparations in several species of snakes. Neurons in ADVR form a homogeneous population. They have large nuclei, scattered cisternae of rough endoplasmic reticulum in their cytoplasm, and bear dendrites from all portions of their somata. The dendrites have a moderate covering of pedunculated spines. Clusters of two to five cells with touching somata can be seen in Nissl, Golgi, and electron microscopic preparations. The area of apposition may contain a series of specialized junctions which resemble gap junctions. Three populations of axons can be identified in rapid Golgi preparations of snake ADVR. Type 1 axons course from the lateral forebrain bundle and bear small varicosities about 1 mu long. Type 2 axons arise from ADVR neurons and bear large varicosities about 5 mu long. The origin of the very thin type 3 axons is not known; they bear small varicosities about 1 mu long. The majority of axon terminals in ADVR are small (1 mu to 2 mu long), contain round synaptic vesicles, and form asymmetric active zones. This type of axon terminates on dendritic spines and shafts and on somata. A small percentage of terminals are large, 5 mu in length, contain round synaptic vesicles, and form asymmetric active zones. This type of axon terminates only on dendritic spines. A small percentage of terminals are small, contain pleomorphic synaptic vesicles, and form symmetric active zones. This type of axon terminates on dendritic shafts and on somata.     

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.