Synaptic development in the crayfish opener muscle

Nerve terminal regions in walking leg opener muscles of several crayfish of different ages (0 to 245 days after hatching) were examined by means of electron microscopy. This muscle is innervated by two axons (excitatory and inhibitory) and at maturity contains three classes of synapse: excitatory and inhibitory neuromuscular synapses, and inhibitory axo-axonal synapses. The muscle itself is initially a syncytium, which gradually becomes subdivided into distinct “muscle fibers” as the animal matures. Innervation was not found in the opener muscle just before or just after hatching, but was present in restricted locations on the inner side of the muscle within a few days of hatching. As the muscle enlarged and became subdivided, innervation appeared in various other locations. Synaptic contacts were located in young stages soon after hatching, and in later stages. Morphological differences characteristic of excitatory and inhibitory nerve terminals could be found even at the earliest stages of innervation. Both excitatory and inhibitory synapses, but particularly the former, showed evidence of progressive enlargement to a final size within the first two months, and no evidence for further enlargement of existing synapses thereafter. Synaptic maturation also involved the appearance of presynaptic “dense bodies” thought to be regions at which transmitter substance is preferentially released. Nerve terminals at different levels of maturation were observed in opener muscles of young crayfish. Clear evidence for differential maturation of the three types of synapse present in this muscle was obtained. The inhibitory neuromuscular synapses attained their final average size and developed their dense bodies sooner than the excitatory neuromuscular synapses. The inhibitory axo-axonal synapses were the last to appear and to mature.     

Synaptic restructuring during long-term facilitation at the crayfish neuromuscular junction

Long-term facilitation was induced by 20-Hz stimulation of the motor axon innervating the opener muscle of the crayfish, Procambarus clarkii. Excitatory postsynaptic potentials remained potentiated for several hours after stimulation. Structural correlates of potentiation were sought. Nerve terminals of the motor axon were fixed for electron microscopy in unstimulated preparations (controls), and during and after 20-Hz stimulation. Synapses were reconstructed from micrographs obtained from serial sections. Synaptic contact area and the number of vesicles at the presynaptic membrane did not change after 20-Hz stimulation, but the latter decreased during stimulation. Presynaptic dense bars ("active zones") decreased in number during and increased after stimulation, while perforated synapses increased after stimulation. Modification of presynaptic structures occurs rapidly and may be linked to long-lasting changes in quantal content of transmission.     

Synaptic repression at crayfish neuromuscular junctions. II. Evidence for calcium involvement

The inability of synaptic junctions to generate normalsized postsynaptic potentials under normal physiological conditions was studied at crayfish neuromuscular synapses. Synaptic repression in the superficial flexor muscle system of the crayfish was induced by surgery: the nerve was cut in the middle of the target field, and the lateral muscle fibers were removed. After this surgery, the remaining medial synapses were unable to generate normal-sized junction potentials (jp) over the medial muscle population. In an attempt to study the mechanism underlying this response, we varied the extracellular calcium concentration of the Ringers solution bathing the preparation, in both repressed and control animals, while monitoring the size of the same junction potential. The junction potential generated by the spontaneous activity of the nerve increased in size with increasing calcium concentrations in control animals, but failed to do so in repressed animals, that is, changes in external calcium concentrations did not affect repressed synapses. However, in the presence of the calcium ionophore A23187, control and repressed synapses both show an increase in the junction potential sizes they generate. Our data suggest that calcium is involved in the mechanisms that underlie synaptic repression in this crustacean neuromuscular system. © 1993 John Wiley & Sons, Inc.     

Synaptic plasticity in a regenerated crayfish phasic motoneuron.

Crustacean neuromuscular systems provide many advantages for the study of synaptic transmission and plasticity. The present study examines aspects of synaptic transmission in the phasic, fast closer excitor (FCE) motoneuron of regenerated crayfish claws. Excitatory postsynaptic potentials (EPSPs) fatigued rapidly and showed poor long-term facilitation (LTF) in the smallest of regenerating claws. EPSPs in larger regenerating claws fatigued less and showed pronounced facilitation. These observations were not the same as those previously made during primary development of this motoneuron (Lnenicka and Atwood, 1985a, J. Neuroscience 5:459-467). Hence, regeneration is not the recapitulation of primary development. In situ stimulation of the FCE is known to lead to long-lasting adaptation of synaptic performance. This adaptation is age dependent; it is expressed in young but not old animals. In the regenerated FCE of old animals, we observed a novel form of long-lasting adaptation to imposed activity: EPSPs showed large initial EPSPs and did not exhibit resistance to fatigue during maintained stimulation. This indicates that aged motoneurons can express adaptive changes to increased activity following axonal regeneration, but that the adaptive changes are the opposite to what is observed in nonregenerated motoneurons.     

Synaptic vesicle recruitment for release explored by Monte Carlo stimulation at the crayfish neuromuscular junction

Neurotransmission at chemically transmitting synapses requires calcium-mediated fusion of synaptic vesicles with the presynaptic membrane. Utilizing ultrastructural information available for the crustacean excitatory neuromuscular junction, we developed a model that employs the Monte Carlo simulation technique to follow the entry and movement of Ca2+ ions at a presynaptic active zone, where synaptic vesicles are preferentially docked for release. The model includes interaction of Ca2+ with an intracellular buffer, and variable separation between calcium channels and vesicle-associated Ca(2+)-binding targets that react with Ca2+ to trigger vesicle fusion. The end point for vesicle recruitment for release was binding of four Ca2+ ions to the target controlling release. The results of the modeling experiments showed that intracellular structures that interfere with Ca2+ diffusion (in particular synaptic vesicles) influence recruitment or priming of vesicles for release. Vesicular recruitment is strongly influenced by the separation distance between an opened calcium channel and the target controlling release, and by the concentration and binding properties of the intracellular buffers, as in previous models. When a single opened calcium channel is very close to the target, a single synaptic vesicle can be recruited. However, many of the single-channel openings actuated by a nerve impulse are likely to be ineffective for release, although they contribute to the buildup of total intracellular Ca2+. Thus, the overall effectiveness of single calcium channels in causing vesicles to undergo exocytosis is likely quite low.     

Facilitation at crayfish neuromuscular junctions

Electrophysical recordings from opener muscle fibers in the crayfishProcambarus clarkii (Fig. 1) show that pre-synaptic facilitation at terminals of the single excitatory axon usually decays in a dual-exponential fashion after a single pulse or after a train of pulses (Figs. 2, 3, 7, 9), as has been reported for frog neuromuscular junctions (Mallart and Martin, 1967) and squid giant synapses (Charlton and Bittner, 1974, 1976). Furthermore, the second component of decay at crayfish synapses is associated with a break in the monotonic decay of the first component, a result which suggests that the decay of facilitation is not due to the simple diffusion of some substance (such as calcium) from specialized release sites.The growth of facilitation at all opener synapses during trains of equalinterval stimuli could not be predicted by assuming that each pulse contributed an equal amount of facilitation which summed linearly with that remaining from all previous stimuli (Figs. 4, 6; Table 2), as reported for synapses in frog and squid. During high frequency stimulation (>40 Hz), those terminals which facilitate dramatically (highF _e synapses) show much greater amounts of facilitation than that predicted by the linear summation model (Figs. 4, 8), whereas other terminals (lowF _e synapses) show much less facilitation than predicted (Fig. 6). The rate of growth of facilitation was often very constant at various stimulus rates in highF _e or mixed type synapses (Figs. 4, 8, 10)-a result not predicted by the linear summation model. Finally, when highF _e synapses were stimulated at different frequencies, the rate of growth of facilitation changed dramatically in a fashion not predictable using linear summation (Mallert and Martin, 1967) or power law (Linder, 1974) models.     

Synaptic repression at crayfish neuromuscular junctions. I. Generation after partial target area removal

Synaptic repression, the inability of synaptic junctions to generate normal-sized postsynaptic potentials under normal physiological conditions, is reported here for crayfish neuromuscular synapses. The synapses in the superficial flexor muscle system of the crayfish change their efficiency in generating a postsynaptic response as a result of a specific alteration in their immediate environment. When the superficial flexor nerve is cut halfway into the target muscle field and the lateral muscle fibers are removed, the intact medial synapses do not generate normal-sized junction potentials (JP) at the 17° –19°C temperature of the Ringers solution. JPs cannot be recorded in 83% of the muscle fibers at 2 weeks after the operation and of the few JPs that can be detected, 80% are smaller than 1 mV in size. By 8 weeks after the operation, JPs were detected in 55% of the muscle fibers, and now only 46% of these are smaller than 1 mV. When the lateral muscle fibers are left in place during the original operation, providing a target area for the cut nerve to grow into, JPs were then detected in 60%–80% of all medial fibers at all time periods after the operation; their size profile, with 10%–25% of the muscle fibers having JP's less than 1 mV, was similar to control values. These results suggest that the efficiency of these synaptic contacts become affected as a result of partial axotomy and removal of the target area of the cut branches of the axons. © 1993 John Wiley & Sons, Inc.     

GABA inactivation at the crayfish neuromuscular junction

Changes in the effective membrane resistance of the abductor muscle of the dactylopodite of the crayfish were used to indicate changes in the GABA concentration in the synaptic cleft. Following bath application of GABA (10^?5 to 5 × 10^?5M), the muscle membrane resistance decreased and then increased slowly over the next few minutes. Renewing the solution or stirring the bath restored the GABA effect. Higher GABA concentrations produced a large stable decrease in membrane resistance. An active uptake system for GABA in the junctional region is suggested by the observation that the slow increase in membrane resistance following GABA application was decreased by cooling to 2°C or by the addition of known GABA uptake blockers such as L -DABA, β-guanidinopropionic acid, or nipecotic acid. The transport inhibitors, PCMBS and chlorpromazine, produced irreversible decreases in muscle membrane resistance, which precluded examining their effects on GABA inactivation. The decrease in GABA effect was not dependent on the external sodium concentration or on the degree of receptor activation. Nipecotic acid, which blocked GABA inactivation, did not affect the decay of the neurally evoked inhibitory junctional potential.     

Effect of doublet impulse sequences in the crayfish claw opener muscles and the computer-simulated neuromuscular synapse

The effects of doublet impulse sequences of the excitatory motor axon on the movement of the claw opener muscles in the crayfish were examined. The excitatory motor axon was stimulated electrically with various patterns of doublet impulse sequences generated by a digital computer. Doublet impulse sequences of stimulation produced a larger sustained movement than an uniform impulse sequences at the same mean rate of stimulation. The movement was largest when the interval between the impulses of a doublet was about 5 ms. This interval generated a movement amplitude 25% greater than that for the uniform impulse sequence. A simple model was formulated to stimulate the neuromuscular synapse of the claw opener muscle. The relationship between stimulation sequences with alternating long and short intervals and responses (firing probabilities) of the neuromuscular synapse at the same mean rate was investigated. The responses was classified into two typical types which are noneffective Type I and effective Type II to the absolute refractory period (ARP). The characteristics which are larger responses with short intervals in Type I and reduction of responses in the ARP region of Type II formed a plateau peak of the experimental results. By incorporating the reduction of end-plate potential (EPP) as a property of nonlinear rule for temporal summation into the model, it was shown that Type I response is maximal with a plateau peak at short interval, agreeing well with the experimental results from the claw opener muscles.     

Synaptic vesicle pools at the frog neuromuscular junction

We have characterized the morphological and functional properties of the readily releasable pool (RRP) and the reserve pool of synaptic vesicles in frog motor nerve terminals using fluorescence microscopy, electron microscopy, and electrophysiology. At rest, about 20% of vesicles reside in the RRP, which is depleted in about 10 s by high-frequency nerve stimulation (30 Hz); the RRP refills in about 1 min, and surprisingly, refilling occurs almost entirely by recycling, not mobilization from the reserve pool. The reserve pool is depleted during 30 Hz stimulation with a time constant of about 40 s, and it refills slowly (half-time about 8 min) as nascent vesicles bud from randomly distributed cisternae and surface membrane infoldings and enter vesicle clusters spaced at regular intervals along the terminal. Transmitter output during low-frequency stimulation (2-5 Hz) is maintained entirely by RRP recycling; few if any vesicles are mobilized from the reserve pool.     

Synaptic plasticity at archicortical and neocortical levels

Research carried out by the author and his collaborators, devoted to analysis of the properties and neurophysiological mechanisms of long-term (for several hours) potentiation, is surveyed. Long-term potentiation of focal potentials and unitary responses of strictly hippocampal structures (areas CA₁ and CA₃) in the unanesthetized rabbit is described. Enhancement of excitatory (EPSPs) and inhibitory (IPSPs) postsynaptic potentials was found after tetanization. No corresponding changes of sensitivity to acetylcholine or acetylcholinesterase activity were found by microiontophoretic and histochemical methods during long-term potentiation. Statistical analysis of EPSPs evoked by microstimulation, based on the quantal hypothesis of synaptic transmission, showed an increase in the number of quanta of transmitter release during potentiation. Long-term potentiation of focal potentials during stimulation of the subcortical white matter in surviving neocortical slices and also long-term potentiation of focal and unitary responses of the sensomotor cortex of the unanesthetized rabbit are described. Potentiation of the "indirect" component of the global response of the pyramidal tract was found. The data suggest the presence of long-term potentiation of monosynaptic neocortical responses. It is concluded that the main mechanism of both hippocampal and neocortical long-term potentiation is increased efficiency of excitatory synapses. It is postulated that synapses modified in this way are used in the formation of memory traces.Brain Institute, All-Union Mental Health Research Center, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 16, No. 5, pp. 651–665, September–October, 1984.     

Synaptic plasticity at hippocampal mossy fibre synapses

The dentate gyrus provides the main input to the hippocampus. Information reaches the CA3 region through mossy fibre synapses made by dentate granule cell axons. Synaptic plasticity at the mossy fibre-pyramidal cell synapse is unusual for several reasons, including low basal release probability, pronounced frequency facilitation and a lack of N-methyl-D-aspartate receptor involvement in long-term potentiation. In the past few years, some of the mechanisms underlying the peculiar features of mossy fibre synapses have been elucidated. Here we describe recent work from several laboratories on the various forms of synaptic plasticity at hippocampal mossy fibre synapses. We conclude that these contacts have just begun to reveal their many secrets.     

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.     

Synaptic dynamics at the neuromuscular junction: mechanisms and models

During development, the neuromuscular junction passes through a stage of extensive polyinnervation followed by a period of wholesale synapse elimination. In this report we discuss mechanisms and interactions that could mediate many of the key aspects of these important developmental events. Our emphasis is on (1) establishing an overall conceptual framework within which the role of many distinct cellular interactions and molecular factors can be evaluated, and (2) generating computer simulations that systematically test the adequacy of different models in accounting for a wide range of biological data. Our analysis indicates that several relatively simple mechanisms are each capable of explaining a variety of experimental observations. On the other hand, no one mechanism can account for the full spectrum of experimental results. Thus, it is important to consider models that are based on interactions among multiple mechanisms. A potentially powerful combination is one based on (1) a scaffold within the basal lamina or in the postsynaptic membrane which is induced by nerve terminals and which serves to stabilize terminals by a positive feedback mechanism; (2) a sprouting factor whose release by muscle fibers is down-regulated by activity and perhaps other factors; and (3) an intrinsic tendency of motor neurons to withdraw some connections while allowing others to grow.     

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.     

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.     

Synaptic plasticity and addiction

Addiction is caused, in part, by powerful and long-lasting memories of the drug experience. Relapse caused by exposure to cues associated with the drug experience is a major clinical problem that contributes to the persistence of addiction. Here we present the accumulated evidence that drugs of abuse can hijack synaptic plasticity mechanisms in key brain circuits, most importantly in the mesolimbic dopamine system, which is central to reward processing in the brain. Reversing or preventing these drug-induced synaptic modifications may prove beneficial in the treatment of one of society's most intractable health problems.