Proliferation and relocation of developing lobster neuromuscular synapses

The development of multiterminal innervation from a single identifiable excitatory motoneuron to the lobster distal accessory flexor muscle (DAFM) was studied by serial section electron microscopy. The number, size, and location of neuromuscular synapses and presynaptic dense bars within the peripheral branching pattern of the axon was determined in cross sections of the DAFM in 1st (24-hr-old)-, 4th (2-week-old)-, and 12th (1-year-old)-stage lobsters. The mean size of synapses remains fairly constant in these three stages but synaptic density, i.e., the number of synapses per unit length of fiber, increased more than 20-fold between the 1st and 4th stages and more than 5-fold between the 4th and 12th stages. Synaptic surface area per fiber length showed a parallel increase. Consequently there is a proliferation of synapses along the length of individual muscle fibers during primary development. Furthermore from the 1st stage where only a few fibers are innervated, synapses proliferate to many more fibers in the 4th and to all fibers in the 12th stage. The neuromuscular synapses are distributed in different proportions within the axonal branching pattern in the three stages. Based on the number and size of synapses and presynaptic dense bars, the main axon and primary branches provide almost equal amounts of innervation in the 1st stage. With further branching in the 4th stage, the main axon accounts for only 20–25% of the innervation; the primary branches for 45% and other finer branches the remainder. By the 12th-stage synapses are found only on branches other than the main axon and its primary offshoots. There is therefore a shift in innervation from the main axon to the primary branches and then to the finer branches during primary development. This shift in innervation involves the formation of new synaptic terminals and the restructuring of existing ones into axonal areas. In this way the multiterminal innervation arising from an identifiable motoneuron is remodeled.     

The electrophysiology and pharmacology of lobster neuromuscular synapses

Effects of drugs on resting potential, membrane resistance, and excitatory and inhibitory postsynaptic potentials (e.p.s.p.'s and i.p.s.p.'s) of lobster muscle fibers were studied using intracellular microelectrodes Acetylcholine, d-tubocurarine, strychnine, and other drugs of respectively related actions on vertebrate synapses were without effects even in 1 per cent solutions (10^- w/v). Gamma-aminobutyric acid (GABA) acted powerfully and nearly maximally at 10^-7 to 10^-6 w/v. Membrane resistance fell two- to tenfold, the resting potential usually increasing slightly. This combination of effects, which indicates activation of inhibitory synaptic membrane, was also produced by other short chain ω-amino acids and related compounds that inactivate depolarizing axodendritic synapses of cat. The conductance change, involving increased permeability to Cl^-, by its clamping action on membrane potential shortened as well as decreased individual e.p.s.p.'s. Picrotoxin in low concentration (ca. 10^-7 w/v) and guanidine in higher (ca. 10^-3 w/v) specifically inactivate inhibitory synapses. GABA and picrotoxin are competitive antagonists. The longer chain ω-amino acids which inactivate hyperpolarizing axodendritic synapses of cat are without effect on lobster neuromuscular synapse. However, one member of this group, carnitine (β-OH-GABA betaine), activated the excitatory synapses, a decreased membrane resistance being associated with depolarzation. The pharmacological properties of lobster neuromuscular synapses and probably also of other crustacean inhibitory synapses appear to stand in a doubly inverted relation to axodendritic synapses of cat.     

Postsynaptic organization and regulation of excitatory synapses

Dynamic regulation of synaptic efficacy is one of the mechanisms thought to underlie learning and memory. Many of the observed changes in efficacy, such as long-term potentiation and long-term depression, result from the functional alteration of excitatory neurotransmission mediated by postsynaptic glutamate receptors. These changes may result from the modulation of the receptors themselves and from regulation of protein networks associated with glutamate receptors. Understanding the interactions in this synaptic complex will yield invaluable insight into the molecular basis of synaptic function. This review focuses on the molecular organization of excitatory synapses and the processes involved in the dynamic regulation of glutamate receptors.     

Basal lamina and the organization of neuromuscular synapses

Fast chemical synapses are comprised of presynaptic and postsynaptic specializations precisely aligned across a protein-filled synaptic cleft. At the vertebrate neuromuscular junction (NMJ), the synaptic cleft contains a structured form of extracellular matrix known as a basal lamina (BL). Synaptic BL is molecularly differentiated from the BL that covers the extrasynaptic region of the myofiber. This review summarizes current understanding of the morphology, composition, and function of the synaptic BL at the vertebrate NMJ. Considerable evidence supports the conclusion that the synaptic BL organizes and maintains pre- and postsynaptic specializations during development and regeneration, and promotes robust neurotransmission in the adult.     

Presynaptic dense bars at neuromuscular synapses of the lobster,Homarus americanus

Summary The threedimensional ultrastructure of presynaptic dense bars was examined by serial section electron microscopy in the excitatory neuromuscular synapses of the accessory flexor muscle in the limbs of larval, juvenile, and adult lobsters. The cross-sectional profile of the dense bar resembles an asymmetric hourglass, the part contacting the presynaptic membrane being larger than that projecting into the terminal. The bar has a height of 55–65 nm and varies in length from 75–600 nm. In its dimensions it resembles the dense projections in the synapses of the CNS of insects and vertebrates. The usual location of these dense bars is at well defined synapses, though a few are found at extrasynaptic sites either in the axon or terminal. In the latter case the bars are close to synapse-bearing regions, particularly in the larval terminals, suggesting that the extrasynaptic bars denote early events in synapse formation. In all cases the bars are intimately associated with electron lucent, synaptic vesicles located on either side, in the indentation of its hourglass-shaped cross sectional profile. The vesicles occur along the length of the bar and contact the presynaptic membrane. Consequently the dense bar may serve to align the vesicles at the presynaptic membrane prior to exocytosis. A similar role has been suggested for the presynaptic dense bodies at the neuromuscular junction of the frog, where synaptic vesicles form a row on either side of this structure.Supported by Muscular Dystrophy Association of Canada and NSERCC. Generous use of laboratory facilities at Woods Hole was provided by the late Fred Lang     

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.     

Differential organization of excitatory and inhibitory synapses within the rat dorsal vagal complex

The dorsal motor nucleus of the vagus (DMV) is pivotal in the regulation of upper gastrointestinal functions, including motility and both gastric and pancreatic secretion. DMV neurons receive robust GABA- and glutamatergic inputs. Microinjection of the GABA(A) antagonist bicuculline (BIC) into the DMV increases pancreatic secretion and gastric motility, whereas the glutamatergic antagonist kynurenic acid (KYN) is ineffective unless preceded by microinjection of BIC. We used whole cell patch-clamp recordings with the aim of unveiling the brain stem neurocircuitry that uses tonic GABA- and glutamatergic synapses to control the activity of DMV neurons in a brain stem slice preparation. Perfusion with BIC altered the firing frequency of 71% of DMV neurons, increasing firing frequency in 80% of the responsive neurons and decreasing firing frequency in 20%. Addition of KYN to the perfusate either decreased (52%) or increased (25%) the firing frequency of BIC-sensitive neurons. When KYN was applied first, the firing rate was decreased in 43% and increased in 21% of the neurons; further perfusion with BIC had no additional effect in the majority of neurons. Our results indicate that there are several permutations in the arrangements of GABA- and glutamatergic inputs controlling the activity of DMV neurons. Our data support the concept of brain stem neuronal circuitry that may be wired in a finely tuned organ- or function-specific manner that permits precise and discrete modulation of the vagal motor output to the gastrointestinal tract.     

Recombinant neuromuscular synapses

The developing neuromuscular junction has provided an important paradigm for studying synapse formation. An outstanding feature of neuromuscular differentiation is the aggregation of acetylcholine receptors (AChRs) at high density in the postsynaptic membrane. While AChR aggregation is generally believed to be induced by the nerve, the mechanisms underlying aggregation remain to be clarified. A 43-kD protein (43k) normally associated with the cytoplasmic aspect of AChR clusters has long been suspected of immobilizing AChRs by linking them to the cytoskeleton. In recent studies, the AChR clustering activity of 43k has, at last, been demonstrated by expressing recombinant AChR and 43k in non-muscle cells. Mutagenesis of 43k has revealed distinct domains within the primary structure which may be responsible for plasma membrane targeting and AChR binding. Other lines of study have provided clues as to how nerve-derived (extracellular) AChR-cluster inducing factors such as agrin might activate 43k-driven postsynaptic membrane specialization.     

Quantitative comparison of low- and high-output neuromuscular synapses from a motoneuron of the lobster (Homarus americanus)

Summary Representative examples of lowand high-output neuromuscular synapses between motoneuron and distal accessory flexor muscle of the lobster were selected on the basis of their mean quantal content, and subsequently analysed by serial section electron microscopy. The high-output terminal has twice as many synapses as the low-output terminal. However, since the mean surface area of synapses is significantly smaller in the high-output terminal than in the low-output one, the total synaptic surface area between the two types of terminals is similar. Also, though the high-output terminal possesses a greater number of presynaptic dense bodies than its low-output counterpart, the mean number per synapse is similar for the two terminals. The terminals, however, differ significantly in the size of their dense bodies. Thus both the mean and total surface area of these bodies is greater in the high-output terminal than in the low-output one. Moreover, the mean ratio of dense body area to synaptic area is significantly greater for the high-output terminal than for its low-output counterpart. This difference in dense body area parallels the difference in quantal content of synaptic transmission between the lowand high-output terminals and supports the hypothesis that presynaptic densities represent the ultrastructural correlates of transmitter mobilization and/or release.Supported by grants from the National Research Council and Muscular Dystrophy Association of Canada to C.K. Govind. D.E. Meiss is a post-doctoral fellow of the Muscular Dystrophy Association of Canada. We thank Eva Yap-Chung for her expert and unfailing technical assistance     

Activity-dependent elimination of neuromuscular synapses

At developing neuromuscular synapses in vertebrates, different motor axon inputs to muscle fibers compete for maintenance of their synapses. Competition results in progressive changes in synaptic structure and strength that lead to the weakening and loss of some inputs, a process that has been called synapse elimination. At the same time, a single input is strengthened and maintained throughout adult life, consistently recruiting muscle fibers to contract even at rapid firing rates. Work over the last decade has led to an understanding of some of the cell biological mechanisms that underlie competition and how these culminate in synapse elimination. We discuss current ideas about how activity modulates neuromuscular synaptic competition, how competition leads to synapse loss, and how these processes are modulated by cell-cell signaling. A common feature of competition at neuromuscular as well as CNS synapses is that temporally correlated activity seems to slow or prevent competition, while uncorrelated activity seems to trigger or enhance competition. Important questions that remain to be addressed include how patterns of motor neuron activity affect synaptic strength, what is the temporal relationship between changes in synaptic strength and structure, and what cellular signals mediate synapse loss. Answers to these questions will expand our understanding of the mechanisms by which activity edits synaptic structure and function, writing permanent changes in neural circuitry.     

AMPA receptor trafficking at excitatory synapses

Excitatory synapses in the CNS release glutamate, which acts primarily on two sides of ionotropic receptors: AMPA receptors and NMDA receptors. AMPA receptors mediate the postsynaptic depolarization that initiates neuronal firing, whereas NMDA receptors initiate synaptic plasticity. Recent studies have emphasized that distinct mechanisms control synaptic expression of these two receptor classes. Whereas NMDA receptor proteins are relatively fixed, AMPA receptors cycle synaptic membranes on and off. A large family of interacting proteins regulates AMPA receptor turnover at synapses and thereby influences synaptic strength. Furthermore, neuronal activity controls synaptic AMPA receptor trafficking, and this dynamic process plays a key role in the synaptic plasticity that is thought to underlie aspects of learning and memory.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage—current relationship of the membrane. By including γ-aminobutyrate (GABA) in the bathing medium, the voltage—current relationship was made linear in the depolarizing direction over a range of 15–20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and asparate functioning synergistically.     

Cooperative interaction of glutamate and aspartate with receptors in the neuromuscular excitatory membrane in walking limbs of the lobster.

When applied to lobster muscle fibers, L-glutamate, L-aspartate, and combinations of the two amino acids can induce membrane depolarization. Under normal conditions, a quantitative analysis of the depolarization response or change in membrane conductance was precluded by nonlinearities in the voltage-current relationship of the membrane. By including gamma-aminobutyrate (GABA) in the bathing medium, the voltage-current relationship was made linear in the depolarizing direction over a range of 15-20 mV from the resting potential. However, a meaningful examination of the increase in membrane conductance caused by glutamate and aspartate was still not possible. Therefore, the depolarization responses caused by the excitatory amino acids were taken as a quantitative reflection of receptor activation in the excitatory postsynaptic membrane. In the presence of GABA, aspartate by itself, at concentrations up to 10 mM, had little excitatory activity, whereas glutamate effected an appreciable membrane depolarization at concentrations of 0.1 to 0.2 mM. Aspartate, at concentrations which exhibited no activity alone, markedly enhanced the excitatory action of glutamate. Aspartate shifted the glutamate dose-response curve to the left, but did not appear to affect the maximum depolarization response elicited by glutamate. These observations are consistent with the concept that aspartate increases the affinity between glutamate and the glutamate binding sites. Limiting slopes of log-dose versus log-response curves for the excitatory action of glutamate suggest that the interaction of glutamate with excitatory receptors is a cooperative process. The possibility exists that individual receptors contain multiple and distinct glutamate and aspartate binding sites. These results support the view that neuromuscular excitation in the lobster is mediated by glutamate and aspartate functioning synergistically.     

Optimal learning with excitatory and inhibitory synapses

Characterizing the relation between weight structure and input/output statistics is fundamental for understanding the computational capabilities of neural circuits. In this work, I study the problem of storing associations between analog signals in the presence of correlations, using methods from statistical mechanics. I characterize the typical learning performance in terms of the power spectrum of random input and output processes. I show that optimal synaptic weight configurations reach a capacity of 0.5 for any fraction of excitatory to inhibitory weights and have a peculiar synaptic distribution with a finite fraction of silent synapses. I further provide a link between typical learning performance and principal components analysis in single cases. These results may shed light on the synaptic profile of brain circuits, such as cerebellar structures, that are thought to engage in processing time-dependent signals and performing on-line prediction.     

Receptor trafficking and the plasticity of excitatory synapses

Newly discovered features of the trafficking of AMPA receptors to and from the postsynaptic membrane of excitatory synapses are now bringing the mechanisms of synaptic plasticity into focus. Recent advances, including the existence of slots, anchors, transport factors and pathways for activity-dependent control, have elucidated the role of the individual AMPA receptor subunits and their binding partners. The latest views describe how subunit type dictates the assembly of heteromeric receptors, and how these heteromers interact with the receptor trafficking machinery and synaptic anchorage factors. Moreover, phosphorylation may play an important role in receptor transport and synaptic turnover.