Aberrant morphology and residual transmitter release at the Munc13-deficient mouse neuromuscular synapse

In cultured hippocampal neurons, synaptogenesis is largely independent of synaptic transmission, while several accounts in the literature indicate that synaptogenesis at cholinergic neuromuscular junctions in mammals appears to partially depend on synaptic activity. To systematically examine the role of synaptic activity in synaptogenesis at the neuromuscular junction, we investigated neuromuscular synaptogenesis and neurotransmitter release of mice lacking all synaptic vesicle priming proteins of the Munc13 family. Munc13-deficient mice are completely paralyzed at birth and die immediately, but form specialized neuromuscular endplates that display typical synaptic features. However, the distribution, number, size, and shape of these synapses, as well as the number of motor neurons they originate from and the maturation state of muscle cells, are profoundly altered. Surprisingly, Munc13-deficient synapses exhibit significantly increased spontaneous quantal acetylcholine release, although fewer fusion-competent synaptic vesicles are present and nerve stimulation-evoked secretion is hardly elicitable and strongly reduced in magnitude. We conclude that the residual transmitter release in Munc13-deficient mice is not sufficient to sustain normal synaptogenesis at the neuromuscular junction, essentially causing morphological aberrations that are also seen upon total blockade of neuromuscular transmission in other genetic models. Our data confirm the importance of Munc13 proteins in synaptic vesicle priming at the neuromuscular junction but indicate also that priming at this synapse may differ from priming at glutamatergic and gamma-aminobutyric acid-ergic synapses and is partly Munc13 independent. Thus, non-Munc13 priming proteins exist at this synapse or vesicle priming occurs in part spontaneously: i.e., without dedicated priming proteins in the release machinery.     

Rate of quantal transmitter release at the mammalian rod synapse.

Under scotopic conditions, the mammalian rod encodes either one photon or none within its integration time. Consequently the signal presented to its synaptic terminal is binary. The synapse has a single active zone that releases neurotransmitter quanta tonically in darkness and pauses briefly in response to a rhodopsin isomerization by a photon. We asked: what minimum tonic rate would allow the postsynaptic bipolar cell to distinguish this pause from an extra-long interval between quanta due to the stochastic timing of release? The answer required a model of the circuit that included the rod convergence onto the bipolar cell and the bipolar cell''s signal-to-noise ratio. Calculations from the model suggest that tonic release must be at least 40 quanta/s. This tonic rate is much higher than at conventional synapses where reliability is achieved by employing multiple active zones. The rod''s synaptic mechanism makes efficient use of space, which in the retina is at a premium.     

Calcium and the tonic release of transmitter at a non-impulsive synapse in the crab

Depolarization-transmitter release coupling was studied in the promotor stretch receptor/motoneuron synapse of the crab. Callinectes sapidus, a preparation in which presynaptic action potentials do not occur. Intracellular microelectrode recordings were made from the presynaptic terminal and from the somata of postsynaptic motoneurons while injecting current pulses into the peripheral stretch receptor dendrite with the aid of the sucrose-gap. 1. For short current pulses, the relationship between presynaptic potential and postsynaptic response was found to be similar to that demonstrated in the giant synapse of the squid stellate ganglion, indicating a common reliance on the properties of voltage-dependent calcium channels. 2. The crab synapse was found to be capable of continuous transmission in the range of seconds and minutes without the pronounced depletion of transmitter seen in the squid, and without inactivation of the release process (i.e., the calcium conductance is non-inactivating). 3. A graded, transient response to depolarising current in the presynaptic fibre was found to be calcium-dependent, and probably to reflect the presence of a separate, inactivating calcium conductance. 4. It was concluded that the graded response of the presynaptic membrane could function in helping to compensate for capacitative distortion of receptor potentials decrementally conducted in the sensory dendrite, and was therefore a specialisation for non-impulsive transmission.     

Presynaptic potentials and facilitation of transmitter release in the squid giant synapse

Presynaptic potentials were studied during facilitation of transmitter release in the squid giant synapse. Changes in action potentials were found to cause some, but not all, of the facilitation during twin-pulse stimulation. During trains of action potentials, there were no progressive changes in presynaptic action potentials which could account for the growth of facilitation. Facilitation could still be detected in terminals which had undergone conditioning depolarization or hyperpolarization. Facilitation could be produced by small action potentials in low [Ca++]o and by small depolarizations in the presence of tetrodotoxin. Although the production of facilitation varied somewhat with presynaptic depolarization, nevertheless, approximately equal amounts of facilitation could be produced by depolarizations which caused the release of very different amounts of transmitter.     

Quantal transmitter release in an identified inhibitory cholinergic synapse of Aplysia

A sustained postsynaptic potential is observed in an identified synapse of Aplysia when the presynaptic neuron is depolarized in the presence of tetrodotoxin (TTX). This prolonged postsynaptic potential appears to be at least in part due to the summation of quantal events. It is still observed when 30 mM CoCl2, which is known to inhibit Ca2+ influx, is added to the external media.     

Increased calmodulin affects cell morphology and mRNA levels of cytoskeletal protein genes.

We have previously described stable mouse C127 cell lines in which a CaM mini-gene has been expressed in a bovine papilloma virus-based expression vector (Rasmussen and Means: EMBO J. 6:3961-3968, 1987). Elevation of CaM to levels five-fold higher than in control cells caused an acceleration in cell cycle progression by reducing the length of the G1 period. When these cell lines were originally isolated it was observed that cells in which CaM levels were increased had a flattened morphology. In this study we have examined the localization of actin, vimentin, and tubulin in these cells as compared to the BPV-transformed control cell line in order to determine if changes in shape were accompanied by differences in the cytoskeletal organization. Cell-cycle-dependent changes in the levels of mRNAs for histone H4, glyceraldehyde-3-phosphate dehydrogenase, beta-actin, vimentin, and beta-tubulin have also been examined. Our results indicate that increased CaM causes differences in the organization of microfilaments, intermediate filaments, and microtubules and that these changes are accompanied by selective differences in the cell-cycle-dependent expression of some mRNAs. Elevated CaM was also correlated with a reduced stability of beta-tubulin mRNA. These studies indicate that CaM has pleiotropic effects on cell function and suggest that stable cell lines with altered CaM levels may provide a useful model system for understanding the molecular basis of CaM-dependent regulation of cellular processes.     

Changes in the topography of transmitter release in a neuromuscular synapse reflect plastic alterations occurring in active zones

The topography of transmitter release along the motor nerve terminals (NT) was studied on the frogcutaneous pectoris muscle under normal conditions and following denervation. Coordinates of release sites (RS) of transmitter quanta were determined by extracellular recording of postsynaptic signals using three microelectrodes. It was shown that RS form groupings that reflect transmitter release in individual active zones (AZ). The topography of transmitter release in the distal parts of the NT under normal conditions was shown to differ from that observed in the proximal parts. The difference consists in a lower probability of transmitter release in AZ and a higher probability of this process between AZ, as well as in a change of release profile in individual AZ. Similar differences were found following denervation. It is suggested that these properties may reflect plastic reorganization occurring in AZ in the course of remodelling of neuromuscular synapse and its degeneration.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 253–260, July–August, 1995.     

Synaptotagmin I: A major Ca2+ sensor for transmitter release at a central synapse

Mice carrying a mutation in the synaptotagmin I gene were generated by homologous recombination. Mutant mice are phenotypically normal as heterozygotes, but die within 48 hr after birth as homozygotes. Studies of hippocampal neurons cultured from homozygous mutant mice reveal that synaptic transmission is severely impaired. The synchronous, fast component of Ca²⁺-dependent neurotransmitter release is decreased, whereas asynchronous release processes, including spontaneous synaptic activity (miniature excitatory postsynaptic current frequency) and release triggered by hypertonic solution or α-latrotoxin, are unaffected. Our findings demonstrate that synaptotagmin I function is required for Ca²⁺-triggering of synchronous neurotransmitter release, but is not essential for asynchronous or Ca²⁺-independent release. We propose that synaptotagmin I is the major low affinity Ca²⁺ sensor mediating Ca²⁺ regulation of synchronous neurotransmitter release in hippocampal neurons.     

Loss of calcium/calmodulin responsiveness in adenylate cyclase of rutabaga,a Drosophila learning mutant

We have isolated and mapped an X-linked recessive mutation in Drosophila that blocks associative learning, and have partially characterized it biochemically. The mutation affects adenylate cyclase activity. Cyclase activity from mutant flies differed from the wild-type enzyme in that it was not stimulated by calcium or calmodulin. Mutant cyclase activity did respond to guanyl nucleotides, fluoride, and monoamines, which suggests that the defect is neither in the hormone receptor nor in either known GTP-binding regulatory protein. The mutation possibly affects the catalytic subunit directly. We postulate that there is at least one other type of adenylate cyclase activity that is unaffected by the mutation and insensitive to calcium/calmodulin.     

Purinergic modulation of transmitter release.

The concept of a purinergic theory for the regulation of the release of neurotransmitters is mainly based on the release of ATP, related nucleotides and adenosine concomitantly with the classical neurotransmitters (Ach and NA) after nerve stimulation, together with the inhibitory effects induced by those substances on the release of those neurotransmitters. As neuromodulators, ATP, related nucleotides and adenosine are in accordance with the classical criteria fulfilled by a neurotransmitter plus the absence of tachyphylaxis to the inhibitory action of purinergic substances, the impossibility to obtain complete blockade of the transmission by using the purinergic compounds and the involvement of Cat²⁺ as a step on the dynamics of the release of neurotransmitters. Furthermore ATP can be responsible for the Wedenski inhibition.     

The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function

Proper synaptic development is critical for establishing all aspects of neural function including learning, memory, and locomotion. Here, we describe the phenotypic consequences of mutations in the wishful thinking (wit) gene, the Drosophila homolog of the vertebrate BMP type II receptor. Mutations in wit result in pharate lethality that can be rescued by expression of a wit transgene in motor neurons but not in muscles. Mutant larvae exhibit small synapses, severe defects in evoked junctional potentials, a lower frequency of spontaneous vesicle release, and an alteration in the ultrastructure of synaptic active zones. These results reveal a novel role for BMP signaling in regulating Drosophila neuromuscular junction synapse assembly and activity and may indicate that similar pathways could govern vertebrate synapse development.     

Effect of changes in action potential shape on calcium currents and transmitter release in a calyx-type synapse of the rat auditory brainstem

We studied the relation between the size of presynaptic calcium influx and transmitter release by making simultaneous voltage clamp recordings from presynaptic terminals, the calyces of Held and postsynaptic cells, the principal cells of the medical nucleus of the trapezoid body, in slices of the rat brainstem. Calyces were voltage clamped with different action potential waveforms. The amplitude of the excitatory postsynaptic currents depended supralinearly on the size of the calcium influx, in the absence of changes in the time-course of the calcium influx. This result is in agreement with the view that at this synapse most vesicles are released by the combined action of multiple calcium channels.     

Presynaptic calcium concentration microdomains and transmitter release.

n-Aequorin J, a luminescent protein which responds to calcium concentration changes in the order of several hundred micromoles, was injected into the preterminal fiber in the squid giant synapse. The activation of the presynaptic terminal leading to release of transmitter was accompanied by light emission at well-defined sites at the active zone in the presynaptic terminal. Location of these light emission sites was very much the same from one stimulus to the next, indicating that light emission was triggered by the inward calcium current occurring at specific and invariant locations. The distribution, size and number of these QEDs (quantum emission domains) coincides well with the location and number of active zones in the presynaptic terminal. The results imply that transmitter release is triggered by very well-localized calcium concentration changes that may be as high as several hundred micromoles.     

Control of polytenic replication in dipteran larvae. I. Increased number of cycles in a mutant strain of Drosophila melanogaster

Previous studies have demonstrated that a consistent maximum number of polytenic replication cycles occurs in the salivary gland nuclei of a wild-type strain of D. melanogaster. Since that number is achieved within the larval period, the DNA synthesis of the prepupal period is believed to be that of propagation of the final cycle. Photometric determinations have been made, in this study, of the salivary gland nuclei of larvae and prepupae of the tu-h strain in which the larval period has been extended as a consequence of delay or failure of pupation. The DNA values indicate that a higher maximum number of polytenic replications is achieved in such nuclei. It is inferred, thereby, that initiation of polytenic replication is a function of the larval state and, since it is terminated by the intervention of metamorphosis, a hormonal dependence is suggested.     

Establishing and sculpting the synapse in Drosophila and C. elegans

Genetic approaches in flies and worms continue to dissect the intricate molecular machinery of chemical synapses. Investigations carried out in the last year provide important new insights into the development and modulation of the presynaptic active zones and postsynaptic receptor fields mediating synaptic function. Mutant screens have identified overlapping gene classes mediating synaptogenesis. The leucocyte common antigen-related receptor tyrosine phosphatase interacts with liprin in the formation of the active zone. Spectrins are essential for the spatial restriction of synaptic proteins to define active zones. Glutamate acts as a negative regulator of its cognate postsynaptic receptor to sculpt receptor field size. Finally, protein translation and degradation regulation emerge as possible key regulators of synaptic efficacy.     

Inactivity produces increases in neurotransmitter release and synapse size.

When hippocampal synapses in culture are pharmacologically silenced for several days, synaptic strength increases. The structural correlate of this change in strength is an increase in the size of the synapses, with all synaptic components--active zone, postsynaptic density, and bouton--becoming larger. Further, the number of docked vesicles and the total number of vesicles per synapse increases, although the number of docked vesicles per area of active zone is unchanged. In parallel with these anatomical changes, the physiologically measured size of the readily releasable pool (RRP) and the release probability are increased. Ultrastructural analysis of individual synapses in which the RRP was previously measured reveals that, within measurement error, the same number of vesicles are docked as are estimated to be in the RRP.     

Autophagy promotes synapse development in Drosophila

Autophagy, a lysosome-dependent degradation mechanism, mediates many biological processes, including cellular stress responses and neuroprotection. In this study, we demonstrate that autophagy positively regulates development of the Drosophila melanogaster larval neuromuscular junction (NMJ). Autophagy induces an NMJ overgrowth phenotype closely resembling that of highwire (hiw), an E3 ubiquitin ligase mutant. Moreover, like hiw, autophagy-induced NMJ overgrowth is suppressed by wallenda (wnd) and by a dominant-negative c-Jun NH₂-terminal kinase (bsk^DN). We show that autophagy promotes NMJ growth by reducing Hiw levels. Thus, autophagy and the ubiquitin–proteasome system converge in regulating synaptic development. Because autophagy is triggered in response to many environmental cues, our findings suggest that it is perfectly positioned to link environmental conditions with synaptic growth and plasticity.     

A tripartite synapse model in Drosophila

Tripartite (three-part) synapses are defined by physical and functional interactions of glia with pre- and post-synaptic elements. Although tripartite synapses are thought to be of widespread importance in neurological health and disease, we are only beginning to develop an understanding of glial contributions to synaptic function. In contrast to studies of neuronal mechanisms, a significant limitation has been the lack of an invertebrate genetic model system in which conserved mechanisms of tripartite synapse function may be examined through large-scale application of forward genetics and genome-wide genetic tools. Here we report a Drosophila tripartite synapse model which exhibits morphological and functional properties similar to those of mammalian synapses, including glial regulation of extracellular glutamate, synaptically-induced glial calcium transients and glial coupling of synapses with tracheal structures mediating gas exchange. In combination with classical and cell-type specific genetic approaches in Drosophila, this model is expected to provide new insights into the molecular and cellular mechanisms of tripartite synapse function.