Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune-mediated neuromuscular disease thought to be caused by autoantibodies against P/Q-type voltage-gated calcium channels (VGCCs), which attack and reduce the number of VGCCs within transmitter release sites (active zones; AZs) at the neuromuscular junction (NMJ), resulting in neuromuscular weakness. However, patients with LEMS also have antibodies to other neuronal proteins, and about 15% of patients with LEMS are seronegative for antibodies against VGCCs. We hypothesized that a reduction in the number of P/Q-type VGCCs alone is not sufficient to explain LEMS effects on transmitter release. Here, we used a computational model to study a variety of LEMS-mediated effects on AZ organization and transmitter release constrained by electron microscopic, pharmacological, immunohistochemical, voltage imaging, and electrophysiological observations. We show that models of healthy AZs can be modified to predict the transmitter release and short-term facilitation characteristics of LEMS and that in addition to a decrease in the number of AZ VGCCs, disruption in the organization of AZ proteins, a reduction in AZ number, a reduction in the amount of synaptotagmin, and the compensatory expression of L-type channels outside the remaining AZs are important contributors to LEMS-mediated effects on transmitter release. Furthermore, our models predict that antibody-mediated removal of synaptotagmin in combination with disruption in AZ organization alone could mimic LEMS effects without the removal of VGCCs (a seronegative model). Overall, our results suggest that LEMS pathophysiology may be caused by a collection of pathological alterations to AZs at the NMJ, rather than by a simple loss of VGCCs. NEW & NOTEWORTHY We used a computational model of the active zone (AZ) in the mammalian neuromuscular junction to investigate Lambert-Eaton myasthenic syndrome (LEMS) pathophysiology. This model suggests that disruptions in presynaptic active zone organization and protein content (particularly synaptotagmin), beyond the simple removal of presynaptic calcium channels, play an important role in LEMS pathophysiology.
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Microphysiological Modeling of the Structure and Function of Neuromuscular Transmitter Release Sites
The general mechanism of calcium-triggered chemical transmitter release from neuronal synapses has been intensely studied, is well-known, and highly conserved between species and synapses across the nervous system. However, the structural and functional details within each transmitter release site (or active zone) are difficult to study in living tissue using current experimental approaches owing to the small spatial compartment within the synapse where exocytosis occurs with a very rapid time course. Therefore, computer simulations offer the opportunity to explore these microphysiological environments of the synapse at nanometer spatial scales and on a sub-microsecond timescale. Because biological reactions and physiological processes at synapses occur under conditions where stochastic behavior is dominant, simulation approaches must be driven by such stochastic processes. MCell provides a powerful simulation approach that employs particle-based stochastic simulation tools to study presynaptic processes in realistic and complex (3D) geometries using optimized Monte Carlo algorithms to track finite numbers of molecules as they diffuse and interact in a complex cellular space with other molecules in solution and on surfaces (representing membranes, channels and binding sites). In this review we discuss MCell-based spatially realistic models of the mammalian and frog neuromuscular active zones that were developed to study presynaptic mechanisms that control transmitter release. In particular, these models focus on the role of presynaptic voltage-gated calcium channels, calcium sensors that control the probability of synaptic vesicle fusion, and the effects of action potential waveform shape on presynaptic calcium entry. With the development of these models, they can now be used in the future to predict disease-induced changes to the active zone, and the effects of candidate therapeutic approaches.
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- Award ID(s):
- 2011616
- NSF-PAR ID:
- 10385680
- Date Published:
- Journal Name:
- Frontiers in Synaptic Neuroscience
- Volume:
- 14
- ISSN:
- 1663-3563
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Long-term potentiation (LTP) is a cellular mechanism of learning and memory that results in a sustained increase in the probability of vesicular release of neurotransmitter. However, previous work in hippocampal area CA1 of the adult rat revealed that the total number of vesicles per synapse decreases following LTP, seemingly inconsistent with the elevated release probability. Here, electron-microscopic tomography (EMT) was used to assess whether changes in vesicle density or structure of vesicle tethering filaments at the active zone might explain the enhanced release probability following LTP. The spatial relationship of vesicles to the active zone varies with functional status. Tightly docked vesicles contact the presynaptic membrane, have partially formed SNARE complexes, and are primed for release of neurotransmitter upon the next action potential. Loosely docked vesicles are located within 8 nm of the presynaptic membrane where SNARE complexes begin to form. Nondocked vesicles comprise recycling and reserve pools. Vesicles are tethered to the active zone via filaments composed of molecules engaged in docking and release processes. The density of tightly docked vesicles was increased 2 h following LTP compared to control stimulation, whereas the densities of loosely docked or nondocked vesicles congregating within 45 nm above the active zones were unchanged. The tethering filaments on all vesicles were shorter and their attachment sites shifted closer to the active zone. These findings suggest that tethering filaments stabilize more vesicles in the primed state. Such changes would facilitate the long-lasting increase in release probability following LTP.
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Abstract Despite its evident importance to learning theory and models, the manner in which the lateral perforant path (LPP) transforms signals from entorhinal cortex to hippocampus is not well understood. The present studies measured synaptic responses in the dentate gyrus (DG) of adult mouse hippocampal slices during different patterns of LPP stimulation. Theta (5 Hz) stimulation produced a modest within‐train facilitation that was markedly enhanced at the level of DG output. Gamma (50 Hz) activation resulted in a singular pattern with initial synaptic facilitation being followed by a progressively greater depression. DG output was absent after only two pulses. Reducing release probability with low extracellular calcium instated frequency facilitation to gamma stimulation while long‐term potentiation, which increases release by LPP terminals, enhanced within‐train depression. Relatedly, per terminal concentrations of VGLUT2, a vesicular glutamate transporter associated with high release probability, were much greater in the LPP than in CA3–CA1 connections. Attempts to circumvent the potent gamma filter using a series of short (three‐pulse) 50 Hz trains spaced by 200 ms were only partially successful: composite responses were substantially reduced after the first burst, an effect opposite to that recorded in field CA1. The interaction between bursts was surprisingly persistent (>1.0 s). Low calcium improved throughput during theta/gamma activation but buffering of postsynaptic calcium did not. In all, presynaptic specializations relating to release probability produce an unusual but potent type of frequency filtering in the LPP. Patterned burst input engages a different type of filter with substrates that are also likely to be located presynaptically.
image Key points The lateral perforant path (LPP)–dentate gyrus (DG) synapse operates as a low‐pass filter, where responses to a train of 50 Hz, γ frequency activation are greatly suppressed.
Activation with brief bursts of γ frequency information engages a secondary filter that persists for prolonged periods (lasting seconds).
Both forms of LPP frequency filtering are influenced by presynaptic, as opposed to postsynaptic, processes; this contrasts with other hippocampal synapses.
LPP frequency filtering is modified by the unique presynaptic long‐term potentiation at this synapse.
Computational simulations indicate that presynaptic factors associated with release probability and vesicle recycling may underlie the potent LPP–DG frequency filtering.
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Jędrzejewska-Szmek, Joanna (Ed.)Chemical synapses exhibit a diverse array of internal mechanisms that affect the dynamics of transmission efficacy. Many of these processes, such as release of neurotransmitter and vesicle recycling, depend strongly on activity-dependent influx and accumulation of Ca 2+ . To model how each of these processes may affect the processing of information in neural circuits, and how their dysfunction may lead to disease states, requires a computationally efficient modelling framework, capable of generating accurate phenomenology without incurring a heavy computational cost per synapse. Constructing a phenomenologically realistic model requires the precise characterization of the timing and probability of neurotransmitter release. Difficulties arise in that functional forms of instantaneous release rate can be difficult to extract from noisy data without running many thousands of trials, and in biophysical synapses, facilitation of per-vesicle release probability is confounded by depletion. To overcome this, we obtained traces of free Ca 2+ concentration in response to various action potential stimulus trains from a molecular MCell model of a hippocampal Schaffer collateral axon. Ca 2+ sensors were placed at varying distance from a voltage-dependent calcium channel (VDCC) cluster, and Ca 2+ was buffered by calbindin. Then, using the calcium traces to drive deterministic state vector models of synaptotagmin 1 and 7 (Syt-1/7), which respectively mediate synchronous and asynchronous release in excitatory hippocampal synapses, we obtained high-resolution profiles of instantaneous release rate, to which we applied functional fits. Synchronous vesicle release occurred predominantly within half a micron of the source of spike-evoked Ca 2+ influx, while asynchronous release occurred more consistently at all distances. Both fast and slow mechanisms exhibited multi-exponential release rate curves, whose magnitudes decayed exponentially with distance from the Ca 2+ source. Profile parameters facilitate on different time scales according to a single, general facilitation function. These functional descriptions lay the groundwork for efficient mesoscale modelling of vesicular release dynamics.more » « less
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Neurotransmission is shaped by extracellular pH. Alkalization enhances pH-sensitive transmitter release and receptor activation, whereas acidification inhibits these processes and can activate acid-sensitive conductances in the synaptic cleft. Previous work has shown that the synaptic cleft can either acidify because of synaptic vesicular release and/or alkalize because of Ca2+extrusion by the plasma membrane ATPase (PMCA). The direction of change differs across synapse types. At the mammalian neuromuscular junction (NMJ), the direction and magnitude of pH transients in the synaptic cleft during transmission remain ambiguous. We set out to elucidate the extracellular pH transients that occur at this cholinergic synapse under near-physiological conditions and identify their sources. We monitored pH-dependent changes in the synaptic cleft of the mouse levator auris longus using viral expression of the pseudoratiometric probe pHusion-Ex in the muscle. Using mice from both sexes, a significant and prolonged alkalization occurred when stimulating the connected nerve for 5 s at 50 Hz, which was dependent on postsynaptic intracellular Ca2+release. Sustained stimulation for a longer duration (20 s at 50 Hz) caused additional prolonged net acidification at the cleft. To investigate the mechanism underlying cleft alkalization, we used muscle-expressed GCaMP3 to monitor the contribution of postsynaptic Ca2+. Activity-induced liberation of intracellular Ca2+in muscle positively correlated with alkalization of the synaptic cleft, whereas inhibiting PMCA significantly decreased the extent of cleft alkalization. Thus, cholinergic synapses of the mouse NMJ typically alkalize because of cytosolic Ca2+liberated in muscle during activity, unless under highly strenuous conditions where acidification predominates.
SIGNIFICANCE STATEMENT Changes in synaptic cleft pH alter neurotransmission, acting on receptors and channels on both sides of the synapse. Synaptic acidification has been associated with a myriad of diseases in the central and peripheral nervous system. Here, we report that in near-physiological recording conditions the cholinergic neuromuscular junction shows use-dependent bidirectional changes in synaptic cleft pH—immediate alkalinization and a long-lasting acidification under prolonged stimulation. These results provide further insight into physiologically relevant changes at cholinergic synapses that have not been defined previously. Understanding and identifying synaptic pH transients during and after neuronal activity provides insight into short-term synaptic plasticity synapses and may identify therapeutic targets for diseases.
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fnsyn.2022.917285
