The synaptic basis of activity-dependent eye-specific competition

Binocular vision requires proper developmental wiring of eye-specific inputs to the brain. Axons from the two eyes initially overlap in the dorsal lateral geniculate nucleus and undergo activity-dependent competition to segregate into target domains. The synaptic basis of such refinement is unknown. Here we used volumetric super-resolution imaging to measure the nanoscale molecular reorganization of developing retinogeniculate eye-specific synapses in the mouse brain. The outcome of binocular synaptic competition was determined by the relative eye-specific maturation of presynaptic vesicle content. Genetic disruption of spontaneous retinal activity prevented subsynaptic vesicle pool maturation, recruitment of vesicles to the active zone, synaptic development and eye-specific competition. These results reveal an activity-dependent presynaptic basis for axonal refinement in the mammalian visual system.

DYNAMICS OF NEUROMUSCULAR TRANSMISSION REPRODUCED BY CALCIUM-DEPENDENT SERIAL TRANSITIONS IN THE VESICLE FUSION COMPLEX

Neuromuscular transmission, from spontaneous release to facilitation and depression was accurately reproduced by a mechanistic kinetic model of sequential maturation transitions in the molecular fusion complex. The model incorporates three predictions. First, sequential calcium-dependent forward transitions take vesicles from docked to pre-primed to primed states, followed by fusion. Second, pre-priming and priming are reversible. Third, fusion and recycling are unidirectional. The model was fed with experimental data from previous studies while the backward (β) and recycling (ρ) rate constant values were fitted. Classical experiments were successfully reproduced when every forward (α) rate constant had the same value, and both backward rate constants were 50-100 times larger. Such disproportion originated an abruptly decreasing gradient of resting vesicles from docked to primed states. Simulations also predict that: i. Spontaneous release reflects primed to fusion spontaneous transitions. ii. Calcium elevations synchronize the series of forward transitions that lead to fusion. iii Facilitation reflects a transient increase of priming following calcium-dependent transitions. iv. Backward transitions and recycling restore the resting state. v. Depression reflects backward transitions and slow recycling after intense release. Such finely-tuned kinetics offers a mechanism for collective non-linear transitional adaptations of a homogeneous vesicle pool to an ever-changing pattern of electrical activity.

Distinct Synaptic Transfer Functions in Same-Type Photoreceptors

Many sensory systems use ribbon-type synapses to transmit their signals to downstream circuits. The properties of this synaptic transfer fundamentally dictate which aspects in the original stimulus will be accentuated or suppressed, thereby partially defining the detection limits of the circuit. Accordingly, sensory neurons have evolved a wide variety of ribbon geometries and vesicle pool properties to best support their diverse functional requirements. However, the need for diverse synaptic functions does not only arise across neuron types, but also within. Here we show that UV-cones, a single type of photoreceptor of the larval zebrafish eye, exhibit striking differences in their synaptic ultrastructure and consequent calcium to glutamate transfer function depending on their location in the eye. We arrive at this conclusion by combining serial section electron microscopy and simultaneous “dual-colour” 2-photon imaging of calcium and glutamate signals from the same synapse in vivo. We further use the functional dataset to fit a cascade-like model of the ribbon synapse with different vesicle pool sizes, transfer rates and other synaptic properties. Exploiting recent developments in simulation-based inference, we obtain full posterior estimates for the parameters and compare these across different retinal regions. The model enables us to extrapolate to new stimuli and to systematically investigate different response behaviours of various ribbon configurations. We also provide an interactive, easy-to-use version of this model as an online tool. Overall, we show that already on the synaptic level of single neuron types there exist highly specialized mechanisms which are advantageous for the encoding of different visual features.

Complexin-1 regulated transition in the assembly of single neuronal SNARE complex

Neurotransmitter release is mediated by the synaptic vesicle exocytosis. Important proteins in this process have been identified including the molecular machine Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, and other regulators. Complexin (Cpx) is one of the vital regulators in this process. The functions of Cpx are proposed to maintain a proper primed vesicle pool by preventing its premature depletion, which facilitates the vesicle fusion in the presence of Ca2+. However, the molecular mechanism remains unclear. Using dual-trap optical tweezers, we detected the interaction of complexin-1 (CpxI) with SNARE. We found that the CpxI stabilizes partially folded SNARE complexes by competing with C-terminal of Vamp protein and interacting with the C-terminal of t-SNARE complex.

Abstract P-43: Effect of Glucocerebrosidase Dysfunction on the Pool of Plasma Exosomes of Patients with Gaucher Disease

Background: Extracellular vesicles (EVs) are small membrane vesicles released from different types of cells. EVs are found in many human biological fluids. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies with the plasma membrane. This type of vesicles is characterized by specific exosomal markers. Exosomes extracted from peripheral body liquids could have specific properties associated with different physiological conditions as well as human disorders, including neurodegenerative diseases. Gaucher disease (GD) – is the most common form of lysosomal storage disorders caused by mutations in the glucocerebrosidase (GBA) gene. Lysosome functionality is critical for the regulation of extracellular vesicle secretion and content. In model animals, the inhibition of glucocerebrosidase has been shown to increase the secretion of extracellular vesicles in brain tissues. Amount evaluation of EVs and their size in the biological fluids of patients with GD has not been early performed; therefore, it is unknown whether lysosomal dysfunction found in GD patients influences the plasma pool of EVs. The aim of this study was to evaluate the amount of blood plasma EVs in patients with GD and their characterization for morphology and size. Methods: EVs were isolated from the blood plasma of 8 GD patients and 8 controls by ultracentrifugation, and were characterized using cryo-electron microscopy (cryo-EM), nanoparticle tracking analysis (NTA), and dynamic light scattering (DLS). Also, the presence of exosomal markers CD9, CD63, CD81, and HSP70 was analyzed by flow cytometry and western blot. Results: Here, it was first shown an increased proportion of exosome fraction in EVs from plasma of GD patients compared to controls by DLS and cryo-EM (p<0.001) that was confirmed by mode size detected by NTA (p<0.02). Moreover, an increased number of double and multilayer vesicles in plasma EVs from GD patients was demonstrated by cryo-EM. We also detected an increase in the expression of exosomal markers on the surface of vesicles from the blood plasma of patients with GD compared to controls. Conclusion: Here, we firstly report that the exosomes obtained from the blood plasma of GD patients have a larger size and altered morphology. Thus, we have shown that lysosomal dysfunction in GD patients leads to a striking alteration of blood plasma extracellular vesicle pool.

Kinetic structure of recycling vesicle pool at the Calyx of Held synapse under in vivo-like activities

Synaptic transmission at mammalian central synapses has ongoing background activity at physiological temperature. The recycling vesicle pool, with proper kinetics, ensures sustained synaptic transmission. However, the kinetic structure of recycling vesicle pool has never been quantitatively analyzed before, and most studies were performed at room temperature and under resting conditions. With the combination of presynaptic capacitance measurement and postsynaptic EPSC recording on calyx of Held synapses at physiological temperature, we studied vesicle recycling under sustained presynaptic stimulation. The kinetics of vesicle reuse was revealed by impeding transmitter refilling with folimycin. We kinetically dissected the recycling vesicle pool as sequentially connected sub-pools and depicted the complete kinetic structure. The sizes and transition rates among these sub-pools were dynamically regulated by neuronal activity, in order to ensure efficient synaptic transmission. Our work highlights the impact of the vesicle recycling machinery on stable and reliable synaptic transmission under variable levels of neuronal activity.Impact statementThe recycling pool of vesicles are kinetically dissected as four populated pools ensuring stable and reliable synaptic transmission

Eye-Region Specific Ribbon Tuning Supports Distinct Modes of Synaptic Transmission in Same-Type Cone-Photoreceptors

SummaryMany sensory systems use ribbon-type synapses to transmit their signals to downstream circuits. The properties of this synaptic transfer fundamentally dictate which aspects in the original stimulus will be accentuated or suppressed, thereby partially defining the detection limits of the circuit. Accordingly, sensory neurons have evolved a wide variety of ribbon geometries and vesicle pool properties to best support their diverse functional requirements. However, the need for diverse synaptic functions does not only arise across neuron types, but also within. Here we show that UV-cones, a single type of photoreceptor of the larval zebrafish eye, exhibit striking differences in their synaptic ultrastructure and consequent calcium to glutamate transfer function depending on their location in the eye. We arrive at this conclusion by combining serial section electron microscopy and simultaneous “dual-colour” 2-photon imaging of calcium and glutamate signals from the same synapse in vivo. We further use the functional dataset to fit a cascade-like model of the ribbon synapse with different vesicle pool sizes, transfer rates and other synaptic properties. Exploiting recent developments in simulation-based inference, we obtain full posterior estimates for the parameters and compare these across different retinal regions. The model enables us to extrapolate to new stimuli and to systematically investigate different response behaviours of various ribbon configurations. We also provide an interactive, easy-to-use version of this model as an online tool. Overall, we show that already on the synaptic level of single neuron types there exist highly specialized mechanisms which are advantageous for the encoding of different visual features.

Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genesCLTAandCLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.

Toxoplasma ferlin1 is a versatile and dynamic mediator of microneme trafficking and secretion

Calcium-dependent exocytosis of the microneme organelles that facilitate host cell invasion is critical for obligate intracellular apicomplexan parasites such as Toxoplasma gondii. Ferlins represent a protein family with roles in exocytosis containing multiple Ca2+-sensing C2 domains. Here we defined the role of T. gondii’s ferlin 1 (FER1) in microneme biology. FER1 localized dynamically to several compartments of the parasite’s secretory pathway as well as to an apical spot near the site of microneme secretion. FER1 function was dissected by overexpression of a variety of N-terminally tagged alleles causing dominant negative phenotypes. This demonstrated FER1 traffics microneme organelles at several discrete steps of their natural trajectories: 1. from ELC to the subpellicular microtubules; 2. along the subpellicular microtubules to the apical end; 3. into the conoid; 4. and inferred from observed retrograde transport from the subpellicular microtubules, recycling of micronemes from mother to daughter parasites. Furthermore, full-length FER1 overexpression results in a squirt of microneme release sufficient for host cell egress. This indicates FER1 facilitates fusion of the most apical, radially organized micronemes with the plasma membrane. Moreover, FER1 acts differentially on the Rab5A/C-dependent and - independent microneme sub-populations. Finally, apical FER1 overlaps with the presence of VP1, a pyrophosphatase proton pump. Integrating all new insights, we propose a model of microneme exocytosis wherein the radial micronemes constitute a readily releasable vesicle pool primed by acidification.