Efficient stimulus-secretion coupling at ribbon synapses requires RIM-binding protein tethering of L-type Ca2+channels

Fast neurotransmitter release from ribbon synapses via Ca2+-triggered exocytosis requires tight coupling of L-type Ca2+channels to release-ready synaptic vesicles at the presynaptic active zone, which is localized at the base of the ribbon. Here, we used genetic, electrophysiological, and ultrastructural analyses to probe the architecture of ribbon synapses by perturbing the function of RIM-binding proteins (RBPs) as central active-zone scaffolding molecules. We found that genetic deletion of RBP1 and RBP2 did not impair synapse ultrastructure of ribbon-type synapses formed between rod bipolar cells (RBCs) and amacrine type-2 (AII) cells in the mouse retina but dramatically reduced the density of presynaptic Ca2+channels, decreased and desynchronized evoked neurotransmitter release, and rendered evoked and spontaneous neurotransmitter release sensitive to the slow Ca2+buffer EGTA. These findings suggest that RBPs tether L-type Ca2+channels to the active zones of ribbon synapses, thereby synchronizing vesicle exocytosis and promoting high-fidelity information transfer in retinal circuits.

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UNC-2 CaV2 channel localization at presynaptic active zones depends on UNC-10/RIM and SYD-2/Liprin-α in Caenorhabditis elegans

10.1101/2021.01.27.428454 ◽

2021 ◽

Author(s):

Kelly H. Oh ◽

Mia Krout ◽

Janet E. Richmond ◽

Hongkyun Kim

Keyword(s):

Active Zone ◽

Calcium Influx ◽

Genetic Screen ◽

Scaffolding Protein ◽

Tight Coupling ◽

Forward Genetic Screen ◽

Precise Control ◽

C Elegans ◽

Vesicle Exocytosis ◽

Active Zones

AbstractPresynaptic active zone proteins couple calcium influx with synaptic vesicle exocytosis. However, the control of presynaptic calcium channel clustering by active zone proteins is not completely understood. In a C. elegans forward genetic screen, we find that UNC-10/RIM (Rab3-interacting molecule) and SYD-2/Liprin-α regulate presynaptic clustering of UNC-2, the CaV2 channel ortholog. We further quantitatively analyzed live animals using endogenously GFP-tagged UNC-2 and active zone components. Consistent with the interaction between RIM and CaV2 in mammals, the intensity and number of UNC-2 channel clusters at presynaptic terminals were greatly reduced in unc-10 mutant animals. To understand how SYD-2 regulates presynaptic UNC-2 channel clustering, we analyzed presynaptic localization of endogenous SYD-2, UNC-10, RIMB-1/RIM-BP (RIM binding protein), and ELKS-1. Our analysis revealed that while SYD-2 is the most critical for active zone assembly, loss of SYD-2 function does not completely abolish presynaptic localization of UNC-10, RIMB-1, and ELKS-1, suggesting an existence of SYD-2-independent active zone assembly. UNC-2 localization analysis in double and triple mutants of active zone components show that SYD-2 promotes UNC-2 clustering by partially controlling UNC-10 localization, and ELKS-1 and RIMB-1 also contribute to UNC-2 channel clustering. In addition, we find that core active zone proteins are unequal in their abundance. While the abundance of UNC-10 at the active zone is comparable to UNC-2, SYD-2 and ELKS-1 are twice more and RIMB-1 four times more abundant than UNC-2. Together our data show that UNC-10, SYD-2, RIMB-1, and ELKS-1 control presynaptic UNC-2 channel clustering in redundant yet distinct manners.Significance StatementPrecise control of neurotransmission is dependent on the tight coupling of the calcium influx through voltage-gated calcium channels (VGCCs) to the exocytosis machinery at the presynaptic active zones. However, how these VGCCs are tethered to the active zone is incompletely understood. To understand the mechanism of presynaptic VGCC localization, we performed a C. elegans forward genetic screen and quantitatively analyzed endogenous active zones and presynaptic VGCCs. In addition to RIM (Rab3-interacting molecule), our study finds that SYD-2/Liprin-α is critical for presynaptic localization of VGCCs. Yet, the loss of SYD-2, the master active zone scaffolding protein, does not completely abolish the presynaptic localization of the VGCC, showing that the active zone is a resilient structure assembled by redundant mechanisms.

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Active zone protein CAST is a component of conventional and ribbon synapses in mouse retina

The Journal of Comparative Neurology ◽

10.1002/cne.20893 ◽

2006 ◽

Vol 495 (4) ◽

pp. 480-496 ◽

Cited By ~ 31

Author(s):

Maki Deguchi-Tawarada ◽

Eiji Inoue ◽

Etsuko Takao-Rikitsu ◽

Marie Inoue ◽

Isao Kitajima ◽

...

Keyword(s):

Active Zone ◽

Mouse Retina ◽

Ribbon Synapses

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Single calcium channel nanodomains drive presynaptic calcium entry at lamprey reticulospinal presynaptic terminals

10.1101/2021.02.02.429388 ◽

2021 ◽

Author(s):

S Ramachandran ◽

S Rodgriguez ◽

M Potcoava ◽

S Alford

Keyword(s):

Active Zone ◽

Neurotransmitter Release ◽

Action Potentials ◽

Information Transfer ◽

Single Channel ◽

Light Sheet ◽

Stochastic Variation ◽

Presynaptic Terminals ◽

Light Sheet Microscopy ◽

Presynaptic Calcium

AbstractSynchronous neurotransmission is central to efficient information transfer in neural circuits, requiring precise coupling between action potentials, Ca2+ entry and neurotransmitter release. However, determinations of Ca2+ requirements for release, which may originate from entry through single voltage-gated Ca2+ channels, remain largely unexplored in simple active zone synapses common in the nervous system. Understanding these requirements is key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurological and neuropsychiatric disorders. Here, we present single channel analysis of evoked active zone Ca2+ entry, using cell-attached patch clamp and lattice light sheet microscopy over active zones at single central lamprey reticulospinal presynaptic terminals. Our findings show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising subtypes N-type (CaV2.2), P/Q-type (CaV2.1) and R-type (CaV2.3), available to gate neurotransmitter release. Significantly, of this pool only 1-6 (mean of 4) channels open upon depolarization. High temporal fidelity lattice light sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations between action potentials and stochastic variation of precise locations of Ca2+ entry within the active zone. Further, Ca2+ channel numbers at each active zone correlate to the number of presynaptic primed synaptic vesicles. Together, our findings indicate 1:1 association of Ca2+ channels with primed vesicles, suggesting Ca2+ entry via as few as one channel may trigger neurotransmitter release.

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Evidence for functional GABAA but not GABAC receptors in mouse cone photoreceptors

Visual Neuroscience ◽

10.1017/s0952523819000038 ◽

2019 ◽

Vol 36 ◽

Cited By ~ 2

Author(s):

Sercan Deniz ◽

Eric Wersinger ◽

Serge Picaud ◽

Michel J. Roux

Keyword(s):

Receptor Antagonist ◽

Gaba Receptors ◽

Information Transfer ◽

Direct Action ◽

Synaptic Cleft ◽

Cell Types ◽

Bipolar Cells ◽

Mouse Retina ◽

Direct Role ◽

Morphological Criteria

AbstractAt the first retinal synapse, horizontal cells (HCs) contact both photoreceptor terminals and bipolar cell dendrites, modulating information transfer between these two cell types to enhance spatial contrast and mediate color opponency. The synaptic mechanisms through which these modulations occur are still debated. The initial hypothesis of a GABAergic feedback from HCs to cones has been challenged by pharmacological inconsistencies. Surround antagonism has been demonstrated to occur via a modulation of cone calcium channels through ephaptic signaling and pH changes in the synaptic cleft. GABAergic transmission between HCs and cones has been reported in some lower vertebrates, like the turtle and tiger salamander. In these reports, it was revealed that GABA is released from HCs through reverse transport and target GABA receptors are located at the cone terminals. In mammalian retinas, there is growing evidence that HCs can release GABA through conventional vesicular transmission, acting both on autaptic GABA receptors and on receptors expressed at the dendritic tips of the bipolar cells. The presence of GABA receptors on mammalian cone terminals remains equivocal. Here, we looked specifically for functional GABA receptors in mouse photoreceptors by recording in the whole-cell or amphotericin/gramicidin-perforated patch clamp configurations. Cones could be differentiated from rods through morphological criteria. Local GABA applications evoked a Cl− current in cones but not in rods. It was blocked by the GABAA receptor antagonist bicuculline methiodide and unaffected by the GABAC receptor antagonist TPMPA [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid]. The voltage dependency of the current amplitude was as expected from a direct action of GABA on cone pedicles but not from an indirect modulation of cone currents following the activation of the GABA receptors of HCs. This supports a direct role of GABA released from HCs in the control of cone activity in the mouse retina.

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Synaptic connectivity of two types of recoverin-labeled cone bipolar cells and glutamic acid decarboxylase immunoreactive amacrine cells in the inner plexiform layer of the rat retina

Visual Neuroscience ◽

10.1017/s0952523899164174 ◽

1999 ◽

Vol 16 (4) ◽

pp. 791-800 ◽

Cited By ~ 9

Author(s):

MYUNG-HOON CHUN ◽

IN-BEOM KIM ◽

SU-JA OH ◽

JIN-WOONG CHUNG

Keyword(s):

Amacrine Cell ◽

Plexiform Layer ◽

Bipolar Cells ◽

Acid Decarboxylase ◽

Inner Plexiform Layer ◽

Rat Retina ◽

Ribbon Synapses ◽

Cell Processes ◽

Cone Bipolar Cells

We investigated the synaptic connectivity of two populations of recoverin-labeled bipolar cells and GABAergic neurons in the inner plexiform layer (IPL) of the rat retina. Two types of cone bipolar cells, type 2 and type 8, were stained with anti-recoverin antibodies, and GABAergic neurons were stained with anti-glutamic acid decarboxylase (GAD) antibodies. Type 2 cone bipolar axons received synaptic input from amacrine cell processes in 177 cases; among these amacrine cell processes, 92 processes (52.0%) were GAD-like immunoreactive. A total of 159 amacrine cell processes, which are presynaptic to type 8 cone bipolar cells, were observed. Among these processes, 117 processes (73.6%) were GAD-like immunoreactive. The postsynaptic elements at the ribbon synapses of recoverin-labeled cone bipolar cells were observed in 482 processes. In both type 2 and type 8 cone bipolar cells, the major output was to amacrine cell processes. At the ribbon synapses of the type 2 cone bipolar cells, 224 of the postsynaptic profiles were amacrine cell processes, 97 processes (43.3%) were GAD-like immunoreactive. In type 8 cone bipolar cells, 45 processes (30.2%) of 149 amacrine cell processes were GAD-like immunoreactive. Our results provide morphological evidence that GABA is a major transmitter involved in the visual processing of type 2 and 8 cone bipolar cells and GABA may have a stronger influence on type 8 cone bipolar cells than type 2 cone bipolar cells in the IPL of the rat retina.

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Multivesicular Release Increases the Efficiency of Information Transmission at Sensory Synapses

10.1101/2021.11.04.467256 ◽

2021 ◽

Author(s):

Ben James ◽

Pawel Piekarz ◽

Jose Moya-Diaz ◽

Leon Lagnado

Keyword(s):

Time Constant ◽

Information Transfer ◽

Ganglion Cells ◽

Classical Model ◽

Sensory Information ◽

Bipolar Cells ◽

Spike Generation ◽

Vesicle Release ◽

Ribbon Synapses ◽

The Impact

The statistics of vesicle release determine how information is transferred in neural circuits. The classical model is of Poisson synapses releasing vesicles independently but ribbon synapses transmit early sensory signals by multivesicular release (MVR) when two or more vesicles are coordinated as a single synaptic event. To investigate the impact of MVR on the spike code we used leaky integrate-and-fire models with inputs simulating the statistics of vesicle release measured experimentally from retinal bipolar cells. Comparing these with models of independent release we find that MVR increases spike generation and the efficiency of information transfer (bits per spike) over a range of conditions that mimic retinal ganglion cells of different time-constant receiving different number of synaptic inputs of different strengths. When a single input drives a neuron with short time-constant, as occurs when hair cells transmit auditory signals, MVR increases information transfer whenever spike generation requires depolarization greater than that caused by a single vesicle. This study demonstrates how presynaptic integration of vesicles by MVR can compensate for less effective summation post-synaptically to increase the efficiency with which sensory information is transmitted at the synapse.

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Protein mutated in paroxysmal dyskinesia interacts with the active zone protein RIM and suppresses synaptic vesicle exocytosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501364112 ◽

2015 ◽

Vol 112 (10) ◽

pp. 2935-2941 ◽

Cited By ~ 25

Author(s):

Yiguo Shen ◽

Woo-Ping Ge ◽

Yulong Li ◽

Arisa Hirano ◽

Hsien-Yang Lee ◽

...

Keyword(s):

Active Zone ◽

Neurotransmitter Release ◽

Autosomal Dominant ◽

Wild Type ◽

Causative Gene ◽

Protein Levels ◽

Synaptic Vesicle Exocytosis ◽

Vesicle Exocytosis ◽

Dopamine Signaling

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.

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PKC-phosphorylation of Liprin-α3 triggers phase separation and controls presynaptic active zone structure

Nature Communications ◽

10.1038/s41467-021-23116-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Javier Emperador-Melero ◽

Man Yan Wong ◽

Shan Shan H. Wang ◽

Giovanni de Nola ◽

Hajnalka Nyitrai ◽

...

Keyword(s):

Phase Separation ◽

Active Zone ◽

Neurotransmitter Release ◽

Hippocampal Neurons ◽

Phosphorylation Site ◽

Double Knockout ◽

Zone Structure ◽

Presynaptic Nerve Terminal ◽

Condensate Formation ◽

And Function

AbstractThe active zone of a presynaptic nerve terminal defines sites for neurotransmitter release. Its protein machinery may be organized through liquid–liquid phase separation, a mechanism for the formation of membrane-less subcellular compartments. Here, we show that the active zone protein Liprin-α3 rapidly and reversibly undergoes phase separation in transfected HEK293T cells. Condensate formation is triggered by Liprin-α3 PKC-phosphorylation at serine-760, and RIM and Munc13 are co-recruited into membrane-attached condensates. Phospho-specific antibodies establish phosphorylation of Liprin-α3 serine-760 in transfected cells and mouse brain tissue. In primary hippocampal neurons of newly generated Liprin-α2/α3 double knockout mice, synaptic levels of RIM and Munc13 are reduced and the pool of releasable vesicles is decreased. Re-expression of Liprin-α3 restored these presynaptic defects, while mutating the Liprin-α3 phosphorylation site to abolish phase condensation prevented this rescue. Finally, PKC activation in these neurons acutely increased RIM, Munc13 and neurotransmitter release, which depended on the presence of phosphorylatable Liprin-α3. Our findings indicate that PKC-mediated phosphorylation of Liprin-α3 triggers its phase separation and modulates active zone structure and function.

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Modulation by Zn2+ of GABA responses in bipolar cells of the mouse retina

Visual Neuroscience ◽

10.1017/s0952523800172098 ◽

2000 ◽

Vol 17 (2) ◽

pp. 273-281 ◽

Cited By ~ 20

Author(s):

M. KANEDA ◽

B. ANDRÁSFALVY ◽

A. KANEKO

Keyword(s):

Axon Terminal ◽

Inhibitory Action ◽

Phosphinic Acid ◽

Amacrine Cells ◽

Bipolar Cells ◽

Mouse Retina ◽

Induced Responses ◽

Inhibitory Interaction ◽

Cell Recording ◽

Recording Conditions

The localization of endogenous Zn2+ in the mouse retina was examined histochemically and the inhibitory action of Zn2+ on GABA-induced responses was studied in bipolar cells isolated from the mouse retina. Accumulation of endogenous Zn2+ was detected in photoreceptors, bipolar, and/or amacrine cells by either the bromopyridylazo-diethylaminophenol method or the dithizone method. Under whole-cell recording conditions, GABA induced a Cl− current in isolated bipolar cells. The current consisted of two components. The first component was inhibited completely by application of 100 μM bicuculline, suggesting that this is a GABAA-receptor mediated current. The second component was inhibited completely by 100 μM 3-aminopropyl-(methyl)-phosphinic acid, suggesting that this is a GABAC-receptor mediated current. GABAC receptors were present at a higher density on the axon terminal than on dendrites. Zn2+ inhibited both GABAA and GABAC receptors. GABAC receptors were more susceptible to Zn2+; the IC50 for the GABAA receptor was 67.4 μM and that for the GABAC receptor was 1.9 μM. These results suggest that Zn2+ modulates the inhibitory interaction between amacrine and bipolar cells, particularly that mediated by the GABAC receptor.

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