Neuropeptidergic regulation of neuromuscular signaling in larval zebrafish alters swimming behavior and synaptic transmission.

Chemical synaptic transmission is modulated to accommodate different activity levels, thus enabling homeostatic scaling in pre- and postsynaptic compartments. In nematodes, cholinergic neurons use neuropeptide signaling to modulate synaptic vesicle content. To explore if this mechanism is conserved in vertebrates, we studied the involvement of neuropeptides in cholinergic transmission at the neuromuscular junction of larval zebrafish.

RIM and RIM-Binding Protein Localize Synaptic CaV2 Channels to Differentially Regulate Transmission in Neuronal Circuits.

At chemical synapses, voltage-gated Ca channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit.

All-optical closed-loop voltage clamp for precise control of muscles and neurons in live animals.

Excitable cells can be stimulated or inhibited by optogenetics. Since optogenetic actuation regimes are often static, neurons and circuits can quickly adapt, allowing perturbation, but not true control. Hence, we established an optogenetic voltage-clamp (OVC).

Photoactivated Adenylyl Cyclases as Optogenetic Modulators of Neuronal Activity.

In the past 15 years, optogenetic methods became invaluable tools in neurobiological research but also in general cell biology. Most prominently, optogenetic methods utilize microbial rhodopsins to elicit neuronal de- or hyperpolarization. However, other optogenetic tools have emerged that allow influencing neuronal function by different approaches.

Synapsin Is Required for Dense Core Vesicle Capture and cAMP-Dependent Neuropeptide Release.

Release of neuropeptides from dense core vesicles (DCVs) is essential for neuromodulation. Compared with the release of small neurotransmitters, much less is known about the mechanisms and proteins contributing to neuropeptide release. By optogenetics, behavioral analysis, electrophysiology, electron microscopy, and live imaging, we show that synapsin SNN-1 is required for cAMP-dependent neuropeptide release in hermaphrodite cholinergic motor neurons.

Rhodopsin-based voltage imaging tools for use in muscles and neurons of .

Genetically encoded voltage indicators (GEVIs) based on microbial rhodopsins utilize the voltage-sensitive fluorescence of all- retinal (ATR), while in electrochromic FRET (eFRET) sensors, donor fluorescence drops when the rhodopsin acts as depolarization-sensitive acceptor. In recent years, such tools have become widely used in mammalian cells but are less commonly used in invertebrate systems, mostly due to low fluorescence yields. We systematically assessed Arch(D95N), Archon, QuasAr, and the eFRET sensors MacQ-mCitrine and QuasAr-mOrange, in the nematode ATR-bearing rhodopsins reported on voltage changes in body wall muscles (BWMs), in the pharynx, the feeding organ [where Arch(D95N) showed approximately 128% ΔF/F increase per 100 mV], and in neurons, integrating circuit activity.

Endophilin A and B Join Forces With Clathrin to Mediate Synaptic Vesicle Recycling in .

Synaptic vesicle (SV) recycling enables ongoing transmitter release, even during prolonged activity. SV membrane and proteins are retrieved by ultrafast endocytosis and new SVs are formed from synaptic endosomes (large vesicles-LVs). Many proteins contribute to SV recycling, e.

Rhodopsin optogenetic toolbox v2.0 for light-sensitive excitation and inhibition in Caenorhabditis elegans.

In optogenetics, rhodopsins were established as light-driven tools to manipulate neuronal activity. However, during long-term photostimulation using channelrhodopsin (ChR), desensitization can reduce effects. Furthermore, requirement for continuous presence of the chromophore all-trans retinal (ATR) in model systems lacking sufficient endogenous concentrations limits its applicability.

Fast cAMP Modulation of Neurotransmission via Neuropeptide Signals and Vesicle Loading.

Cyclic AMP (cAMP) signaling augments synaptic transmission, but because many targets of cAMP and protein kinase A (PKA) may be involved, mechanisms underlying this pathway remain unclear. To probe this mechanism, we used optogenetic stimulation of cAMP signaling by Beggiatoa-photoactivated adenylyl cyclase (bPAC) in Caenorhabditis elegans motor neurons. Behavioral, electron microscopy (EM), and electrophysiology analyses revealed cAMP effects on both the rate and on quantal size of transmitter release and led to the identification of a neuropeptidergic pathway affecting quantal size.

RIC-3 phosphorylation enables dual regulation of excitation and inhibition of Caenorhabditis elegans muscle.

Brain function depends on a delicate balance between excitation and inhibition. Similarly, Caenorhabditis elegans motor system function depends on a precise balance between excitation and inhibition, as C. elegans muscles receive both inhibitory, GABAergic and excitatory, cholinergic inputs from motor neurons.

A photosensitive degron enables acute light-induced protein degradation in the nervous system.

Acutely inducing degradation enables studying the function of essential proteins. Available techniques target proteins post-translationally, via ubiquitin or by fusing destabilizing domains (degrons), and in some cases degradation is controllable by small molecules. Yet, they are comparably slow, possibly inducing compensatory changes, and do not allow localized protein depletion.

A consistent muscle activation strategy underlies crawling and swimming in Caenorhabditis elegans.

Although undulatory swimming is observed in many organisms, the neuromuscular basis for undulatory movement patterns is not well understood. To better understand the basis for the generation of these movement patterns, we studied muscle activity in the nematode Caenorhabditis elegans. Caenorhabditis elegans exhibits a range of locomotion patterns: in low viscosity fluids the undulation has a wavelength longer than the body and propagates rapidly, while in high viscosity fluids or on agar media the undulatory waves are shorter and slower.

In vivo synaptic recovery following optogenetic hyperstimulation.

Local recycling of synaptic vesicles (SVs) allows neurons to sustain transmitter release. Extreme activity (e.g.

RAB-5 and RAB-10 cooperate to regulate neuropeptide release in Caenorhabditis elegans.

Neurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C.

Specific expression of channelrhodopsin-2 in single neurons of Caenorhabditis elegans.

Optogenetic approaches using light-activated proteins like Channelrhodopsin-2 (ChR2) enable investigating the function of populations of neurons in live Caenorhabditis elegans (and other) animals, as ChR2 expression can be targeted to these cells using specific promoters. Sub-populations of these neurons, or even single cells, can be further addressed by restricting the illumination to the cell of interest. However, this is technically demanding, particularly in free moving animals.

Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans.

Essentially any behavior in simple and complex animals depends on neuronal network function. Currently, the best-defined system to study neuronal circuits is the nematode Caenorhabditis elegans, as the connectivity of its 302 neurons is exactly known. Individual neurons can be activated by photostimulation of Channelrhodopsin-2 (ChR2) using blue light, allowing to directly probe the importance of a particular neuron for the respective behavioral output of the network under study.

Optogenetic analysis of GABAB receptor signaling in Caenorhabditis elegans motor neurons.

In the nervous system, a perfect balance of excitation and inhibition is required, for example, to enable coordinated locomotion. In Caenorhabditis elegans, cholinergic and GABAergic motor neurons (MNs) effect waves of contralateral muscle contraction and relaxation. Cholinergic MNs innervate muscle as well as GABAergic MNs, projecting to the opposite side of the body, at dyadic synapses.

Optogenetic long-term manipulation of behavior and animal development.

Channelrhodopsin-2 (ChR2) is widely used for rapid photodepolarization of neurons, yet, as it requires high-intensity blue light for activation, it is not suited for long-term in vivo applications, e.g. for manipulations of behavior, or photoactivation of neurons during development.

PACα--an optogenetic tool for in vivo manipulation of cellular cAMP levels, neurotransmitter release, and behavior in Caenorhabditis elegans.

Photoactivated adenylyl cyclase α (PACα) was originally isolated from the flagellate Euglena gracilis. Following stimulation by blue light it causes a rapid increase in cAMP levels. In the present study, we expressed PACα in cholinergic neurons of Caenorhabditis elegans.

An ER-resident membrane protein complex regulates nicotinic acetylcholine receptor subunit composition at the synapse.

Nicotinic acetylcholine receptors (nAChRs) are homo- or heteropentameric ligand-gated ion channels mediating excitatory neurotransmission and muscle activation. Regulation of nAChR subunit assembly and transfer of correctly assembled pentamers to the cell surface is only partially understood. Here, we characterize an ER transmembrane (TM) protein complex that influences nAChR cell-surface expression and functional properties in Caenorhabditis elegans muscle.

The conserved RIC-3 coiled-coil domain mediates receptor-specific interactions with nicotinic acetylcholine receptors.

RIC-3 belongs to a conserved family of proteins influencing nicotinic acetylcholine receptor (nAChR) maturation. RIC-3 proteins are integral membrane proteins residing in the endoplasmic reticulum (ER), and containing a C-terminal coiled-coil domain (CC-I). Conservation of CC-I in all RIC-3 family members indicates its importance; however, previous studies could not show its function.

Optogenetic analysis of synaptic function.

We introduce optogenetic investigation of neurotransmission (OptIoN) for time-resolved and quantitative assessment of synaptic function via behavioral and electrophysiological analyses. We photo-triggered release of acetylcholine or gamma-aminobutyric acid at Caenorhabditis elegans neuromuscular junctions using targeted expression of Chlamydomonas reinhardtii Channelrhodopsin-2. In intact Channelrhodopsin-2 transgenic worms, photostimulation instantly induced body elongation (for gamma-aminobutyric acid) or contraction (for acetylcholine), which we analyzed acutely, or during sustained activation with automated image analysis, to assess synaptic efficacy.

Multimodal fast optical interrogation of neural circuitry.

Our understanding of the cellular implementation of systems-level neural processes like action, thought and emotion has been limited by the availability of tools to interrogate specific classes of neural cells within intact, living brain tissue. Here we identify and develop an archaeal light-driven chloride pump (NpHR) from Natronomonas pharaonis for temporally precise optical inhibition of neural activity. NpHR allows either knockout of single action potentials, or sustained blockade of spiking.