Simultaneous spectral illumination of microplates for high-throughput optogenetics and photobiology.

The biophysical characterization and engineering of optogenetic tools and photobiological systems has been hampered by the lack of efficient methods for spectral illumination of microplates for high-throughput analysis of action spectra. Current methods to determine action spectra only allow the sequential spectral illumination of individual wells. Here we present the open-source RainbowCap-system, which combines LEDs and optical filters in a standard 96-well microplate format for simultaneous and spectrally defined illumination.

Efficient generation of a self-organizing neuromuscular junction model from human pluripotent stem cells.

The complex neuromuscular network that controls body movements is the target of severe diseases that result in paralysis and death. Here, we report the development of a robust and efficient self-organizing neuromuscular junction (soNMJ) model from human pluripotent stem cells that can be maintained long-term in simple adherent conditions. The timely application of specific patterning signals instructs the simultaneous development and differentiation of position-specific brachial spinal neurons, skeletal muscles, and terminal Schwann cells.

A bistable inhibitory OptoGPCR for multiplexed optogenetic control of neural circuits.

Information is transmitted between brain regions through the release of neurotransmitters from long-range projecting axons. Understanding how the activity of such long-range connections contributes to behavior requires efficient methods for reversibly manipulating their function. Chemogenetic and optogenetic tools, acting through endogenous G-protein coupled receptor (GPCRs) pathways, can be used to modulate synaptic transmission, but existing tools are limited in sensitivity, spatiotemporal precision, or spectral multiplexing capabilities.

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling.

Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H, Na, K and anions have been discovered or engineered, Ca-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca permeability and improve their Ca affinity by targeted mutagenesis at the central selectivity filter.

Optogenetics at the presynapse.

Optogenetic actuators enable highly precise spatiotemporal interrogation of biological processes at levels ranging from the subcellular to cells, circuits and behaving organisms. Although their application in neuroscience has traditionally focused on the control of spiking activity at the somatodendritic level, the scope of optogenetic modulators for direct manipulation of presynaptic functions is growing. Presynaptically localized opsins combined with light stimulation at the terminals allow light-mediated neurotransmitter release, presynaptic inhibition, induction of synaptic plasticity and specific manipulation of individual components of the presynaptic machinery.

Aberrant Phase Separation of FUS Leads to Lysosome Sequestering and Acidification.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets and .

Recruitment of release sites underlies chemical presynaptic potentiation at hippocampal mossy fiber boutons.

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin.

Efficient optogenetic silencing of neurotransmitter release with a mosquito rhodopsin.

Information is carried between brain regions through neurotransmitter release from axonal presynaptic terminals. Understanding the functional roles of defined neuronal projection pathways requires temporally precise manipulation of their activity. However, existing inhibitory optogenetic tools have low efficacy and off-target effects when applied to presynaptic terminals, while chemogenetic tools are difficult to control in space and time.

Somatostatin interneurons activated by 5-HT receptor suppress slow oscillations in medial entorhinal cortex.

Serotonin (5-HT) is one of the major neuromodulators present in the mammalian brain and has been shown to play a role in multiple physiological processes. The mechanisms by which 5-HT modulates cortical network activity, however, are not yet fully understood. We investigated the effects of 5-HT on slow oscillations (SOs), a synchronized cortical network activity universally present across species.

SynaptoPAC, an optogenetic tool for induction of presynaptic plasticity.

Optogenetic manipulations have transformed neuroscience in recent years. While sophisticated tools now exist for controlling the firing patterns of neurons, it remains challenging to optogenetically define the plasticity state of individual synapses. A variety of synapses in the mammalian brain express presynaptic long-term potentiation (LTP) upon elevation of presynaptic cyclic adenosine monophosphate (cAMP), but the molecular expression mechanisms as well as the impact of presynaptic LTP on network activity and behavior are not fully understood.

Potassium channel-based optogenetic silencing.

Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK.

VGLUT2 Functions as a Differential Marker for Hippocampal Output Neurons.

The subiculum is the gatekeeper between the hippocampus and cortical areas. Yet, the lack of a pyramidal cell-specific marker gene has made the analysis of the subicular area very difficult. Here we report that the vesicular-glutamate transporter 2 (VGLUT2) functions as a specific marker gene for subicular burst-firing neurons, and demonstrate that VGLUT2-Cre mice allow for Channelrhodopsin-2 (ChR2)-assisted connectivity analysis.

Cannabinoid type 2 receptors mediate a cell type-specific self-inhibition in cortical neurons.

Endogenous cannabinoids are diffusible lipid ligands of the main cannabinoid receptors type 1 and 2 (CBR and CBR). In the central nervous system endocannabinoids are produced in an activity-dependent manner and have been identified as retrograde modulators of synaptic transmission. Additionally, some neurons display a cell-autonomous slow self-inhibition (SSI) mediated by endocannabinoids.

Optogenetic Tools for Subcellular Applications in Neuroscience.

The ability to study cellular physiology using photosensitive, genetically encoded molecules has profoundly transformed neuroscience. The modern optogenetic toolbox includes fluorescent sensors to visualize signaling events in living cells and optogenetic actuators enabling manipulation of numerous cellular activities. Most optogenetic tools are not targeted to specific subcellular compartments but are localized with limited discrimination throughout the cell.

RIM-binding protein 2 regulates release probability by fine-tuning calcium channel localization at murine hippocampal synapses.

The tight spatial coupling of synaptic vesicles and voltage-gated Ca channels (Cas) ensures efficient action potential-triggered neurotransmitter release from presynaptic active zones (AZs). Rab-interacting molecule-binding proteins (RIM-BPs) interact with Ca channels and via RIM with other components of the release machinery. Although human RIM-BPs have been implicated in autism spectrum disorders, little is known about the role of mammalian RIM-BPs in synaptic transmission.

Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis.

SEE ZEKERIDOU AND LENNON DOI101093/AWW213 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Little is known about the cerebrospinal fluid autoantibody repertoire. Antibodies against the NR1 subunit of the NMDAR are thought to be pathogenic; however, direct proof is lacking as previous experiments could not distinguish the contribution of further anti-neuronal antibodies.

Optogenetic acidification of synaptic vesicles and lysosomes.

Acidification is required for the function of many intracellular organelles, but methods to acutely manipulate their intraluminal pH have not been available. Here we present a targeting strategy to selectively express the light-driven proton pump Arch3 on synaptic vesicles. Our new tool, pHoenix, can functionally replace endogenous proton pumps, enabling optogenetic control of vesicular acidification and neurotransmitter accumulation.

Vesicular Synaptobrevin/VAMP2 Levels Guarded by AP180 Control Efficient Neurotransmission.

Neurotransmission depends on synaptic vesicle (SV) exocytosis driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation of vesicular synaptobrevin/VAMP2 (Syb2). Exocytic fusion is followed by endocytic SV membrane retrieval and the high-fidelity reformation of SVs. Syb2 is the most abundant SV protein with 70 copies per SV, yet, one to three Syb2 molecules appear to be sufficient for basal exocytosis.

Clathrin regenerates synaptic vesicles from endosomes.

Ultrafast endocytosis can retrieve a single, large endocytic vesicle as fast as 50-100 ms after synaptic vesicle fusion. However, the fate of the large endocytic vesicles is not known. Here we demonstrate that these vesicles transition to a synaptic endosome about one second after stimulation.

Ultrafast endocytosis at mouse hippocampal synapses.

To sustain neurotransmission, synaptic vesicles and their associated proteins must be recycled locally at synapses. Synaptic vesicles are thought to be regenerated approximately 20 s after fusion by the assembly of clathrin scaffolds or in approximately 1 s by the reversal of fusion pores via 'kiss-and-run' endocytosis. Here we use optogenetics to stimulate cultured hippocampal neurons with a single stimulus, rapidly freeze them after fixed intervals and examine the ultrastructure using electron microscopy--'flash-and-freeze' electron microscopy.

Novel APP/Aβ mutation K16N produces highly toxic heteromeric Aβ oligomers.

Here, we describe a novel missense mutation in the amyloid precursor protein (APP) causing a lysine-to-asparagine substitution at position 687 (APP770; herein, referred to as K16N according to amyloid-β (Aβ) numbering) resulting in an early onset dementia with an autosomal dominant inheritance pattern. The K16N mutation is located exactly at the α-secretase cleavage site and influences both APP and Aβ. First, due to the K16N mutation APP secretion is affected and a higher amount of Aβ peptides is being produced.

Homeostatic regulation of NCAM polysialylation is critical for correct synaptic targeting.

During development, axonal projections have a remarkable ability to innervate correct dendritic subcompartments of their target neurons and to form regular neuronal circuits. Altered axonal targeting with formation of synapses on inappropriate neurons may result in neurodevelopmental sequelae, leading to psychiatric disorders. Here we show that altering the expression level of the polysialic acid moiety, which is a developmentally regulated, posttranslational modification of the neural cell adhesion molecule NCAM, critically affects correct circuit formation.