Gottschalk A et al. (2006) J Neurosci Methods "Visualization of integral and peripheral cell surface proteins in live Caenorhabditis elegans."

Schafer WR, Gottschalk A

[J Neurosci Methods, 2006]

To study the abundance of specific receptors and other cell surface proteins at synapses, it would be advantageous to specifically label these proteins only when inserted in the plasma membrane. We describe a method that allows to fluorescently label cell surface proteins in live and behaving animals, namely in the nematode Caenorhabditis elegans. Proteins such as subunits of the levamisole sensitive nicotinic acetylcholine receptor (nAChR) were epitope-tagged at their extracellular C-termini, and fluorescent antibodies against those tags were injected into the body fluid. These antibodies specifically labelled synaptic regions on the cell surface of muscles and neurons, and simultaneous use of different tags facilitated co-localization studies. Quantification of the fluorescence is possible, as verified by demonstrating that mutations in ric-3 and unc-38, which cause behavioural resistance to cholinergic agonists, strongly reduce or even abolish nAChR cell surface expression. We also used this method to visualize the extracellular peripheral membrane protein ODR-2, which is related to a neurotoxin-like protein regulating vertebrate neuronal nAChRs. Likewise, fluorescent alpha-bungarotoxin, when injected, bound to certain nAChRs in the pharynx and the nervous system. This showed that, theoretically, any molecular interaction of sufficient affinity may be used to specifically label cell surface structures in live nematodes.

Gottschalk A et al. (2005) EMBO J "Identification and characterization of novel nicotinic receptor-associated proteins in Caenorhabditis elegans."

Gottschalk A, Anderson SD, Schafer WR, Schedletzky T, Yates JR, Almedom RB

[EMBO J, 2005]

Nicotinic acetylcholine receptors (nAChRs) mediate fast excitatory neurotransmission in neurons and muscles. To identify nAChR accessory proteins, which may regulate their expression or function, we performed tandem affinity purification of the levamisole-sensitive nAChR from Caenorhabditis elegans, mass spectrometry of associated components, and RNAi-based screening for effects on in vivo nicotine sensitivity. Among the proteins identified was the calcineurin A subunit TAX-6, which appeared to function as a negative regulator of nAChR activity. We also identified five proteins not previously linked to nAChR function, whose inactivation conferred nicotine resistance, implicating them as positive regulators of nAChR activity. Of these, the copine NRA-1 colocalized with the levamisole receptor at neuronal and muscle plasma membranes, and, when mutated, caused reduced synaptic nAChR expression. Loss of SOC-1, which acts in receptor tyrosine kinase (RTK) signaling, also reduced synaptic levamisole receptor levels, as did mutations in the fibroblast growth factor receptor EGL-15, and another RTK, CAM-1. Thus, tandem affinity purification is a viable approach to identify novel proteins regulating neurotransmitter receptor activity or expression in model systems like C. elegans.

Glock C et al. (2015) Methods Mol Biol "Microbial Rhodopsin Optogenetic Tools: Application for Analyses of Synaptic Transmission and of Neuronal Network Activity in Behavior."

Glock C, Gottschalk A, Nagpal J

[Methods Mol Biol, 2015]

Optogenetics was introduced as a new technology in the neurosciences about a decade ago (Zemelman et al., Neuron 33:15-22, 2002; Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005; Zemelman et al., Proc Natl Acad Sci USA 100:1352-1357, 2003). It combines optics, genetics, and bioengineering to render neurons sensitive to light, in order to achieve a precise, exogenous, and noninvasive control of membrane potential, intracellular signaling, network activity, or behavior (Rein and Deussing, Mol Genet Genomics 287:95-109, 2012; Yizhar et al., Neuron 71:9-34, 2011). As C. elegans is transparent, genetically amenable, has a small nervous system mapped with synapse resolution, and exhibits a rich behavioral repertoire, it is especially open to optogenetic methods (White et al., Philos Trans R Soc Lond B Biol Sci 314:1-340, 1986; De Bono et al., Optogenetic actuation, inhibition, modulation and readout for neuronal networks generating behavior in the nematode Caenorhabditis elegans, In: Hegemann P, Sigrist SJ (eds) Optogenetics, De Gruyter, Berlin, 2013; Husson et al., Biol Cell 105:235-250, 2013; Xu and Kim, Nat Rev Genet 12:793-801, 2011). Optogenetics, by now an "exploding" field, comprises a repertoire of different tools ranging from transgenically expressed photo-sensor proteins (Boyden et al., Nat Neurosci 8:1263-1268, 2005; Nagel et al., Curr Biol 15:2279-2284, 2005) or cascades (Zemelman et al., Neuron 33:15-22, 2002) to chemical biology approaches, using photochromic ligands of endogenous channels (Szobota et al., Neuron 54:535-545, 2007). Here, we will focus only on optogenetics utilizing microbial rhodopsins, as these are most easily and most widely applied in C. elegans. For other optogenetic tools, for example the photoactivated adenylyl cyclases (PACs, that drive neuronal activity by increasing synaptic vesicle priming, thus exaggerating rather than overriding the intrinsic activity of a neuron, as occurs with rhodopsins), we refer to other literature (Weissenberger et al., J Neurochem 116:616-625, 2011; Steuer Costa et al., Photoactivated adenylyl cyclases as optogenetic modulators of neuronal activity, In: Cambridge S (ed) Photswitching proteins, Springer, New York, 2014). In this chapter, we will give an overview of rhodopsin-based optogenetic tools, their properties and function, as well as their combination with genetically encoded indicators of neuronal activity. As there is not "the" single optogenetic experiment we could describe here, we will focus more on general concepts and "dos and don'ts" when designing an optogenetic experiment. We will also give some guidelines on which hardware to use, and then describe a typical example of an optogenetic experiment to analyze the function of the neuromuscular junction, and another application, which is Ca(2+) imaging in body wall muscle, with upstream neuronal excitation using optogenetic stimulation. To obtain a more general overview of optogenetics and optogenetic tools, we refer the reader to an extensive collection of review articles, and in particular to volume 1148 of this book series, "Photoswitching Proteins."

Fischer E et al. (2017) Sci Rep "An optogenetic arrhythmia model to study catecholaminergic polymorphic ventricular tachycardia mutations."

Schuler C, Fischer E, Gottschalk A

[Sci Rep, 2017]

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a condition of abnormal heart rhythm (arrhythmia), induced by physical activity or stress. Mutations in ryanodine receptor 2 (RyR2), a Ca2+ release channel located in the sarcoplasmic reticulum (SR), or calsequestrin 2 (CASQ2), a SR Ca2+ binding protein, are linked to CPVT. For specific drug development and to study distinct arrhythmias, simple models are required to implement and analyze such mutations. Here, we introduced CPVT inducing mutations into the pharynx of Caenorhabditis elegans, which we previously established as an optogenetically paced heart model. By electrophysiology and video-microscopy, we characterized mutations in csq-1 (CASQ2 homologue) and unc-68 (RyR2 homologue). csq-1 deletion impaired pharynx function and caused missed pumps during 3.7Hz pacing. Deletion mutants of unc-68, and in particular the point mutant UNC-68(R4743C), analogous to the established human CPVT mutant RyR2(R4497C), were unable to follow 3.7Hz pacing, with progressive defects during long stimulus trains. The pharynx either locked in pumping at half the pacing frequency or stopped pumping altogether, possibly due to UNC-68 leakiness and/or malfunctional SR Ca2+ homeostasis. Last, we could reverse this 'worm arrhythmia' by the benzothiazepine S107, establishing the nematode pharynx for studying specific CPVT mutations and for drug screening.

Costa WS et al. (2014) Methods Mol Biol "Photoactivated adenylyl cyclases as optogenetic modulators of neuronal activity."

Gottschalk A, Liewald J, Costa WS

[Methods Mol Biol, 2014]

In recent years, optogenetic methods became invaluable tools, particularly in neurobiological research. 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. In this chapter we describe the use of photoactivated adenylyl cyclases (PACs) as modulators of neuronal activity. Using Caenorhabditis elegans as a model organism, this chapter shows how to measure the effect of PAC photoactivation by behavioral and electrophysiological assays, as well as their significance to neurobiology.

Wabnig S et al. (2015) PLoS One "High-Throughput All-Optical Analysis of Synaptic Transmission and Synaptic Vesicle Recycling in Caenorhabditis elegans."

Wabnig S, Yu SC, Gottschalk A, Liewald JF

[PLoS One, 2015]

Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct 'signature' of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3-5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous screens for synaptic function genes, based on behavioral or pharmacological assays, allowed no distinction of the stage of the SV cycle in which a protein might act. We generated a strain enabling RNAi specifically only in cholinergic neurons, thus resulting in healthier animals and avoiding lethal phenotypes resulting from knockdown elsewhere. RNAi of control genes resulted in Ca2+ measurements that were consistent with results obtained in the respective genomic mutants, albeit to a weaker extent in most cases, and could further be confirmed by opto-electrophysiological measurements for mutants of some of the genes, including synaptojanin. We screened 95 genes that were previously implicated in cholinergic transmission, and several controls. We identified genes that clustered together with known SV recycling genes, exhibiting a similar signature of their Ca2+ dynamics. Five of these genes (C27B7.7, erp-1, inx-8, inx-10, spp-10) were further assessed in respective genomic mutants; however, while all showed electrophysiological phenotypes indicative of reduced cholinergic transmission, no obvious SV recycling phenotypes could be uncovered for these genes.

Stirman JN et al. (2012) Nat Protoc "A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents."

Husson SJ, Stirman JN, Lu H, Gottschalk A, Crane MM

[Nat Protoc, 2012]

Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer-sized structures in both stationary and moving objects. The initial steps of the protocol describe how to modify an off-the-shelf video projector by insertion of optical filters and modification of projector optics. Subsequent steps involve altering the microscope's epifluorescence optical train as well as alignment and characterization of the system. When fully assembled, the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 m at medium magnifications. Compared with other custom-assembled systems and commercially available products, this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days.

Stirman JN et al. (2010) J Neurosci Methods "High-throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics."

Brauner M, Stirman JN, Lu H, Gottschalk A

[J Neurosci Methods, 2010]

Over the past several years, optogenetic techniques have become widely used to help elucidate a variety of neuroscience problems. The unique optical control of neurons within a variety of organisms provided by optogenetics allows researchers to probe neural circuits and investigate neuronal function in a highly specific and controllable fashion. Recently, optogenetic techniques have been introduced to investigate synaptic transmission in the nematode Caenorhabditis elegans. For synaptic transmission studies, although quantitative, this technique is manual and very low-throughput. As it is, it is difficult to apply this technique to genetic studies. In this paper, we enhance this new tool by combining it with microfluidics technology and computer automation. This allows us to increase the assay throughput by several orders of magnitude as compared to the current standard approach, as well as improving standardization and consistency in data gathering. We also demonstrate the ability to infuse drugs to worms during optogenetic experiments using microfluidics. Together, these technologies will enable high-throughput genetic studies such as those of synaptic function.

McKay JP et al. (2004) Genetics "eat-2 and eat-18 are required for nicotinic neurotransmission in the Caenorhabditis elegans pharynx."

Schafer WR, Avery L, McKay JP, Gottschalk A, Raizen DM

[Genetics, 2004]

Mutations in eat-2 and eat-18 cause the same defect in C. elegans feeding behaviour: the pharynx is unable to pump rapidly in the presence of food. EAT-2 is a nicotinic acetylcholine receptor subunit that functions in the pharyngeal muscle. It is localized to the synapse between pharyngeal muscle and the main pharyngeal excitatory motor neuron MC, and it is required for MC stimulation of pharyngeal muscle. eat-18 encodes a small protein that has no homology to previously characterized proteins. It has a single transmembrane domain and a short extracellular region. Allele-specific genetic interactions between eat-2 and eat-18 suggest that EAT-18 interacts physically with the EAT-2 receptor. While eat-2 appears to be required specifically for MC neurotransmission, eat-18 also appears to be required for the function of other nicotinic receptors in the pharynx. In eat-18 mutants, the gross localization of EAT-2 at the MC synapse is normal, suggesting that it is not required for trafficking. These data indicate that eat-18 could be a novel component of the pharyngeal nicotinic receptor.

Schultheis C et al. (2011) J Neurophysiol "Optogenetic analysis of GABAB receptor signaling in Caenorhabditis elegans motor neurons."

Brauner M, Gottschalk A, Schultheis C, Liewald JF

[J Neurophysiol, 2011]

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. Only a few connections exist from GABAergic to cholinergic MNs, emphasizing that GABA signaling is mainly directed toward muscle. Yet, a GABA(B) receptor comprising GBB-1 and GBB-2 subunits, expressed in cholinergic MNs, was shown to affect locomotion, likely by feedback inhibition of cholinergic MNs in response to spillover GABA. In the present study, we examined whether the GBB-1/2 receptor could also affect short-term plasticity in cholinergic MNs with the use of channelrhodopsin-2-mediated photostimulation of GABAergic and cholinergic neurons. The GBB-1/2 receptor contributes to acute body relaxation, evoked by photoactivation of GABAergic MNs, and to effects of GABA on locomotion behavior. Loss of the plasma membrane GABA transporter SNF-11, as well as acute photoevoked GABA release, affected cholinergic MN function in opposite directions. Prolonged stimulation of GABA MNs had subtle effects on cholinergic MNs, depending on stimulus duration and gbb-2. Thus GBB-1/2 receptors serve mainly for linear feedback inhibition of cholinergic MNs but also evoke minor plastic changes.