Schultheis C et al. (2011) PLoS One "Optogenetic long-term manipulation of behavior and animal development."

Liewald JF, Gottschalk A, Schultheis C, Bamberg E, Nagel G

[PLoS One, 2011]

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. We used "slow" ChR2 variants with mutations in the C128 residue, that exhibit delayed off-kinetics and increased light sensitivity in Caenorhabditis elegans. Following a 1 s light pulse, we could photodepolarize neurons and muscles for minutes (and with repeated brief stimulation, up to days) with low-intensity light. Photoactivation of ChR2(C128S) in command interneurons elicited long-lasting alterations in locomotion. Finally, we could optically induce profound changes in animal development: Long-term photoactivation of ASJ neurons, which regulate larval growth, bypassed the constitutive entry into the "dauer" larval state in daf-11 mutants. These lack a guanylyl cyclase, which possibly renders ASJ neurons hyperpolarized. Furthermore, photostimulated ASJ neurons could acutely trigger dauer-exit. Thus, slow ChR2s can be employed to long-term photoactivate behavior and to trigger alternative animal development.

Schultheis, C. et al. (2013) International Worm Meeting "Unbiased optogenetic circuit mapping: AVK interneurons, a case study."

Schultheis, C., Wabnig, S., Erbguth, K., Gottschalk, A., Brauner, M.

[International Worm Meeting, 2013]

C. elegans has only 302 neurons, for many of which still no function is known. Optogenetic tools offer the possibility to map the function of each neuron in behavior, provided they are specifically activated in these neurons. We began to map the function of previously uncharacterized neurons. As a first example, we analyzed AVK interneurons, taking advantage of the availability of an AVK-specific promoter. Photoinhibiting AVK interneurons using halorhodopsin strongly affected behavior: Animals reduced swimming cycles by 50%, and showed deeper body bends on solid. In contrast, photostimulation using ChR2 had no obvious effects. Genetic ablation of AVK by caspase expression or acute ablation using miniSOG (minimal singlet O2 generator), as well as specific expression of tetanus toxin (abolishing chemical transmission), recapitulated the hyperpolarization phenotypes. This indicates that AVK tonically releases a transmitter to mediate normal locomotion. This transmitter may be FLP-1 neuropeptides, as flp-1 knockdown specifically in AVK had similar effects on swimming behavior, AVK::FLP-1 expression in flp-1 mutants recued the phenotypes, and AVK::FLP-1::mCherry was detectable in coelomocytes, verifying that AVK releases FLP-1 peptides. Also, lack of FLP-1 or of AVK led to increased local dwelling of the animals. AVK neurons span the length of the animal and may be proprioceptive, as body bending during semi-restrained locomotion caused Ca2+ fluctuations in the AVK nervering process. AVK may act antagonistically to the proprioceptive DVA neuron, photoactivation of which increases body bends, while photoinhibition of DVA causes decreased bending. Both AVK and DVA are likely modulated by dopamine (DA), released from PDE neurons, which are the most prominent presynaptic partners of both AVK and DVA. Importantly, photostimulated DA transmission leads to reduced bending angles and a slowing of locomotion. We analyzed which DA receptors mediate this modulation in AVK and DVA. Furthermore, we currently analyze neurons acting downstream of AVK for their roles in this novel pathway impacting on locomotion control in C. elegans. Our approach should contribute to a holistic understanding of the C. elegans nervous system.

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.

Schultheis C et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Using Channelrhodopsin-2 Slow Mutants To Complement The Optogenetics Toolbox"

Nagel G, Gottschalk A, Schultheis C

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Channelrhodopsin-2 (ChR2) is a blue light-gated cation-channel from the green algae Chlamydomonas reinhardtii (Nagel et al. 2003). The optogenetic approach uses heterologous expression of ChR2 for in-vivo light-dependent depolarization of neurons or muscles, for functional characterization of synaptic transmission and of neuronal networks (Nagel et al. 2005; Liewald et al. 2008; Liu et al. 2009). However, the required high light intensities for photoactivation do not allow long-term applications, as permanent illumination can cause phototoxicity, thus hampering applications e.g. during development, where long-term altered neuronal activity may influence certain processes or pathways. Recently, it was shown that mutation of the Cys128 residue of ChR2 vastly delays the inactivation of the channel (Berndt et al. 2009). Hence a short light pulse of reduced intensity is sufficient to render these "slow mutants" open for several minutes. Furthermore, slow mutants can be inactivated with green light. To test these ChR2 slow mutants in C. elegans, we expressed them in body wall muscle cells. We found that a brief (1 sec) blue light pulse with reduced intensity (200x less than for WT-ChR2) triggers body contraction for several minutes. A subsequent green light pulse was able to terminate the contraction. Similar results were obtained for photoactivation of ChR2 slow mutants in cholinergic and GABAergic motorneurons to trigger long-term synaptic transmission. In C. elegans two pairs of command interneurons (PVC and AVB) are required to trigger forward locomotion (Zheng et al. 1999). Backward command interneurons (AVA, AVE and AVD) appear to be less active, depending on sensory neuron input, which explains the dominant forward movement. Through photoactivation of ChR2(C128S) in both types of command interneurons, we could permanently disrupt the dominance of forward command interneurons, which significantly increased the ratio of backward movement until a green light pulse terminated ChR2 activity. Our results show that the ChR2 slow mutants can be employed to investigate long-term behavioural effects with minimum light delivery and that this activity can be precisely terminated using green light. We are now investigating possibilities to alter neuronal function in the long-term during animal development.

Schmitt C et al. (2012) PLoS One "Specific expression of channelrhodopsin-2 in single neurons of Caenorhabditis elegans."

Husson SJ, Gottschalk A, Schultheis C, Liewald JF, Schmitt C

[PLoS One, 2012]

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. Thus, it would be helpful if expression of ChR2 could be restricted to single neurons or neuron pairs, as even wide-field illumination would photostimulate only this particular cell. To this end we adopted the use of Cre or FLP recombinases and conditional ChR2 expression at the intersection of two promoter expression domains, i.e. in the cell of interest only. Success of this method depends on precise knowledge of the individual promoters' expression patterns and on relative expression levels of recombinase and ChR2. A bicistronic expression cassette with GFP helps to identify the correct expression pattern. Here we show specific expression in the AVA reverse command neurons and the aversive polymodal sensory ASH neurons. This approach shall enable to generate strains for optogenetic manipulation of each of the 302 C. elegans neurons. This may eventually allow to model the C. elegans nervous system in its entirety, based on functional data for each neuron.

Brauner M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Heterosynaptic Inhibition of Cholinergic Motor Neurons via Metabotropic GABAB Receptors is not Plastic"

Brauner M, Gottschalk A, Liewald J F, Schultheis C

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

In the ventral nerve cord, cholinergic (ACh) and GABAergic neurons coordinate locomotion by mediating contralateral contraction and relaxation of ventral and dorsal muscles. ACh-neurons that innervate ventral muscle also trigger GABA-neurons projecting to the dorsal side at dyadic synapses. Very few recurrent synapses from GABA-neurons to ACh-neurons are known, so it is unclear if they are crucial. Dittman et al (2008) showed that metabotropic GABAB receptors, made of GBB-1 and GBB-2 subunits and expressed by ACh-neurons, influence locomotion, likely by heterosynaptic inhibition, detecting spill-over GABA and mediating feedback inhibition. Using Channelrhodopsin-2 (ChR2) to photo-stimulate cholinergic (transgene zxIs6) or GABAergic (transgene zxIs3) neurons, we can evoke contraction or relaxation of the animal, as all body wall muscles are simultaneously stimulated or inhibited, respectively (Liewald et al, 2008). The response is graded, with a more or less linear correlation between light-intensity and photo-evoked post-synaptic currents (photo-ePSCs). When we stimulated GABA-neurons in unc-49(e407) mutants lacking ionotropic GABAA receptors, photo-ePSCs were completely abolished and responses strongly reduced, though some body relaxation remained. This could be mediated by the metabotropic GBB-1/-2 receptor in ACh-neurons. Consistently, in unc-49; gbb-2(tm1165) or unc-49; gbb-1(tm1406) double mutants no relaxation resulted at all. We asked whether also feedback inhibition is merely graded, or whether it could induce plastic changes in ACh-neurons, e.g. increased inhibition, the more ACh is released onto GABA-neurons. This would evoke more spillover-GABA, detected by GBB-1/-2. Stimulus trains and continuous photo-stimulation of ACh-neurons did not reveal any time-dependent altered body contractions, neither in wild type nor in gbb-2 mutants, which was also seen when both zxIs6 and zxIs3 were present and photo-triggered at once: Apart from an overall reduced contraction (due to concomitantly photo-released GABA), no plastic changes were apparent over time. Thus GBB-1/-2 receptors serve to reduce release from ACh-neurons, but do not mediate short- or long-term plasticity at the NMJ.

Husson SJ et al. (2012) PLoS One "Microbial light-activatable proton pumps as neuronal inhibitors to functionally dissect neuronal networks in C. elegans."

Schultheis C, Gottschalk A, Liewald JF, Lu H, Husson SJ, Stirman JN

[PLoS One, 2012]

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. In analogy, other excitable cells can be inhibited by expressing Halorhodopsin from Natronomonas pharaonis (NpHR) and subsequent illumination with yellow light. However, inhibiting C. elegans neurons using NpHR is difficult. Recently, proton pumps from various sources were established as valuable alternative hyperpolarizers. Here we show that archaerhodopsin-3 (Arch) from Halorubrum sodomense and a proton pump from the fungus Leptosphaeria maculans (Mac) can be utilized to effectively inhibit excitable cells in C. elegans. Arch is the most powerful hyperpolarizer when illuminated with yellow or green light while the action spectrum of Mac is more blue-shifted, as analyzed by light-evoked behaviors and electrophysiology. This allows these tools to be combined in various ways with ChR2 to analyze different subsets of neurons within a circuit. We exemplify this by means of the polymodal aversive sensory ASH neurons, and the downstream command interneurons to which ASH neurons signal to trigger a reversal followed by a directional turn. Photostimulating ASH and subsequently inhibiting command interneurons using two-color illumination of different body segments, allows investigating temporal aspects of signaling downstream of ASH.

Stirman J N et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "High-Throughput Study of Synaptic Transmission Enabled by Optogenetics and Microfluidics"

Lu H, Schultheis C, Stirman J N, Wabnig S, Gottschalk A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Over the past several years, optogenetic techniques have become widely used to help elucidate a variety of neuroscience problems. For example, the blue light gated cation channel channelrhodopsin-2 (ChR2) enables unique optical control of neurons within a variety of organisms, allowing 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 C. elegans (Liewald et al., 2008, Liu et al., 2009): ChR2 is expressed and photoactivated in motor-neurons, and the resulting post-synaptic currents are either directly recorded, or analyzed indirectly as a change of body length. For synaptic transmission studies, although quantitative, this technique is manual and very low-throughput. In this study, we enhance this new tool by combining it with microfluidic technology and computer automation (including image analysis). This allows us to increase the assay throughput by several orders of magnitude as compared to the current approach, as well as improving standardization and consistency in data gathering. We show that the automated microfluidic approach yield the same quantitative data as manual approaches. We also demonstrate the ability to administer drugs to worms during optogenetic experiments using microfluidics enabling drug interactions with optically activated neural circuits to be studied. We are combining these technologies with RNAi for high throughput genetic screening of synaptic transmission. In preliminary studies, we have found that RNAi mediated knockdown of selected genes can phenocopy the optogenetic elicited behavior of deletion alleles of the same genes. Integrating our microfluidic device with an automated liquid handling system allowed us to perform a pilot study on over 75 genes, and showed that this approach has a vast increase in speed and data acquisition. Together, optogenetics, microfluidics, and system/image automation will enable high-throughput genetic studies of synaptic function.

Stirman JN et al. (2011) Nat Methods "Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans."

Crane MM, Schultheis C, Husson SJ, Gottschalk A, Wabnig S, Lu H, Stirman JN

[Nat Methods, 2011]

The ability to optically excite or silence specific cells using optogenetics has become a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but it has only been shown in motionless animals without the ability to observe behavior output. We present a real-time, multimodal illumination technology that allows both tracking and recording the behavior of freely moving C. elegans while stimulating specific cells that express channelrhodopsin-2 or MAC. We used this system to optically manipulate nodes in the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology enhances our ability to control, alter, observe and investigate how neurons, muscles and circuits ultimately produce behavior in animals using optogenetics.

Weissenberger S et al. (2011) J Neurochem "PAC--an optogenetic tool for in vivo manipulation of cellular cAMP levels, neurotransmitter release, and behavior in Caenorhabditis elegans."

Gottschalk A, Schultheis C, Nagel G, Liewald JF, Weissenberger S, Erbguth K

[J Neurochem, 2011]

Photoactivated adenylyl cyclase (PAC) 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 PAC in cholinergic neurons of Caenorhabditis elegans. Photoactivation led to a rise in swimming frequency, speed of locomotion, and a decrease in the number of backward locomotion episodes. The extent of the light-induced behavioral effects was dependent on the amount of PAC that was expressed. Furthermore, electrophysiological recordings from body wall muscle cells revealed an increase in miniature post-synaptic currents during light stimulation. We conclude that the observed effects were caused by cAMP synthesis because of photoactivation of pre-synaptic PAC which subsequently triggered acetylcholine release at the neuromuscular junction. Our results demonstrate that PAC can be used as an optogenetic tool in C. elegans for straightforward in vivo manipulation of intracellular cAMP levels by light, with good temporal control and high cell specificity. Thus, using PAC allows manipulation of neurotransmitter release and behavior by directly affecting intracellular signaling.