Jeffrey N. Stirman

Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans

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.

Autonomous screening of C. elegans identifies genes implicated in synaptogenesis

Morphometric studies in multicellular organisms are generally performed manually because of the complexity of multidimensional features and lack of appropriate tools for handling these organisms. Here we present an integrated system that identifies and sorts Caenorhabditis elegans mutants with altered subcellular traits in real time without human intervention. We performed self-directed screens 100 times faster than manual screens and identified both genes and phenotypic classes involved in synapse formation.Morphometric studies in multicellular organisms are mostly performed manually because of the complexity of multidimensional features and lack of appropriate tools for handling these organisms. Here we present an integrated system to autonomously (i.e. without human supervision) identify and sort mutants with altered subcellular traits in real-time. We performed self-directed screens of synapse formation 100× faster and found both novel genes and phenotypic classes previously unidentified in extensive manual screens.

Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain

Two-photon calcium imaging provides an optical readout of neuronal activity in populations of neurons with subcellular resolution. However, conventional two-photon imaging systems are limited in their field of view to ∼1 mm2, precluding the visualization of multiple cortical areas simultaneously. Here, we demonstrate a two-photon microscope with an expanded field of view (>9.5 mm2) for rapidly reconfigurable simultaneous scanning of widely separated populations of neurons. We custom designed and assembled an optimized scan engine, objective, and two independently positionable, temporally multiplexed excitation pathways. We used this new microscope to measure activity correlations between two cortical visual areas in mice during visual processing.

High-throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics

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.

A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents

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 microscopes 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 μm 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.

Optogenetic analysis of a nociceptor neuron and network reveals ion channels acting downstream of primary sensors

BACKGROUND Nociception generally evokes rapid withdrawal behavior in order to protect the tissue from harmful insults. Most nociceptive neurons responding to mechanical insults display highly branched dendrites, an anatomy shared by Caenorhabditis elegans FLP and PVD neurons, which mediate harsh touch responses. Although several primary molecular nociceptive sensors have been characterized, less is known about modulation and amplification of noxious signals within nociceptor neurons. First, we analyzed the FLP/PVD network by optogenetics and studied integration of signals from these cells in downstream interneurons. Second, we investigated which genes modulate PVD function, based on prior single-neuron mRNA profiling of PVD. RESULTS Selectively photoactivating PVD, FLP, and downstream interneurons via Channelrhodopsin-2 (ChR2) enabled the functional dissection of this nociceptive network, without interfering signals by other mechanoreceptors. Forward or reverse escape behaviors were determined by PVD and FLP, via integration by command interneurons. To identify mediators of PVD function, acting downstream of primary nocisensor molecules, we knocked down PVD-specific transcripts by RNAi and quantified light-evoked PVD-dependent behavior. Cell-specific disruption of synaptobrevin or voltage-gated Ca(2+) channels (VGCCs) showed that PVD signals chemically to command interneurons. Knocking down the DEG/ENaC channel ASIC-1 and the TRPM channel GTL-1 indicated that ASIC-1 may extend PVDs dynamic range and that GTL-1 may amplify its signals. These channels act cell autonomously in PVD, downstream of primary mechanosensory molecules. CONCLUSIONS Our work implicates TRPM channels in modifying excitability of and DEG/ENaCs in potentiating signal output from a mechano-nociceptor neuron. ASIC-1 and GTL-1 homologs, if functionally conserved, may denote valid targets for novel analgesics.

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. 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.

Bending Amplitude-- a New Quantitative Assay of C. elegans Locomotion: Identification of Phenotypes for Mutants in Genes Encoding Muscle Focal Adhesion Components

The nematode Caenorhabditis elegans uses striated muscle in its body wall for locomotion. The myofilament lattice is organized such that all the thin filament attachment structures (dense bodies, analogous to Z-disks) and thick filament organizing centers (M-lines) are attached to the muscle cell membrane. Thus, the force of muscle contraction is transmitted through these structures and allows locomotion of the worm. Dense bodies and M-lines are compositionally similar to focal adhesions and costameres, and are based on integrin and associated proteins. Null mutants for many of the newly discovered dense body and M-line proteins do not have obvious locomotion defects when observed casually, or when assayed by counting the number of times a worm moves back and forth in liquid. We hypothesized that many of these proteins, located as they are in muscle focal adhesions, function in force transmission, but we had not used an appropriate or sufficiently sensitive assay to reveal this function. Recently, we have developed a new quantitative assay of C. elegans locomotion that measures the maximum bending amplitude of an adult worm as it moves backwards. The assay had been used to reveal locomotion defects for null mutants of genes encoding ATN-1 (α-actinin) and PKN-1 (protein kinase N). Here, we describe the details of this method, and apply it to 21 loss of function mutants in 17 additional genes, most of which encode components of muscle attachment structures. As compared to wild type, mutants in 11 genes were found to have less ability to bend, and mutants in one gene were found to have greater ability to bend. Loss of function mutants for eight proteins had been reported to have normal locomotion (ZYX-1 (zyxin), ALP-1 (Enigma), DIM-1, SCPL-1), or locomotion that was not previously investigated (FRG-1 (FRG1), KIN-32 (focal adhesion kinase), LIM-8), or had only slightly decreased locomotion (PFN-3 (profilin)).

α-Actinin Is Required for the Proper Assembly of Z-Disk/Focal-Adhesion-Like Structures and for Efficient Locomotion in Caenorhabditis elegans

The actin binding protein α-actinin is a major component of focal adhesions found in vertebrate cells and of focal-adhesion-like structures found in the body wall muscle of the nematode Caenorhabditis elegans. To study its in vivo function in this genetic model system, we isolated a strain carrying a deletion of the single C. elegans α-actinin gene. We assessed the cytological organization of other C. elegans focal adhesion proteins and the ultrastructure of the mutant. The mutant does not have normal dense bodies, as observed by electron microscopy; however, these dense-body-like structures still contain the focal adhesion proteins integrin, talin, and vinculin, as observed by immunofluorescence microscopy. Actin is found in normal-appearing I-bands, but with abnormal accumulations near muscle cell membranes. Although swimming in water appeared grossly normal, use of automated methods for tracking the locomotion of individual worms revealed a defect in bending. We propose that the reduced motility of α-actinin null is due to abnormal dense bodies that are less able to transmit the forces generated by actin/myosin interactions.

A touchscreen based global motion perception task for mice

Global motion perception is a function of higher, or extrastriate, visual system circuitry. These circuits can be engaged in visually driven navigation, a behavior at which mice are adept. However, the properties of global motion perception in mice are unclear. Therefore, we developed a touchscreen-based, two-alternative forced choice (2AFC) task to explore global motion detection in mice using random dot kinematograms (RDK). Performance data was used to compute coherence thresholds for global motion perception. The touchscreen-based task allowed for parallel training and testing with multiple chambers and minimal experimenter intervention with mice performing hundreds of trials per session. Parameters of the random dot kinematograms, including dot size, lifetime, and speed, were tested. Mice learned to discriminate kinematograms whose median motion direction differed by 90 degrees in 7-24days after a 10-14day pre-training period. The average coherence threshold (measured at 70% correct) in mice for this task was 22±5%, with a dot diameter of 3.88mm and speed of 58.2mm/s. Our results confirm the ability of mice to perform global motion discriminations, and the touchscreen assay provides a flexible, automated, and relatively high throughput method with which to probe complex visual function in mice.