Mori I et al. (2000) Japanese Worm Meeting "An approach toward imaging activities of the neuronal network: a progress report 2000"

Mori I, Kimura KD

[Japanese Worm Meeting, 2000]

One of the big missing pieces for current neurobiology is to understand how a neuronal network works as a whole, instead of just a part of a big network. Apparently, our worm, C. elegans is the ideal system to solve the problem because, as well known, it has the description of its wiring diagram, many sophisticated molecular and genetic techniques, and so on. To monitor many neuronal activities in the network simultaneously, we have set up a system for calcium imaging of the worm neurons with cameleon, a GFP-based calcium sensor (ref. 1). Calcium changes in pharyngeal or body wall muscles have been monitored with cameleon (ref. 2 - 4). Our system is composed of W-View optics and HiSCA fast scan cooled CCD camera (Hamamatsu). The optics separates an image into two images based on spectrum information with dichroic mirror, and projects the two images to an imaging detector side by side. Thus, it enables simultaneous recording of two different fluorescence images from CFP (FRET donor) and YFP (acceptor) of cameleon. We expressed cameleon (YC2.1, ref. 5) specifically in AFD thermo-sensing neuron with gcy-8 promoter (a gift from B. Wedel and D. Garbers). The fluorescence intensities of CFP and YFP were reasonably strong, and we are trying to detect ratio change (YFP/CFP) during temperature change. We will report our strategy, results, and current problems. We hope this will help those who also want to apply the system to other aspects of the worm biology. References: 1. Miyawaki et al., Nature 388 (1997). 2. Kerr et al., 1999 IWM. 3. Centonzel et al., 1999 IWM. 4. Yu and Bargmann, 1999 IWM. 5. Miyawaki et al., PNAS 96 (1999).

Kotaro Kimura et al. (2003) International Worm Meeting "Transient response in AFD thermosensory neuron by rapid temperature change."

Ikue Mori, Kotaro Kimura

[International Worm Meeting, 2003]

Little is known about how environmental signals are received, integrated and assessed in intact neuronal network to regulate behavior and/or adaptation of animals. To understand how actual signals are conveyed in the network, we are interested in monitoring neuronal activity of C. elegans with calcium imaging. The thermosensory circuit is of particular interest, because it is one of a few circuits whose sensory and interneurons are identified and little information is available as to how activity of AFD thermosensory neuron is regulated by temperature. Cameleon, a GFP-based calcium sensor, can substitute calcium concentration into fluorescence ratio between CFP and YFP in the macromolecule via flourescence resonance energy transfer (Ref. 1). Using cameleon, calcium imaging of worm touch and ASH sensory neurons have been reported by Schafer lab and other groups in recent worm meetings. To begin our analysis, we made an attempt to apply camelon system on calcium imaging of the thermosensory circuit. We found transient cameleon response associated with rapid temperature change in AFD thermosensory neuron. The fluorescence ratio in AFD cell body increases transiently when the temperature is increased from 15 deg to 25 deg within 20 seconds, and the ratio decreases when the temperature is decreased from 25 deg to 15 deg within same period. Magnitude of the ratio change during the temperature increase is larger than that of the decrease. However, the ratios at 15, 20 or 25 deg are at similar level when the worms have been kept at each temperature for minutes. These results suggest that activity of AFD may be dependent on temperature change and not on net temperature. We are currently working on obtaining a proof that the ratio change is due to calcium change specific to AFD. We thank Atsushi Miyawaki (RIKEN) for YC2.12 and Takeshi Ishihara (NIG) for AFD-specific promoter. Ref. 1: Miyawaki et al., Nature 388, 882 (1997)

K Kimura et al. (1999) International C. elegans Meeting "An approach toward imaging activities of the neural network"

I Mori, K Matsumoto, K Kimura

[International C. elegans Meeting, 1999]

All neural connections have been revealed through the extensive EM analysis 1 , which should be the structural basis of all neural functions. Further, as demonstrated for the touch response and thermotaxis behavior, laser ablation studies in some cases lead to identify the neural circuit consisting of a set of neurons, which is required for the particular behavior 2, 3 . We still do not know, however, how actual neural signals are conveyed in the network. For example, it remains to be elucidated as to how different sensory signals are transmitted in the network to generate appropriate behaviors. To address these questions, we aim to monitor the activities of the neural network using an optical method. The optical method allows monitoring multipoint simultaneously, and it is not destructive. Among optically detectable changes shown to associate with neuronal excitation, the calcium change is one of the best studied because of its massive magnitude, relative to other changes. Already, cameleon, a GFP-based calcium sensor 4 , has been successfully used to monitor calcium transients associated with pharyngeal pumping 5 . We are currently setting up an optical system for cameleon to monitor calcium changes in neurons in the thermotaxis circuit. In the neural model for thermotaxis, the counterbalancing regulation by the two downstream interneurons was proposed to be critical for the migration to a preferred temperature 3 . It is still unknown, however, whether signals transmitted between the connected neurons in the circuit are excitatory or inhibitory. Recently, we have shown that thermotaxis can be used to study memory and learning (see the abstract by Mohri et al). What kind of changes is occurring in the network during learning? With the availability of the neural model, we believe that the thermotaxis circuit provides an ideal simple network to apply the cameleon-based imaging. Through this analysis, we hope not only to understand the actual neural regulation of thermotaxis, but also to help develop a system for imaging neuronal activities of the C. elegans nervous system as a whole. We thank Dr. Miyawaki for cameleon constructs. 1. CGC Bibliography #938, 2. CGC #765, 3. CGC #2214, 4. Miyawaki et al., Nature 388 (1997), 5. Kerr & Schafer, '98 WCWM

Kimura KD et al. (2004) Curr Biol "The C. elegans thermosensory neuron AFD responds to warming."

Kimura KD, Miyawaki A, Mori I, Matsumoto K

[Curr Biol, 2004]

The mechanism of temperature sensation is far less understood than the sensory response to other environmental stimuli such as light, odor, and taste. Thermotaxis behavior in C. elegans requires the ability to discriminate temperature differences as small as similar to0.05degreesC and to memorize the previously cultivated temperature [1, 2]. The AFD neuron is the only major thermosensory neuron required for the thermotaxis behavior [3]. Genetic analyses have revealed several signal transduction molecules that are required for the sensation and/or memory of temperature information in the AFD neuron [4-7], but its physiological properties, such as its ability to sense absolute temperature or temperature change, have been unclear. We show here that the AFD neuron responds to warming. Calcium concentration in the cell body of AFD neuron is increased transiently in response to warming, but not to absolute temperature or to cooling. The transient response requires the activity of the TAX-4 cGMP-gated cation channel, which plays an essential role in the function of the AFD neuron [5]. Interestingly, the AFD neuron further responds to step-like warming above a threshold that is set by temperature memory. We suggest that C. elegans provides an ideal model to genetically and physiologically reveal the molecular mechanism for sensation and memory of temperature information.

Shimozono S et al. (2004) EMBO Rep "Slow Ca2+ dynamics in pharyngeal muscles in Caenorhabditis elegans during fast pumping."

Miyawaki A, Kirino Y, Mori I, Shimozono S, Fukano T, Kimura KD

[EMBO Rep, 2004]

The pharyngeal muscles of Caenorhabditis elegans are composed of the corpus, isthmus and terminal bulb from anterior to posterior. These components are excited in a coordinated fashion to facilitate proper feeding through pumping and peristalsis. We analysed the spatiotemporal pattern of intracellular calcium dynamics in the pharyngeal muscles during feeding. We used a new ratiometric fluorescent calcium indicator and a new optical system that allows simultaneous illumination and detection at any two wavelengths. Pumping was observed with fast, repetitive and synchronous spikes in calcium concentrations in the corpus and terminal bulb, indicative of electrical coupling throughout the muscles. The posterior isthmus, however, responded to only one out of several pumping spikes to produce broad calcium transients, leading to peristalsis, the slow and gradual motion needed for efficient swallows. The excitation-calcium coupling may be uniquely modulated in this region at the level of calcium channels on the plasma membrane.

M Dekkers et al. (2004) European Worm Meeting "IN VIVO IMAGING OF GUSTATORY SIGNALLING IN C. ELEGANS"

M Dekkers, G Jansen

[European Worm Meeting, 2004]

We are interested in the molecular and cellular mechanisms that govern gustatory plasticity. Using behavioural assays, we have identified many signalling molecules that are involved in this behaviour. In addition, we have identified several pairs of sensory neurons that mediate gustatory plasticity: the ASE neurons provide an attractive signal in response to NaCl, which is balanced by inhibitory inputs from the ASI neurons and the nociceptive neurons ASH and ADL. In a parallel approach we use genetically encoded probes to image the responses of single cells upon stimulation. We drive expression of these imaging constructs in the cells mentioned above using cell-specific promoters. The primary reporter construct we use is the Cameleon construct (Miyawaki et al 1999), which uses Fluorescence Resonance Energy Transfer (FRET) to visualise changes in the calcium levels in the cytosol. We have optimised imaging using an sra-6-prom::Cameleon line (kindly provided by the Schafer lab, with feedback), which is expressed in ASH. Besides the Cameleon construct we are also testing a construct that is a measure for PIP2 hydrolysis in the membrane (van der Wal et al 2001) and a pH sensitive derivate of GFP, pHluorin (Miesenbck et al.1998), as a reporter for vesicle release in the synaptic cleft. Together with the data from the behavioural assays, this will allow us to further dissect the intercellular and intracellular signalling routes leading to gustatory behaviour.

Kerr RA et al. (1998) West Coast Worm Meeting "IMAGING CALCIUM IN EXCITABLE CELLS IN C. ELEGANS"

Schafer WR, Tsien RY, Kerr RA

[West Coast Worm Meeting, 1998]

Electrophysiology in C. elegans has traditionally been very difficult. One way of circumventing the problems with conventional electrode recording is imaging genetically encoded fluorescent probes. Cameleons are genetically-encoded ratiometric fluorescent calcium sensors, based on calmodulin and GFP [Miyawaki et al, Nature 388:882-887]. By using calcium levels as an indirect indicator of electrical activity, we can thus investigate the behavior of exitable cells. We have imaged calcium transients in a subset of excitable cells in C. elegans. We have used the myo-2 promoter to express cameleon in the pharyngeal muscle of C. elegans and imaged using two-photon confocal microscopy. Worms in which pumping has been stimulated by application of serotonin show a strong correlation between visible contraction and the cameleon-reported calcium transients. When pumping is inhibited by application of ivermectin, neither visible pumping nor calcium transients are observed. Ratio changes corresponding to contractions are approximately threefold greater than background noise under the current setup. Cameleon is thus a viable sensor for pharyngeal muscle activity. We are currently in the process of imaging smaller excitable cells including neurons (with cameleon under the control of the pan-neuronal promoter unc-119). Target regions of interest include HSNs, which control egg-laying, the ventral nerve cord, and other neurons that generate rhythmic behavior. Current progress on imaging calcium changes in neurons will be described.

Yosuke Miyanishi et al. (2010) East Asia Worm Meeting "Functional imaging of neurons for 2-nonanone avoidance"

Yosuke Miyanishi, Kotaro Kimura

[East Asia Worm Meeting, 2010]

The enhancement of sensory responses after prior exposure to a stimulus is a fundamental mechanism of neural function in animals. Its molecular basis, however, has not been studied in as much depth as the reduction of sensory responses, such as adaptation or habituation. We have found that the avoidance behavior of C. elegans in response to a repellent odor 2-nonanone is enhanced rather than reduced after preexposure to the odors: The preexposed animals migrated farther away from the odor source than control animals did, and this enhancement effect of preexposure was maintained for at least 1 hr after the conditioning (KD Kimura, K. Fujita, I. Katsura, in revision). Genetic and pharmacological analyses revealed that the enhancement of 2-nonanone avoidance requires dopamine signaling via D2-like dopamine receptor dop-3, suggesting a new form of sensory response enhancement that is regulated by dopamine signaling in the animals.To understand neural basis for the enhancement 2-nonanone avoidance, we are setting up a ratiometric imaging system for monitoring calcium or voltage changes in the neurons using cameleon (Miyawaki et al., 1997) or Mermaid (Tsutsui et al., 2008). For the delivery of 2-nonanone during the imaging, we are planning to use the microfluidic chamber for gas stimuli in collaboration with the de Bono lab (Persson et al., 2009). We thank Drs. E. Busch and M. de Bono (MRC) for the chamber and M. Tomioka and Y. Iino (U. Tokyo) for suggestions on microfluidics for liquids.

CI Bargmann (1999) International C. elegans Meeting "Imaging of neuronal activity in olfactory circuits in C. elegans"

CI Bargmann

[International C. elegans Meeting, 1999]

timyu@cgl.ucsf.edu We are interested in studying the neural circuits underlying chemotaxis behavior in C. elegans . To this end, we are attempting to image neuronal activity in intact worms using cameleons -- fluorescent indicators, based on spectral variants of GFP and calmodulin, that allow ratiometric imaging of calcium concentrations (Miyawaki et al, Nature 388 882). To work out the best conditions for this experiment, we have attempted to record calcium changes in body wall muscle during locomotion. Under control of the myo-3 promoter, yellow cameleon-2 is abundantly expressed in body wall muscle. Dual-emission wavelength imaging of myo-3::ycam2 worms during sinusoidal locomotion reveals ratio ( F535/F480 ) increases in body wall muscle preceding muscle contraction, consistent with calcium increases during excitation-contraction coupling. Initial attempts to express yellow cameleon-2 in chemosensory neurons resulted in low levels of expression, even when injected at high concentrations. We therefore engineered worm-optimized yellow cameleons by interrupting cameleon coding sequences with artificial C. elegans introns. Addition of five introns to yellow cameleon resulted in a robust increase in expression as assayed under the mec-7 promoter. Currently, we are expressing our modified yellow cameleon-2 in AWA, AWB and AWC, chemosensory neurons that have been shown to mediate attraction or repulsion from a variety of volatile odorants. The sensory transduction channels in these neurons are predicted to allow calcium entry after odorant stimulation. Progress on recording calcium changes in response to odorant application will be described.

Yu TW et al. (2000) West Coast Worm Meeting "Characterizing the Neural Circuitry of Chemotaxis to Volatile Odorants"

Gallegos ME, Gray J, Yu TW, Bargmann CI

[West Coast Worm Meeting, 2000]

The C. elegans chemotaxis circuit offers the possibility of applying genetics to understand a functionally complex neural circuit. Further characterization of this circuit, as well as an in vivo physiological assay, would help relate new results to knowledge of circuitry in other systems. Anatomically, the chemotaxis circuit can be defined as having three main layers: sensory neurons, interneurons, and motorneurons. The sensory input to the chemotaxis circuit is relatively well-understood. As a first step in characterizing the remainder of the circuit, we are using laser ablations to identify functionally important interneurons, motorneurons, and muscles. We are also developing new behavioral assays to more specifically control chemotaxis circuit input and more directly assay circuit output. By flowing odorants over an airtight plate, we can create a temporal gradient of volatile odorant. It has recently been shown that ASE-mediated (water-soluble) chemotaxis is accomplished by modulating the frequency of pirouettes (i.e., omega bends and reversals) (3). Using a video camera to record worm movement during odorant application and subsequently counting omega bends and reversals, we have begun to characterize the kinetics of the pirouette response to volatile odorants. Finally, we will apply imaging techniques to assay physiology. We have constructed a myo3::cameleon (1) that expresses in body wall muscles. We will use myo3::cameleon worms to identify muscles important for omega bends and will follow up by ablating the corresponding muscles and motorneurons. We hope to extend this analysis to neurons in order to characterize circuit activity during olfactory stimulation and adaptation. We are also exploring other imaging technologies. 1. Miyawaki et al. (1997) Nature. 388, 882-887. 2. Pierce-Shimomura et al. (1999) J Neurosci. 19(21), 9557-69.