With its compact nervous system of only 302 neurons and 8,000 synaptic connections, the nematode worm Caenorhabditis elegans is the only animal for which a complete nervous system has been anatomically mapped. Moreover, despite the small size, it can produce a rich repertoire of simple and more complex behaviours and it is a powerful experimental model organism to study animal development and neurobiology. It therefore represents an excellent platform to solve one of the central challenges of neuroscience: uncover how the nervous system extracts information from the environment and generate behaviour in response to external changes.
A high-resolution imaging technique able to efficiently achieve unbiased, fast volumetric imaging of neurons together with the use of transgenic animals expressing pan-neuronal, genetically encoded calcium indicators, and microfluidics, allows to combine whole brain imaging with complex sensory stimulation patterns, from chemical stimuli to temperature or oxygen changes.
Here we show transgenic nematodes expressing a pan-neuronal, genetically encoded fluorescent Ca2+ indicator confined in a microfluidic device. Fluorescent signals derive from nuclear GCaMP5K (NLS-GCaMP5K) that is present in the nuclei and absent from the neuropil. In order to paralyze muscles and suppress motion, the nematodes were previously exposed to 1 mM levamisole, an acetylcholine receptor–specific agonist. After setting-up this condition, we were able to monitor the neuronal activity under the desired experimental condition (e.g. exposure to specific chemical stimuli). In particular, the time-lapse imaging was performed with X-Light V3 Confocal Spinning Disk for one hour with a time frame of 5 seconds.
X-light V3 acquisition
(NIKON Plan Apo Lambda 20X air, 0.75 NA)
3D volume reconstruction
Regarding the microfluidic device we used, it is a revised version of a previous design by Albrecht and Bargmann (High-content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments; Nat Methods 8(7):599–605, 2011) and was fabricated using classical soft-lithography techniques. A mold of the microchannels network was obtained by spin coating 50 μm thick layers of SU-8 3050 photoresist on a glass substrate and using a traditional optical lithography to develop the channel pattern. A single high-resolution Glass-mask were used and the microfluidic network had a uniform height of 50 μm. After development in SU-8 developer, the master was treated with a thermal high temperature process. A mixture of 10:1 polydimethylsiloxane (PDMS) was used in a casting process on the SU-8 mold and cured at 80 °C for one hour. Layer of PDMS were cut, access wells punched using a gauge needle, and PDMS unit was bonded to a glass substrate via oxygen plasma treatment.
In conclusion, combination of high-quality microscopy, microfluidic and genetic techniques make possible to reveal the neural basis of brain computations and decision-making by establishing the fundamental relationships between anatomical connectivity and function.