Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

Abstract

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 µm and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Homeostatic feedback modulates the development of two-state patterned activity in a model serotonin motor circuit inCaenorhabditis elegans

“…Ratiometric Ca 2+ recordings were performed on freely behaving animals mounted between a glass coverslip and chunk of NGM agar as previously described (Collins and Koelle, 2013; Li et al, 2013; Collins et al, 2016; Ravi et al, 2018). Recordings were collected on an inverted Leica TCS SP5 confocal microscope using the 8 kHz resonant scanner at ~20 fps at 256×256 pixel resolution, 12-bit depth and ≥2X digital zoom using a 20x Apochromat objective (0.7 NA) with the pinhole opened to ~20 μm.…”

“…Ratiometric analysis (GCaMP5:mCherry) for all Ca 2+ recordings was performed after background subtraction using Volocity 6.3.1 as described (Collins et al, 2016; Ravi et al, 2018). The egg-laying active state was operationally defined as the period one minute prior to the first egg-laying event and ending one minute after the last (in the case of a typical active phase where 3-4 eggs are laid in quick succession).…”

The sex-specific VC neurons are mechanically activated motor neurons that facilitate serotonin-induced egg laying inC. elegans

“…Ratiometric Ca 2+ imaging of the vulval muscles and VC neurons in freely behaving animals was 730 performed as previously described methods (Ravi, Nassar, et al, 2018). Late L4 hermaphrodites were staged and then imaged h later by being moved to an NGM agar chunk and a glass coverslip being placed over.…”

“…Colibri.2 470 nm and 590 nm LEDs were used to coexcite GCaMP5 and mCherry fluorescence which was captured at 20 Hz onto a Hamamatsu ORCA Flash-4.0V2 sCMOS camera after channel separation using a Gemini image splitter. A custom script in Bonsai was used to measure stage position at each frame of the recording which was added to centroid position of the fluorescence object to measure animal speed in Ca 2+ imaging experiments (Lopes et al, 2015;Ravi, Nassar, et al, 2018). Each animal was recorded until it entered an active egg-laying phase (up to 1 h), after which a 10-minute segment centered around the onset of egg laying was extracted from the full recoding for analysis.…”

“…Each animal was recorded until it entered an active egg-laying phase (up to 1 h), after which a 10-minute segment centered around the onset of egg laying was extracted from the full recoding for analysis. Two-channel fluorescence (GCaMP5/mCherry) image sequences were processed and analyzed in Fiji (Schindelin et al, 2012), Volocity (PerkinElmer), and a custom script for MATLAB (MathWorks) as previously described (Ravi, Nassar, et al, 2018).…”