4.4 Article

Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

Journal

JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
Volume -, Issue 132, Pages -

Publisher

JOURNAL OF VISUALIZED EXPERIMENTS
DOI: 10.3791/56911

Keywords

Neuroscience; Issue 132; Caenorhabditis elegans; calcium; behavior; fluorescence; imaging; Ca2+reporters

Funding

  1. NINDS [R01 NS086932]
  2. NIGMS IMSD program [R25 GM076419]
  3. NIH Office of Research Infrastructure Programs [P40 OD010440]

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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 similar to 1 mu m 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.

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