Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans.

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.

Tc-99m red blood cell bleeding scan in a pregnant woman presenting with hematemesis: a brief review of indications and guidelines for radionuclide scans during pregnancy.

A 6-month pregnant 35-year-old woman with hematemesis and dark blood in her stool was evaluated for location of an active gastrointestinal bleeding site. The findings from 2 esophagogastroduodenoscopies and a colonoscopy were negative. The patient was referred for a Tc-99m red blood cell scintigraphy, for location of an active bleeding site. As the patient was 6 months pregnant, the necessity of the case was discussed with the patient's referring physician and after determining that this study was necessary, the risk of radiation exposure to the fetus was discussed with the radiation safety officer and the patient. The procedure was performed after the patient signed informed consent regarding the considerations of radiation safety to the fetus. The Tc-99m red blood cell bleeding scan clearly shows activity in the fetus in addition to a large arteriovenous malformation in the patient. The visualized fetus prompted this report to discuss radiation risks concerns and a brief review of indications and guidelines for performing nuclear medicine procedures in pregnant patients.