Simultaneous Voltage and Calcium Imaging from Primary Sensory Neurons in Live Mice
Abstract
Pain is a very common symptom that affects more military personnel and Veterans than the general population. Available treatments often provide inadequate pain control and unfavorable side effects. Therefore, it is important to develop a clearer understanding of the pain mechanisms in order to develop better therapeutics. A clear understanding of pain mechanisms requires powerful tools to reveal the pain generation/exacerbation signals from pain-sensing neurons. In the study proposed here, mice will be engineered to produce two kinds of fluorescent signals: red fluorescence from a red-glowing protein called RedCaMP, which is a calcium indicator, and green fluorescence from a green-glowing protein called ASAP4, which is a voltage indicator. We will perform the surgery on the back of mice having both RedCaMP and ASAP4 indicators. Dorsal root ganglia, also called DRG, where pain-sensing neurons are located will be exposed for imaging experiments. In this experiment, a beam of laser light will be shined on the surface of DRG neuron to let RedCaMP and ASAP emit red and green fluorescence, then we will be able to capture both color fluorescence from the pain-sensory neuron at the same time by an advanced microscope, called confocal microscope, which can capture images at very fast scanning rate (1 kHz, 1 ms per image). The green fluorescence intensity represents potential or voltage levels in the neuronal membrane, and the red fluorescence intensity indicates calcium levels in the neuronal cytosol. Both voltage and calcium are very important indicators for neuronal activity. As the neuronal membrane potential becomes more positive or depolarized, or intracellular calcium levels becomes higher, the neuron turns into an excitable or activated state. By visualizing the real-time fluorescence changes from both voltage and calcium indicators, we are able to know the status of pain-sensing neurons at any given time under physiological and pathological conditions. For example, if we gently press the hindpaw of a normal mouse, we may see a small and transient increase in fluorescence in pain-sensing neurons, suggesting neurons are generating non-painful signals, but if we give the same press on the injured hindpaw, we may see a strong and long-lasting increase in fluorescence in those neurons, suggesting neurons are generating painful signals for brain perception. Due to the fast voltage changes in the neuronal membrane, which normally occurred in the millisecond range, previous investigations could only use calcium indicator, which acts much slower to infer neuronal activity. However, calcium indicator imaging has many misleading aspects as calcium is a second messenger and has inherently slow response kinetics; it could not truly reflect the fast and dynamic voltage changes in the neuronal membrane. This project aims to develop an innovative approach to track both voltage and calcium changes in pain-sensing neurons of live mice. Successful completion of the proposed research will help us to understand a missing link between voltage and calcium signals in the peripheral nervous system. The new discoveries made with this novel approach will strengthen the interpretation of calcium imaging studies that have been widely and routinely used in many laboratories around the world. Most importantly, our study will allow us to uncover the cellular mechanisms of pain, especially chronic pain.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210076
Entities
People
- Yan Zhang
Organizations
- United States Army
- University of Texas Health Science Center at San Antonio