Investigation of Pain Mechanisms by Calcium Imaging Approaches

Abstract

Due to the complex circuitry and plethora of cell types involved in somatosensation, it is becoming increasingly important to be able to observe cellular activity at the population level. In addition, since cells rely on an intricate variety of extracellular factors, it is important to strive to maintain the physiological environment. Many electrophysiological techniques require the implementation of artificially-produced physiological environments and it can be difficult to assess the activity of many cells simultaneously. Moreover, imaging Ca²⁺ transients using Ca²⁺-sensitive dyes often requires in vitro preparations or in vivo injections, which can lead to variable expression levels. With the development of more sensitive genetically-encoded Ca²⁺ indicators (GECIs) it is now possible to observe changes in Ca²⁺ transients in large populations of cells at the same time. Recently, groups have used a GECI called GCaMP to address fundamental questions in somatosensation. Researchers can now induce GCaMP expression in the mouse genome using viral or gene knock-in approaches and observe the activity of populations of cells in the pain pathway such as dorsal root ganglia (DRG), spinal neurons, or glia. This approach can be used in vivo and thus maintains the organism's biological integrity. The implementation of GCaMP imaging has led to many advances in our understanding of somatosensation. Here, we review the current findings in pain research using GCaMP imaging as well as discussing potential methodological considerations.

Keywords: DRG; GCaMP imaging; Neural circuit; Pain pathways; Spinal cord.

Fig. 1

Representative in vivo DRG images from a Pirt-cre;Rosa26-flox-stop-flox-GCaMP6s heterozygous mouse. A Background GCaMP fluorescence in the absence of stimulation to the hind paw. Arrows indicate spontaneously firing neurons. B ROIs manually traced for neurons from panels C and D responding to noxious heat (red), brush (green), or both heat and brush (yellow). C Representative Ca²⁺ transient in response to placing the ipsilateral hindpaw in a beaker of 48 °C water. Cell diameters of responding neurons range from 11.3 to 33.1 μm. Mean cell diameter = 21.0 ± 0.3 μm. D Representative Ca²⁺ response to applying gentle brushing to the dorsal aspect of the ipsilateral hind paw. Cell diameters of responding neurons range from 18.7 to 44.6 μm. Mean cell diameter = 29.8 ± 1.1 μm.