Use of Genetically Encoded Calcium Indicators (GECIs) Combined with Advanced Motion Tracking Techniques to Examine the Behavior of Neurons and Glia in the Enteric Nervous System of the Intact Murine Colon

Abstract

Genetically encoded Ca2+ indicators (GECIs) have been used extensively in many body systems to detect Ca2+ transients associated with neuronal activity. Their adoption in enteric neurobiology has been slower, although they offer many advantages in terms of selectivity, signal-to-noise and non-invasiveness. Our aims were to utilize a number of cell-specific promoters to express the Ca2+ indicator GCaMP3 in different classes of neurons and glia to determine their effectiveness in measuring activity in enteric neural networks during colonic motor behaviors. We bred several GCaMP3 mice: (1) Wnt1-GCaMP3, all enteric neurons and glia; (2) GFAP-GCaMP3, enteric glia; (3) nNOS-GaMP3, enteric nitrergic neurons; and (4) ChAT-GCaMP3, enteric cholinergic neurons. These mice allowed us to study the behavior of the enteric neurons in the intact colon maintained at a physiological temperature, especially during the colonic migrating motor complex (CMMC), using low power Ca2+ imaging. In this preliminary study, we observed neuronal and glial cell Ca2+ transients in specific cells in both the myenteric and submucous plexus in all of the transgenic mice variants. The number of cells that could be simultaneously imaged at low power (100–1000 active cells) through the undissected gut required advanced motion tracking and analysis routines. The pattern of Ca2+ transients in myenteric neurons showed significant differences in response to spontaneous, oral or anal stimulation. Brief anal elongation or mucosal stimulation, which evokes a CMMC, were the most effective stimuli and elicited a powerful synchronized and prolonged burst of Ca2+ transients in many myenteric neurons, especially when compared with the same neurons during a spontaneous CMMC. In contrast, oral elongation, which normally inhibits CMMCs, appeared to suppress Ca2+ transients in some of the neurons active during a spontaneous or an anally evoked CMMC. The activity in glial networks appeared to follow neural activity but continued long after neural activity had waned. With these new tools an unprecedented level of detail can be recorded from the enteric nervous system (ENS) with minimal manipulation of tissue. These techniques can be extended in order to better understand the roles of particular enteric neurons and glia during normal and disordered motility.

Structurally defined signaling in neuro‐glia units in the enteric nervous system

“…In addition, electrical stimulation of enteric neurons resulted in prompt GCaMP3 rises in enteric glial cells within myenteric ganglia. These results are in line with what we observed using Wnt1|GCaMP3 mice(Fung et al, 2017) and with the Ca 2+ indicator dye Fluo-4(Boesmans, Martens, et al, 2013) and are comparable to other studies using GCaMP3(Hennig et al, 2015) and GCaMP5G-tdT (McClain & Gulbransen, 2017). Notably, the differences with other studies in terms of expression pattern and response kinetics could be due to the…”

A Novel Striated Muscle-Specific Myosin-Blocking Drug for the Study of Neuromuscular Physiology

“…The main type was the standard deviation map, which was generated by calculating the standard deviation of fluorescence intensity at every pixel immediately prior to the application of the stimulus (0.5–1.0 s) extending to 30–50 s depending on the stimulus used. Standard deviations were color coded (“Fire” CLUT) to more accurately portray the relative intensity of the signal in muscle cells in relation to background noise (Hennig et al, 2015). To better appreciate the heterogeneity of muscle fiber responses during different stimulation paradigms, ST maps were colored and overlaid and summary images were created using different statistical procedures including average intensity and the standard deviation of intensity (e.g., Figures 6B,C ).…”

Activity-induced Ca2+signaling in perisynaptic Schwann cells is mediated by P2Y1receptors and regulates muscle fatigue

“…Image sequences were recorded at 25 frames per second, and were exported as 8-bit TIFF files into custom-written software (Volumetry G8d). A Gauss filter (3x3 pixel, sd = 1.0) was applied to reduce camera noise, and motion-correction routines were used to stabilize neural and Schwann cell elements in the movie (see Hennig et al, 2015). Changes in background fluorescence were stabilized by subtracting the average intensity near the main phrenic nerve branch.…”