Sox10-PC::G5-tdT transgenic mice are an effective model to study glial [Ca2+]i responses specifically in the enteric nervous system. A: model of our experimental paradigm comparing Fluo-4 and GCaMP5G imaging in the colonic myenteric plexus. Note that Fluo-4 loads both enteric neurons and glia, whereas GCaMP5G expression is isolated to enteric glia in our genetic model. B: representative epifluorescence images showing the specific expression of tdT (red, left) in S100β-immunoreactive enteric glia (grayscale, middle) in the colonic myenteric plexus of Sox10-PC::G5-tdT mice (overlay, right). C: representative still images from a Ca2+ imaging experiment showing GCaMP5G fluorescence (grayscale, middle 2) in enteric glia (identified by tdT fluorescence; red, left) in a myenteric ganglion from a Sox10-PC::G5-tdT mouse. GCaMP5G fluorescence is low at rest (baseline) and increases robustly when glial [Ca2+]i responses are stimulated by ADP (100 μM; peak). Note that [Ca2+]i responses are limited to tdT-positive glial cells (overlay, right). D: representative traces of [Ca2+]i levels in enteric glia (black traces) within a myenteric ganglion (averaged response of all glia within ganglion, overlaid in green) from Sox10-PC::G5-tdT mice exposed to an ADP dose-response curve (10 µM, 100 µM, and 1 mM, subsequently). ΔF/F, change in fluorescence. E: representative traces comparing the mean [Ca2+]i responses of myenteric glia with ADP recorded using GCaMP5G fluorescence of n = 20 glia from a single myenteric ganglion from Sox10-PC::G5-tdT mice (magenta) or traditional Fluo-4 loading of n = 17 glia from a single myenteric ganglion from background control animals (green). F: GCaMP5G fluorescence reports an average peak [Ca2+]i response that is comparable with those reported by Fluo-4. Peak responses are the average of all glia within a myenteric ganglion exposed to ADP of n = 6–7 ganglia from at least 3 mice. Scale bars = 10 μM.
The effect of fluoroacetate (FA) on averaged ganglionic glial [Ca2+]i responses triggered by ADP in Sox10-PC::G5-tdT mice. A: representative traces showing the average [Ca2+]i response of all glia within a myenteric ganglion driven by ADP (100 μM) in control samples (magenta) or samples exposed to FA (5 mM, blue). The control trace is the averaged response of n = 20 glia within a single myenteric ganglion, and the FA trace is the averaged response of n = 26 glia within a single myenteric ganglion. B: quantification of the effect of FA on peak glial [Ca2+]i responses driven by ADP. Data are expressed as an averaged response of all glia within a myenteric ganglion (averaged ganglionic response) of n = 9 ganglia from at least 5 mice.
The effect of fluoroacetate (FA) on [Ca2+]i responses in individual enteric glial cells triggered by ADP in Sox10-PC::G5-tdT mice. A: representative traces showing [Ca2+]i responses in enteric glial cells within a myenteric ganglion that were evoked by exposure to ADP (green-shaded areas). Magenta traces show the responses of glial cells in a control ganglion (left); blue traces show the responses of glial cells in a ganglion exposed to 5 mM FA (right). Each trace represents the responses of 1 glial cell. B–F: quantification of the effects of FA on the percentage of glia [tdT-positive cells (tdT+)] responding to ADP (B), the peak [Ca2+]i response of cells still exhibiting a response (C), the half time to peak of glial [Ca2+]i responses (D), the full width at half max (FWHM; E), and the activation profile of glial [Ca2+]i responses (F). Data are representative of recordings in n = 90 and 160 glial cells (FA and control, respectively) from at least 5 mice. *P < 0.05, **P < 0.01, ****P < 0.0001, Student's t-test.
The effect of fluoroacetate (FA) on hemichannel-dependent dye uptake by myenteric glia in the mouse colon. A and B: representative epifluorescence images showing ethidium bromide (EtBr) fluorescence in whole-mount preparations of the myenteric plexus from the mouse colon exposed to buffer (A) or ADP (B). Glial cells within myenteric ganglia (outlined by dashed lines) normally display a low amount of dye uptake (A) that increases robustly when stimulated with ADP (B). C: quantification of the effects of FA, the connexin-43 mimetic peptide 43Gap26, the Ca2+ chelator EGTA, and ADP on mean glial cell EtBr fluorescence in whole-mount preparations of the mouse myenteric plexus. Scale bar (B) = 10 μm and applies to A and B. Measurements are representative of n = 225 glia to 373 glial cells from at least 3 mice. ****P < 0.0001 compared with buffer, #P < 0.05 compared with ADP, ANOVA.
The effects of fluoroacetate (FA) on ganglionic cell density and glial expression of key proteins. A–C and A′–C′: representative epifluorescence images of S100β immunoreactivity (green, A and A′), glial fibrillary acidic protein (GFAP) immunoreactivity (magenta, B and B′), and overlay (C and C′) in myenteric ganglia from control whole mounts (A, B, and C) and whole mounts exposed to FA (A′, B′, and C′). Note that S100β immunoreactivity decreases in glia exposed to FA (green, A′) and that GFAP immunoreactivity increases in glia exposed to FA (magenta, B′). D: quantification of ganglionic S100β, GFAP, and connexin-43 (Cx43) immunoreactivity, expressed as the fluorescence density [arbitrary fluorescence units per square micrometer (au μm−2)], and the packing density of myenteric glial and neurons [number of S100β+ glia and HuC/D+ neurons per square millimeter (mm−2)] expressed as the percentage of buffer control levels. Scale bar (C′) = 10 μm and applies to A–C and A′–C′. E: quantification of glial cells coexpressing S100β and GFAP. Measurements were obtained by sampling ganglia from n = 4 animals. *P < 0.05, Student's t-test.
Cover: Electrophysiological and morphological measurements were obtained simultaneously from a single corticospinal neuron. These data served as constraints on evolutionary optimization, generating a family of corticospinal models. A three-dimensional reconstruction serves as the backbone for a pseudo-color visualization of synaptic efficiency as a function of dendritic location, simulated in a single biophysical model selected from the family of optimal individuals. Excitatory synapses at yellow dendritic locations resulted in the largest depolarizations at the soma, while the same synaptic activation at purple locations generated only weak somatic depolarizations. This visualization is surrounded by scatter plots representing the evolutionary optimization: biophysical models optimized across different fitness functions demonstrate tradeoffs between full high-dimensional error (y-axis) and individual error scores (individual x-axes; clockwise order from top
left: subthreshold error, instantaneous firing rate error, spike-shape error, average firing rate error). Color based on 5 error percentiles in increasing instantaneous firing-rate error (purple, red, dark orange, light orange, yellow). From Neymotin SA, Suter BA, Dura-Bernal S, Shepherd GMG, Migliore M, Lytton WW. Optimizing computer models of corticospinal neurons to replicate in vitro dynamics. J Neurophysiol; doi:10.1152/jn.00570.2016.