The acute inhibition of enteric glial metabolism with fluoroacetate alters calcium signaling, hemichannel function, and the expression of key proteins

Abstract

Glia play key roles in the regulation of neurotransmission in the nervous system. Fluoroacetate (FA) is a metabolic poison widely used to study glial functions by disrupting the tricarboxylic acid cycle enzyme aconitase. Despite the widespread use of FA, the effects of FA on essential glial functions such as calcium (Ca²⁺) signaling and hemichannel function remain unknown. Therefore, our goal was to assess specifically the impact of FA on essential glial cell functions that are involved with neurotransmission in the enteric nervous system. To this end, we generated a new optogenetic mouse model to study specifically the effects of FA on enteric glial Ca²⁺ signaling by crossing PC::G5-tdTomato mice with Sox10::creER^T2 mice. FA did not change the peak glial Ca²⁺ response when averaged across all glia within a ganglion. However, FA decreased the percent of responding glia by 30% (P < 0.05) and increased the peak Ca²⁺ response of the glial cells that still exhibited a response by 26% (P < 0.01). Disruption of Ca²⁺ signaling with FA impaired the activity-dependent uptake of ethidium bromide through connexin-43 (Cx43) hemichannels (P < 0.05) but did not affect baseline Cx43-dependent dye uptake. FA did not cause overt glial or neurodegeneration, but glial cells significantly increased glial fibrillary acid protein by 56% (P < 0.05) following treatment with FA. Together, these data show that the acute impairment of glial metabolism with FA causes key changes in glial functions associated with their roles in neurotransmission and phenotypic changes indicative of reactive gliosis.

New & noteworthy: Our study shows that the acute impairment of enteric glial metabolism with fluoroacetate (FA) alters specific glial functions that are associated with the modification of neurotransmission in the gut. These include subtle changes to glial agonist-evoked calcium signaling, the subsequent disruption of connexin-43 hemichannels, and changes in protein expression that are consistent with a transition to reactive glia. These changes in glial function offer a mechanistic explanation for the effects of FA on peripheral neuronal networks.

Keywords: autonomic nervous system; enteric glial cells; enteric nervous system; glia; intestine.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.