Ca2+ responses in enteric glia are mediated by connexin-43 hemichannels and modulate colonic transit in mice

Abstract

Background & aims: In the enteric nervous system, neurotransmitters initiate changes in calcium (Ca(2+) responses) in glia, but it is not clear how this process affects intestinal function. We investigated whether Ca(2+)-mediated responses in enteric glia are required to maintain gastrointestinal function.

Methods: We used in situ Ca(2+) imaging to monitor glial Ca(2+) responses, which were manipulated with pharmacologic agents or via glia-specific disruption of the gene encoding connexin-43 (Cx43) (hGFAP::CreER(T2+/-)/Cx43(f/f) mice). Gastrointestinal function was assessed based on pellet output, total gut transit, colonic bead expulsion, and muscle tension recordings. Proteins were localized and quantified by immunohistochemistry, immunoblot, and reverse transcription polymerase chain reaction analyses.

Results: Ca(2+) responses in enteric glia of mice were mediated by Cx43 hemichannels. Cx43 immunoreactivity was confined to enteric glia within the myenteric plexus of the mouse colon; the Cx43 inhibitors carbenoxolone and 43Gap26 inhibited the ability of enteric glia to propagate Ca(2+) responses. In vivo attenuation of Ca(2+) responses in the enteric glial network slowed gut transit overall and delayed colonic transit--these changes are also observed during normal aging. Altered motility with increasing age was associated with reduced glial Ca(2+)-mediated responses and changes in glial expression of Cx43 messenger RNA and protein.

Conclusions: Ca(2+)-mediated responses in enteric glia regulate gastrointestinal function in mice. Altered intercellular signaling between enteric glia and neurons might contribute to motility disorders.

Keywords: ADP; ATP; Aging; CBX; Ca(2+); Cx43; EG; ENS; ER(T2); GFAP; Intestinal Nervous System; Mouse Model; Purines; adenosine diphosphate; adenosine triphosphate; calcium; carbenoxolone; connexin-43; enteric glia; enteric nervous system; glial fibrillary acidic protein; i-cKO; inducible and conditional knockout; mRNA; messenger RNA; moa; months of age; mutated estrogen receptor.

Figure 1

EG express Cx43. (A) GFAP (green) and Cx43 (magenta) immunoreactivity (ir) in the mouse colon myenteric plexus (scale bar = 20 μm). (A’–A”) Panels at right show enlarged view of boxed areas in A. Note connexin-43–ir puncta in near vicinity of (A’) of or colocalized with (A”) GFAP–ir EG (arrows). Images are representative of labeling in a minimum of 4 animals. Tamoxifen treatment induces tdTomato reporter expression in EG (red, B’) without altering Cx43–ir (green, B) in the hGFAP::Cre^ERT2+/−/tdTomato^+/− reporter mouse. (B””) Overlay of B-B”. (C) Cx43-ir (green) is diminished following tamoxifen–mediated specific glial ablation of Cx43 in hGFAP::Cre^ERT2+/−/Cx43^f/f/tdTomato^+/− mice. (C’) tdTomato reporter expression in EG (red). (C”) Overlay of C-C’. B and C are presented using the same fluorescence dynamic scale (a linear green scale ranging 100-300 fluorescence intensity units). Scale bar in C” = 10 μm and applies to B-C”.

Figure 2

Purine–evoked Ca²⁺ waves between EG depend on Cx43. (A) Model of purine–evoked Ca²⁺ responses through the EG network. (B) Representative traces of Ca²⁺ response evoked by ADP (30 s, 100 μM) in the presence or absence of the Cx43 antagonist, carbenoxolone (CBX; 50 μM; blue shaded area) in 21 EG within a myenteric ganglion (gray traces, average in green). Note that CBX limits the number of responding glia and on average, the Ca²⁺ response is lost. Glial responses recover upon washout of drug. (C) Representative Ca²⁺ response evoked by ADP in 13 EG within a myenteric ganglion (gray traces, average in green) in the presence or absence of the specific Cx43 mimetic peptide, 43Gap26 (20 μM). Effect of CBX and 43Gap26 on (D) the number of glia responding to ADP per ganglion and (E) peak glial Ca²⁺ responses (n=5-14, ***P=.0001, ANOVA).

Figure 3

EG Ca²⁺ waves evoked by enteric neuron–to–glia communication depend on Cx43. (A) Model depicting how pharmacological activation of enteric neurons initiates Ca²⁺ responses in EG. (B) Representative traces of average Ca²⁺ response in glia (green traces) and neurons (blue traces) within a myenteric ganglion following stimulation of enteric neuron–to–glia communication with the P2X7 agonist, BzATP (100 μM). Note that both neurons and glia respond in control (solid traces) but glial responses are lost when Cx43 is inhibited with CBX (50 μM; dashed traces). (C) Representative Ca²⁺ wave evoked through 12 glia within a myenteric ganglion (gray traces, average in green) by stimulating enteric neuron–glia communication with BzATP in the presence or absence of CBX.

Figure 4

Specific deletion of glial Cx43 impairs gastrointestinal transit to a similar extent as physiological aging. Whole gut and colonic transit times (A; n=6-8, median ± interquartile range, *P=.0097, Mann-Whitney U-Test), fecal pellet composition analysis (B; n=8-9, mean ± SEM, *P<0.05, Student's t-test) and isometric muscle tension recordings (summary data on left for contractions in C, relaxations in D; representative traces at right show the effect of 20 Hz (500 milliamps) EFS on contractile and relaxation responses; n=5, *P<0.05, two-way ANOVA) measured in tamoxifen–treated background (Backgr: pooled WT and Cx43^f/f) and inducible and conditional, glial–specific, Cx43 knockout (i-cKO) mice. Whole gut and colonic transit (E; n=3-8, mean ± SEM, **P=.0041, two-tailed t test and *P=.0011, Mann-Whitney U-Test, respectively) and endogenous fecal pellet output (F; n=4, mean ± SEM, P=.793, two-way ANOVA) measured in mice at from 2–12 m.o.a.

Figure 5

Purine–evoked Ca²⁺ waves between EG are lost with aging. (A) Representative Ca²⁺ response evoked by ADP in 9 EG (gray traces, average in green) within a myenteric ganglion from a 12 m.o.a. mouse. (B) Quantification of the number of neurons (blue) or glia (green) responding per myenteric ganglion to ADP in 2 (light shaded bars) and 12 m.o.a. mice (dark shaded bars; n = 6, mean ± SEM; ****P<.0001 between 2 and 12 m.o.a. glia, P=.1018 between 2 and 12 m.o.a. neurons, two-tailed t test). (C) Quantification of peak Ca²⁺ responses initiated by ADP in neurons (blue) or glia (green) in 2 (light shaded bars) and 12 (dark shaded bars) m.o.a. mice (n = 6, mean ± SEM; *P=.011 between 2 and 12 m.o.a. glia, *P=.0352 between 2 and 12 m.o.a. neurons, two-tailed t test).

Figure 6

EG Ca²⁺ waves evoked by enteric neuron–to–glia communication are lost with aging. (A) Numbers of neurons (blue) or glia (green) responding per myenteric ganglion to BzATP (100 μM) at 2 (light shaded bars) and 12 (dark shaded bars) m.o.a. (n = 6, mean ± SEM; ****P<.0001 between 2 and 12 m.o.a. glia, **P=.0068 between 2 and 12 m.o.a. neurons, two-tailed t test). (B) Peak Ca²⁺ response magnitude initiated by ADP in neurons (blue) or glia (green) at 2 (light shaded bars) and 12 (dark shaded bars) m.o.a. (n = 6, mean ± SEM; *P=.0136 between 2 and 12 m.o.a. glia, *P=.0219 between 2 and 12 m.o.a. neurons, two-tailed t test). (C) Neuron (blue) and glial (green) packing density within myenteric ganglia with age (n = 4, mean ± SEM; glia ***P=.0007, neurons ***P=.0006, ANOVA).

Figure 7

Cx43 expression with age. (A) Mean Cx43-ir intensity with age (n = 4, mean ± SEM). (A’-A’”) Representative ganglia showing variable Cx43-ir (transformed to heatmap images to depict intensity, arbitrary fluorescence units [AFU]) at 5 m.o.a. (scale bar = 60 μm). (B) Representative Western blots of Cx43 and β-actin (loading control) from the colons of 2 and 15 m.o.a. mice. (B’) Cx43 protein expression at 2 and 15 m.o.a. expressed as the ratio of Cx43 to β-actin (mean ± SEM). (C) Cx43 mRNA levels (normalized to GAPDH) at 2, 5 and 12 m.o.a. (n = 4, mean ± SEM, ANOVA, *P≤.5).