Enteric glia are targets of the sympathetic innervation of the myenteric plexus in the guinea pig distal colon

Abstract

Astrocytes respond to synaptic activity in the CNS. Astrocytic responses are synapse specific and precisely regulate synaptic activity. Glia in the peripheral nervous system also respond to neuronal activity, but it is unknown whether glial responses are synapse specific. We addressed this issue by examining the activation of enteric glia by distinct neuronal subpopulations in the enteric nervous system. Enteric glia are unique peripheral glia that surround enteric neurons and respond to neuronally released ATP with increases in intracellular calcium ([Ca2+]i). Autonomic control of colonic function is mediated by intrinsic (enteric) and extrinsic (sympathetic, parasympathetic, primary afferent) neural pathways. Here we test the hypothesis that a defined population of neurons activates enteric glia using a variety of techniques to ablate or stimulate components of the autonomic innervation of the colon. Our findings demonstrate that, in the male guinea pig colon, activation of intrinsic neurons does not stimulate glial [Ca2+]i responses and fast enteric neurotransmission is not necessary to initiate glial responses. However, ablating extrinsic innervation significantly reduces glial responses to neuronal activation. Activation of primary afferent fibers does not activate glial [Ca2+]i responses. Selectively ablating sympathetic fibers reduces glial activation to a similar extent as total extrinsic denervation. Neuronal activation of glia follows the same frequency dependence as sympathetic neurotransmitter release, but the only sympathetic neurotransmitter that activates glial [Ca2+]i responses is ATP, suggesting that sympathetic fibers release ATP to activate enteric glia. Therefore, enteric glia discern activity in adjacent synaptic pathways and selectively respond to sympathetic activation.

Figure 1.

Electrical stimulation of interganglionic myenteric fiber tracts does not require fast nicotinic transmission to stimulate [Ca²⁺]_i responses in enteric glia. A, Schematic of experiment. FTS activates enteric, sympathetic, parasympathetic, and primary afferent nerves, which then release substances that stimulate responses in enteric glia and enteric neurons. B, Averaged glial response (mean ± SEM) of 44 glial regions within a myenteric ganglion illustrates that glial responses to FTS are not blocked by hexamethonium (Hex). C, At rest (baseline), a myenteric ganglion in an LMMP whole-mount preparation from the distal colon of a guinea pig (outlined by dashed line) displays low Fluo-4 fluorescence. C′, After a 5 min blockade of nicotinic receptors with 100 μm hexamethonium, electrical stimulation (3 s, 5 V, 20 Hz) of an interganglionic fiber tract stimulates Ca²⁺ responses in a proportion of glia within the ganglion (peak response pseudocolored in green). C″, Bath application of 100 μm ATP elicits a robust glial Ca²⁺ response (peak response pseudocolored in magenta). C‴, Overlay of the peak FTS and peak ATP responses demonstrates that many glia respond to both ATP and FTS.

Figure 2.

Nicotinic stimulation of enteric neurons is not sufficient to elicit Ca²⁺ responses in enteric glia. A, A myenteric ganglion (outlined by dashed line) displays low Fluo-4 fluorescence at rest. A′, Bath application of the nicotinic agonist epibatidine (300 nm) elicits a robust Ca²⁺ response in enteric neurons (peak response pseudocolored magenta), whereas ATP elicits a Ca²⁺ response dominated by enteric glia (peak response pseudocolored green) (A″). A‴, Overlay of the epibatidine response (A′) and ATP response (A″) demonstrates that ATP-responding glia do not respond to nicotinic stimulation of neurons with Ca²⁺ responses. B, Trace showing the averaged response of enteric glia (35 cells; blue) and neurons (6 cells; red) within a myenteric ganglion (error bars omitted for clarity). Neurons respond to nicotinic stimulation with robust Ca²⁺ responses that are inhibited by 100 μm hexamethonium (Hex). Only very small Ca²⁺ are detected in enteric glia, possibly as a result of overlapping neuronal elements.

Figure 3.

Stimulation of primary afferent nerves with capsaicin stimulates Ca²⁺ responses in enteric neurons but not enteric glia. A, Trace showing the averaged response of enteric glia (45 cells; gray) and neurons (5 cells; black) within a myenteric ganglion (error bars omitted for clarity). Application of capsaicin (50 μm) stimulates Ca²⁺ responses only in neurons, whereas application of ATP (100 μm) elicits responses in both neurons and glia. B, Summary data showing that glia (averaged glia response from n = 19 ganglia) do not respond to capsaicin (ANOVA, p > 0.05). However, capsaicin (Caps) significantly (ANOVA, ***p < 0.0001) stimulates neuronal responses that are ∼30% as large as neuronal ATP responses (averaged neuronal responses from n = 10 ganglia).

Figure 4.

Chemical sympathectomy and extrinsic denervation reduce FTS responses in enteric glia to a similar extent. A, A′, Chemical sympathectomy. TH-IR sympathetic nerve fibers densely innervate the myenteric plexus of the guinea pig colon under normal conditions (A). Treatment with 6-OHDA reduces the number of TH-IR fibers within the myenteric plexus, indicating a loss of sympathetic innervation. Scale bar: A′, 100 μm. B–C′, Surgical extrinsic denervation. In control tissue (taken ∼5 cm oral to the site of denervation), PGP9.5-IR myenteric neurons (green in B) are surrounded by TH-IR (red in B′) extrinsic fibers. C, C′, Surgical denervation significantly reduces the amount of TH-IR extrinsic fibers (red in C′), whereas PGP9.5-IR myenteric neurons remain intact (green in C). Scale bars: B′, C′, 50 μm.

Figure 5.

ATP is the only postganglionic sympathetic neurotransmitter that stimulates Ca²⁺ responses in enteric glia. A, Baseline Fluo-4 fluorescence in a myenteric ganglion (outlined by dotted line). A′, Application of NE (100 μm) causes a Ca²⁺ response in cells lining blood vessels (arrows in A′), resulting in a constriction of the vessel. However, no Ca²⁺ responses occur in enteric glia within the myenteric ganglia. A″, Application of ATP (100 μm) elicits a robust Ca²⁺ response in enteric glia. B, Trace showing the averaged response (mean ± SEM) of 35 glial regions within a myenteric ganglion. No responses are detected with application of NE. C, Averaged response (mean ± SEM) of 34 glial regions within a myenteric ganglion demonstrating that enteric glia do not respond to SST (1 μm). D, Enteric glia also do not respond to NPY (1 μm). Trace showing averaged response (mean ± SEM) of 51 glial regions within a myenteric ganglion.

Figure 6.

Model of neuron-to-glia transmission in the myenteric plexus of the guinea pig distal colon. Stimulation of primary afferent fibers releases neuropeptides and ATP (CGRP, calcitonin gene-related peptide; SP, substance P), resulting in stimulation of enteric neurons. Activation of presynaptic enteric neurons release acetylcholine (ACh) and ATP that act on fast nicotinic (nAChR) and purinergic (P2XR) receptors on postsynaptic enteric neurons. Sympathetic activation initially releases ATP that acts at P2Y₄ receptors on enteric glia, eliciting glial Ca²⁺ responses. The slower sustained sympathetic response releases NE that acts on presynaptic adrenergic receptors (AR) on enteric neurons.