Abstract
In the brainstem nucleus of the solitary tract (NTS), primary vagal afferent neurons express the transient receptor potential vanilloid subfamily member 1 (TRPV1) at their central terminals where it contributes to quantal forms of glutamate release. The endogenous membrane lipid anandamide (AEA) is a putative TRPV1 agonist in the brain, yet the extent to which AEA activation of TRPV1 has a neurophysiological consequence is not well established. We investigated the ability of AEA to activate TRPV1 in vagal afferent neurons in comparison to capsaicin (CAP). Using ratiometric calcium imaging and whole-cell patch clamp recordings we confirmed that AEA excitatory activity requires TRPV1, binds competitively at the CAP binding site, and has low relative affinity. While AEA-induced increases in peak cytosolic calcium were similar to CAP, AEA-induced membrane currents were significantly smaller. Removal of bath calcium increased the AEA current with no change in peak CAP currents revealing a calcium sensitive difference in specific ligand activation of TRPV1. Both CAP- and AEA-activated TRPV1 currents maintained identical reversal potentials, arguing against a major difference in ion selectivity to resolve the AEA differences in signaling. In contrast with CAP, AEA did not alter spontaneous glutamate release at NTS synapses. We conclude: (1) AEA activation of TRPV1 is markedly different from CAP and produces different magnitudes of calcium influx from whole-cell current; and (2) exogenous AEA does not alter spontaneous glutamate release onto NTS neurons. As such, AEA may convey modulatory changes to calcium-dependent processes, but does not directly facilitate glutamate release.
Highlights
Primary vagal afferent neurons relay satiety information to the brain and initiate coordinated autonomic and gastrointestinal reflex pathways (Saper, 2002; Berthoud, 2008)
We initially determined if the stimulatory effects of AEA on dissociated vagal afferent neurons were mediated by transient receptor potential vanilloid subfamily member 1 (TRPV1) as previously reported (Zygmunt et al, 1999; Smart et al, 2000) or via another cellular mechanism
In this study we investigated the ability of the endogenous cannabinoid AEA to directly bind to and gate native TRPV1 ion channels in primary vagal afferent neurons
Summary
Primary vagal afferent neurons relay satiety information to the brain and initiate coordinated autonomic and gastrointestinal reflex pathways (Saper, 2002; Berthoud, 2008). Most vagal afferents are unmyelinated or lightly myelinated C- and Aδ-fibers which extensively express the transient receptor potential vanilloid subfamily member 1 (TRPV1) throughout the neuron (Holzer, 1991). TRPV1 activation at the central endings selectively controls quantal forms of glutamate release including action-potential independent spontaneous release and action-potential. Capsaicin (CAP) activation of TRPV1 dramatically increases the frequency of spontaneous vesicle fusion and release, presumably through the direct influx of calcium ions (Andresen et al, 2012). TRPV1-facilitated glutamate release targets a discrete pool of vesicles under some experimental conditions (Peters et al, 2010). Sustained TRPV1 activation dramatically increases spontaneous release while depleting other vesicle release pathways; including action-potential driven synchronous release (Doyle et al, 2002). The extent to which TRPV1 fluxed calcium is required for TRPV1 to control glutamate release is unknown
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