TRPV1 enhances cholecystokinin signaling in primary vagal afferent neurons and mediates the central effects on spontaneous glutamate release in the NTS.

The gut peptide cholecystokinin (CCK) is released during feeding and promotes satiation by increasing excitation of vagal afferent neurons that innervate the upper gastrointestinal tract. Vagal afferent neurons express CCK1 receptors (CCK1Rs) in the periphery and at central terminals in the nucleus of the solitary tract (NTS). While the effects of CCK have been studied for decades, CCK receptor signaling and coupling to membrane ion channels are not entirely understood. Previous findings have implicated L-type voltage-gated calcium channels as well as transient receptor potential (TRP) channels in mediating the effects of CCK, but the lack of selective pharmacology has made determining the contributions of these putative mediators difficult. The nonselective ion channel transient receptor potential vanilloid subtype 1 (TRPV1) is expressed throughout vagal afferent neurons and controls many forms of signaling, including spontaneous glutamate release onto NTS neurons. Here we tested the hypothesis that CCK1Rs couple directly to TRPV1 to mediate vagal signaling using fluorescent calcium imaging and brainstem electrophysiology. We found that CCK signaling at high concentrations (low-affinity binding) was potentiated in TRPV1-containing afferents and that TRPV1 itself mediated the enhanced CCK1R signaling. While competitive antagonism of TRPV1 failed to alter CCK1R signaling, TRPV1 pore blockade or genetic deletion (TRPV1 KO) significantly reduced the CCK response in cultured vagal afferents and eliminated its ability to increase spontaneous glutamate release in the NTS. Together, these results establish that TRPV1 mediates the low-affinity effects of CCK on vagal afferent activation and control of synaptic transmission in the brainstem.NEW & NOTEWORTHY Cholecystokinin (CCK) signaling via the vagus nerve reduces food intake and produces satiation, yet the signaling cascades mediating these effects remain unknown. Here we report that the capsaicin receptor transient receptor potential vanilloid subtype 1 (TRPV1) potentiates CCK signaling in the vagus and mediates the ability of CCK to control excitatory synaptic transmission in the nucleus of the solitary tract. These results may prove useful in the future development of CCK/TRPV1-based therapeutic interventions.

Contributing mechanisms underlying desensitization of cholecystokinin-induced activation of primary nodose ganglia neurons.

Cholecystokinin (CCK) is a gut-derived peptide that potently promotes satiety and facilitates gastric function in part by activating G protein-coupled CCK1 receptors on primary vagal afferent neurons. CCK signaling is dynamic and rapidly desensitizes, due to decreases in either receptor function and the resulting signal cascade, ion channel effectors, or both. Here we report a decay-time analytical approach using fluorescent calcium imaging that relates peak and steady-state calcium responses in dissociated vagal afferent neurons, enabling discrimination between receptor and ion channel effector functions. We found desensitization of CCK-induced activation was predictable, consistent across cells, and strongly concentration dependent. The decay-time constant (tau) was inversely proportional to CCK concentration, apparently reflecting the extent of receptor activation. To test this possibility, we directly manipulated the ion channel effector(s) with either decreased bath calcium or the broad-spectrum pore blocker ruthenium red. Conductance inhibition diminished the magnitude of the CCK responses without altering decay kinetics, confirming changes in tau reflect changes in receptor function selectively. Next, we investigated the contributions of the PKC and PKA signaling cascades on the magnitude and decay-time constants of CCK calcium responses. While inhibition of either PKC or PKA increased CCK calcium response magnitude, only general PKC inhibition significantly decreased the decay-time constant. These findings suggest that PKC alters CCK receptor signaling dynamics, while PKA alters the ion channel effector of the CCK response. This analytical approach should prove useful in understanding receptor/effector changes underlying acute desensitization of G-protein coupled signaling and provide insight into CCK receptor dynamics.

Direct Anandamide Activation of TRPV1 Produces Divergent Calcium and Current Responses.

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.

Ethyl Vanillin Activates TRPA1.

The nonselective cation channel transient receptor potential ankryn subtype family 1 (TRPA1) is expressed in neurons of dorsal root ganglia and trigeminal ganglia and also in vagal afferent neurons that innervate the lungs and gastrointestinal tract. Many TRPA1 agonists are reactive electrophilic compounds that form covalent adducts with TRPA1. Allyl isothiocyanate (AITC), the common agonist used to identify TRPA1, contains an electrophilic group that covalently binds with cysteine residues of TRPA1 and confers a structural change on the channel. There is scientific motivation to identify additional compounds that can activate TRPA1 with different mechanisms of channel gating. We provide evidence that ethyl vanillin (EVA) is a TRPA1 agonist. Using fluorescent calcium imaging and whole-cell patch-clamp electrophysiology on dissociated rat vagal afferent neurons and TRPA1-transfected COS-7 cells, we discovered that EVA activates cells also activated by AITC. Both agonists display similar current profiles and conductances. Pretreatment with A967079, a selective TRPA1 antagonist, blocks the EVA response as well as the AITC response. Furthermore, EVA does not activate vagal afferent neurons from TRPA1 knockout mice, showing selectivity for TRPA1 in this tissue. Interestingly, EVA appears to be pharmacologically different from AITC as a TRPA1 agonist. When AITC is applied before EVA, the EVA response is occluded. However, they both require intracellular oxidation to activate TRPA1. These findings suggest that EVA activates TRPA1 but via a distinct mechanism that may provide greater ease for study in native systems compared with AITC and may shed light on differential modes of TRPA1 gating by ligand types.

Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.

[This corrects the article DOI: 10.1371/journal.pone.0159277.].

Redundant Postsynaptic Functions of SynCAMs 1-3 during Synapse Formation.

Investigating the roles of synaptogenic adhesion molecules during synapse formation has proven challenging, often due to compensatory functions between additional family members. The synaptic cell adhesion molecules 1-3 (SynCAM1-3) are expressed both pre- and postsynaptically, share highly homologous domains and are synaptogenic when ectopically presented to neurons; yet their endogenous functions during synaptogenesis are unclear. Here we report that SynCAM1-3 are functionally redundant and collectively necessary for synapse formation in cultured hippocampal neurons. Only triple knockdown (KD) of SynCAM1-3 using highly efficient, chained artificial microRNAs (amiRNAs) reduced synapse density and increased synapse area. Electrophysiological recordings of quantal release events supported an increase in synapse size caused by SynCAM1-3 depletion. Furthermore, a combinatorial, mosaic lentiviral approach comparing wild type (WT) and SynCAM1-3 KD neurons in the same culture demonstrate that SynCAM1-3 set synapse number and size through postsynaptic mechanisms. The results demonstrate that the redundancy between SynCAM1-3 has concealed their synaptogenic function at the postsynaptic terminal.

A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications.

Recombination-based cloning is a quick and efficient way to generate expression vectors. Recent advancements have provided powerful recombinant DNA methods for molecular manipulations. Here, we describe a novel collection of three-fragment MultiSite Gateway cloning system-compatible vectors providing expanded molecular tools for vertebrate research. The components of this toolkit encompass a broad range of uses such as fluorescent imaging, dual gene expression, RNA interference, tandem affinity purification, chemically-inducible dimerization and lentiviral production. We demonstrate examples highlighting the utility of this toolkit for producing multi-component vertebrate expression vectors with diverse primary research applications. The vectors presented here are compatible with other Gateway toolkits and collections, facilitating the rapid generation of a broad range of innovative DNA constructs for biological research.

Improved knockdown from artificial microRNAs in an enhanced miR-155 backbone: a designer's guide to potent multi-target RNAi.

Artificial microRNA (amiRNA) sequences embedded in natural microRNA (miRNA) backbones have proven to be useful tools for RNA interference (RNAi). amiRNAs have reduced off-target and toxic effects compared to other RNAi-based methods such as short-hairpin RNAs (shRNA). amiRNAs are often less effective for knockdown, however, compared to their shRNA counterparts. We screened a large empirically-designed amiRNA set in the synthetic inhibitory BIC/miR-155 RNA (SIBR) scaffold and show common structural and sequence-specific features associated with effective amiRNAs. We then introduced exogenous motifs into the basal stem region which increase amiRNA biogenesis and knockdown potency. We call this modified backbone the enhanced SIBR (eSIBR) scaffold. Using chained amiRNAs for multi-gene knockdown, we show that concatenation of miRNAs targeting different genes is itself sufficient for increased knockdown efficacy. Further, we show that eSIBR outperforms wild-type SIBR (wtSIBR) when amiRNAs are chained. Finally, we use a lentiviral expression system in cultured neurons, where we again find that eSIBR amiRNAs are more potent for multi-target knockdown of endogenous genes. eSIBR will be a valuable tool for RNAi approaches, especially for studies where knockdown of multiple targets is desired.

Netrin signaling breaks the equivalence between two identified zebrafish motoneurons revealing a new role of intermediate targets.

BACKGROUND: We previously showed that equivalence between two identified zebrafish motoneurons is broken by interactions with identified muscle fibers that act as an intermediate target for the axons of these motoneurons. Here we investigate the molecular basis of the signaling interaction between the intermediate target and the motoneurons. PRINCIPAL FINDINGS: We provide evidence that Netrin 1a is an intermediate target-derived signal that causes two equivalent motoneurons to adopt distinct fates. We show that although these two motoneurons express the same Netrin receptors, their axons respond differently to Netrin 1a encountered at the intermediate target. Furthermore, we demonstrate that when Netrin 1a is knocked down, more distal intermediate targets that express other Netrins can also function to break equivalence between these motoneurons. SIGNIFICANCE: Our results suggest a new role for intermediate targets in breaking neuronal equivalence. The data we present reveal that signals encountered during axon pathfinding can cause equivalent neurons to adopt distinct fates. Such signals may be key in diversifying a neuronal population and leading to correct circuit formation.