Expression of VGLUT-1 and VGLUT-2 in the DCN and the VCN.

<p>(<b>A</b>) Photomicrographs of VGLUT-1 and VGLUT-2 staining in the DCN with the layers being individually labelled (ML molecular layer, FL fusiform layer, DL deep layer). The overlay shows that VGLUT-1 is mainly present in the ML whereas VGLUT-2 is mainly present in the DL. (<b>C</b>) (<b>B</b>). Photomicrographs of VGLUT-1 and VGLUT-2 staining in the VCN. The overlay shows that VGLUT-1 is mainly expressed in the VCN in comparison to VGLUT-2. Scale bar: 200 µm. All slices are 20 µm thick. (<b>C</b>) Histograms representing the fluorescence intensity of VGLUT-1 and VGLUT-2 in the DCN layers, the MCD and the shell region. * p<0.05, *** P<0.001, NS non significant. (<b>D</b>) Histograms representing the puncta density of VGLUT-1 and VGLUT-2 labelled terminals in the DCN layers, the MCD and the shell region, *** P<0.001. ML: molecular layer; FL: fusiform cell layer; DL: deep layer.</p

Sagittal brainstem slice showing a retrograde labelling of the lateral vestibular nucleus (LVN) following injection of dextran amine in the dorsal cochlear nucleus (DCN) (A,C) and an anterograde labelling of the DCN following injection of dextran amine in the LVN (B,D).

<p>(<b>A</b>) Overlay of a brightfield and fluorescence photomicrograph at 3 hours post injection of dextran amine showing the position of the LVN and the DCN relative to the spinal vestibular nucleus (SpVe) and the nucleus Y (y). The fluorescence in the DCN shows the injection site. (<b>B</b>) The LVN is labeled as a result of retrograde transport of dextran amine. (<b>C</b>) Overlay of a brightfield and fluorescence photomicrograph showing the injection site in the LVN. (<b>D</b>) Labeled terminals in the DCN as a result of anterograde transport of dextran amine. Scale bar: (A) and (B) 200 µm, (C) and (D) 20 µm. All slices are 120 µm thick. ML: molecular layer; FL: fusiform cell layer; DL: deep layer.</p

MRGPR-X1 induce EGR-1 via ERK-1/2 and CCR2 via NFAT in primary DRG neurons.

<p>BAM8-22-induced (2 µM) calcium signals in rat DRG neurons co-expressing MRGPR-X1 and aequorin or solely aequorin are presented in (A). BAM8-22-induced (2 µM, 8 h) activation of the NFAT (B) or TCF/SRF (C) reporter is shown in rat DRG neurons transiently co-expressing MRGPR-X1. RTQ-PCR experiments were performed with cDNAs derived from serum-starved MRGPR-X1 expressing rat DRG neurons stimulated or not with BAM8-22 (2 µM) for 6 h (D) or 40 min (E). CsA (1 µM, 30 min) was used to block calcineurin in (B and D) or PD-184352 (10 µM, 30 min) to inhibit ERK-1/2 activity in (C and E). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

Reconstruction of retrograde cell body labelling in the LVN following an injection of dextran amine in the DCN.

<p>Injections were performed on coronal (left) and sagittal (right) brainstem preparations and serial sections of 100 µm were performed. The black area represents the injection site in the DCN and the red dots represent labelled cell bodies including those in the LVN (grey area). LVN: lateral vestibular nucleus; DCN: dorsal cochlear nucleus; VCN: ventral cochlear nucleus; icp: inferior cerebellar peduncle; sp5: spinal trigeminal tract; sp5O: spinal trigeminal nucleus; 8vn: vestibular route of 8^th nerve; SpVe: spinal vestibular nucleus; LR4V: lateral recess 4^th ventricle; Cu: cuneate nucleus.</p

VGLUT-2 immunoreactivity increases after acoustic overexposure (AOE) triggering hearing deficit.

<p>(<b>A</b>) Auditory brainstem response (ABR) recordings are elicited by a tone pip of 24 kHz and 94 dB SPL. The top traces show ABRs obtained at day 0 in a control subject and prior to AOE. The bottom traces show ABRs obtained at day 5 in both subjects. After AOE, the ABR is characterised by a flat trace and wave I (shown by the dotted line) is absent. (<b>B</b>) Summary plot representing the ABR threshold shifts between day 0 and day 5 in response to the tone pip frequency. * p<0.05. (<b>C</b>) Expression of VGLUT-1 (left) and VGLUT-2 (middle) in DCN coronal slices originating from a control subject (top) and after AOE (bottom). The overlay of VGLUT-1 and VGLUT-2 is shown in the right panels. (<b>D</b>) Histograms representing the fluorescence intensity of VGLUT-1 and VGLUT-2 in the DCN layers, the magnocellular domain of the VCN MCD and the shell region, *** P<0.001, NS non significant. ML: molecular layer; FL: fusiform cell layer; DL: deep layer. After AOE, the VGLUT-1 immunoreactivity decreases and the VGLUT-2 immunoreactivity increases in all DCN layers and the MCD. VGLUT-1 is decreased after AOE in the shell but VGLUT-2 expression was unaffected. (<b>E</b>) Examples of synaptic boutons originating from the LVN labelled with VGLUT-2 in control condition (left) and after AOE (middle). Note the presence of multiple VGLUT-2 labelled terminals after AOE. Results are summarised in the histogram (right). * p<0.05, Scale bar: (C) 100 µm, (E) 5 µm.</p

MRGPR-X1 induce CCR2 via NFAT in F11-MRGPR-X1 cells.

<p>RTQ-PCR experiments were performed with cDNAs derived from serum-starved F11-MRGPR-X1 cells stimulated or not with BAM8-22 (1 µM) for 6 h (A-C) using specific primers for 5 distinct genes as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058756#pone-0058756-t001" target="_blank">table 1</a>. The name of the analyzed gene is listed beneath the corresponding bar. Genes with a p value <0.05 are show in black bars. In (B and C) cells were treated or not with CsA (1 µM, 30 min). Relative BAM8-22-induced gene expression was normalized to β-actin and calculated using the ΔΔCp method. Data from 4 independent experiments were compiled and expressed as the mean ± S.E.M. BAM8-22-induced (1 µM, 20 h) CCR2 protein expression in F11-MRGPR-X1 cells was assessed by flow cytometry (D) or by CCL2-promoted (100 ng/ml) inhibition of FSK-induced (5 µM) cAMP accumulation (E). In (D) one representative experiment is shown. Accumulation of the data from 5 independent experiments revealed an increase in the number of CCR2 positive cells by 10.8±1.4% after BAM8-22 stimulation. In (E, left panel) one representative experiment is shown and in (E, right panel) data from 5 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells. Dagger signs indicate a significant difference between BAM8-22-stimulated inhibitor-treated and untreated cells.</p

Coronal brainstem slice showing a retrograde labelling of LVN following injection of dextran amine in the DCN (A,C) and an anterograde labelling of the DCN following injection of dextran amine in the LVN (B,D).

<p>(<b>A</b>) Overlay of a brightfield and fluorescence photomicrograph at 3 hours post injection of dextran amine showing the position of the DCN relatively to the inferior cerebellar peduncle (icp) and the cerebellum. The fluorescence in the DCN shows the injection site. (<b>B</b>) The LVN is labeled as a result of retrograde transport of dextran amine. (<b>C</b>) Overlay of a brightfield and fluorescence photomicrograph showing the position of the dextran amine injection site in the LVN. (<b>D</b>) Labeled terminals in the DCN as a result of anterograde transport of dextran amine. Scale bar: (A) and (B) 200 µm, (C) and (D) 20 µm. All slices are 120 µm thick.</p

MRGPR-X1-induced signaling in DRG neurons and CTMCs.

<p>A cartoon illustrating the signaling circuit by which MRGPR-X1 affect chemokine signaling in DRG neurons and connective tissue mast cells is given.</p

MRGPR-X1-induced CCL2 release in LAD-2 mast cells.

<p>(A) MRGPR-X1 (40 cycles) or β-actin (30 cycles) mRNA expression was determined in LAD-2 cells by RT-PCR. As a negative control, RT-PCR was conducted without addition of cDNA (H₂O). (B) Calcium signals in fura2-loaded LAD-2 cells are shown after injection of BAM8-22 (5 µM) or ionomycin (5 µM) or HBS as positive or negative control, respectively. (C) CCL2 release in LAD-2 cells after stimulation with BAM8-22 (5 µM, 18 h) was determined by ELISA. Data from 4 independent experiments performed in triplicates were compiled and expressed as the mean ± S.E.M. Asterisks indicate a significant difference to not stimulated cells.</p

Glutamatergic post-synaptic currents (EPSCs) elicited in identified DCN cells by stimulating the LVN in a sagittal slice.

<p>(<b>A</b>) Photomicrograph of a DCN fusiform cell filled with lucifer yellow (top) and whole cell voltage clamp recording of this fusiform cell while stimulating the LVN (bottom). (<b>B</b>) Photomicrograph of a DCN granule cell filled with lucifer yellow (top) and whole cell voltage clamp recording of this granule cell while stimulating the LVN (bottom). Both cells were held at −68 mV and the LVN was stimulated at 0.3 Hz. Glutamatergic EPSCs are represented in black and are blocked by 50 µm D-AP5 and 10 µm NBQX (traces in red). Each trace represents an average of 10–20 single traces. The arrowhead represents the artifact of stimulus that has been removed for clarity. Scale bar: (A) 50 µm, (B) 20 µm.</p