Figure 1 Expression of ERG1 channels in LC neurons. (A–D) Expression of Erg1a (A), Erg1b (B), Erg2 (C) and Erg3 (D) proteins in the LC and adjacent areas, as visualized through immunohistochemistry. (E) represents a stained section where the primary antibody was omitted. (F–I) Expression of Erg1a (F) and Erg1b (H) mRNAs in the LC and adjacent areas (sagittal sections), via non-radioactive in situ hybridization using anti-sense probes. Sections processed in parallel with sense probes (G,I) showed no signal. High magnification images of LC stained with the anti-TH (K) and anti-ERG1 (L) antibodies. Images of neurons stained with DAPI (J) and merged (M) are reported. Note that cells within the LC nucleus (indicated as LC) were heavily stained by the anti-TH specific antibody (green signal). Staining with anti-ERG1 antibody (red signal) was also present. Scale bars (A–I) = 100 μm, (J–M) image size is 185 × 185 μm. Abbreviations: 4V, 4th ventricle; Bar, Barrington’s nucleus; IntP, interposed cerebellar nucleus, posterior part; LC, Locus coeruleus; LDTg, laterodorsal tegmental nucleus; MVePC, medial vestibular nucleus, parvicellular part; PkC, Purkinje cells; SpVe, spinal vestibular nucleus.

Figure 2 Anatomical localization of LC nucleus. (A) Sagittal section of the murine whole brain, each color distinguishes different regions in the brain, with black showing the localization of the LC nucleus in the pons of brainstem. (B) Cartoon showing the boundaries of the LC nucleus in red at the level of the Vth ventricle, compared to other surrounding structures in pink, where sections were dissected.

Figure 3 WAY increased LC neuron firing frequency. (A) Plot showing LC neuron firing rate (%) increased after WAY application and recovered back to baseline levels during the wash-out period. One-way ANOVA was performed on the mean of data points taken from a 60 s period representing the peak effect of each condition. * p < 0.05; n = 6 neurons each from a different mouse. (B) Firing frequency from a representative LC neuron. The firing frequency of a 60 s period in control/baseline conditions, during WAY application and during the wash-out. (C) Bar graph showing data from the neuron in (B). One-way ANOVA was performed on the mean data points enclosed by the red rectangles shown in (B); **** p < 0.0001. (D) Raster plots of the data shown in (B).

Figure 4 WAY alters LC neuronal firing pattern. (A) Bar graph showing a decrease in mean inter-spike interval (ISI) with WAY application (* p < 0.05, ns = no significant difference, n = 6), that was reversed by wash-out. (B–D) Representative data from a single neuron, showing ISI scatterplots (B), ISI distributions fitted with the Gaussian equation Y = Amplitude × exp(−0.5 × ((X-Mean)/SD)2) (C) and ISI auto-correlograms (D) calculated in baseline conditions (blue), during WAY application (orange) and after drug wash-out (green), from a 200 s representative period of a single recording. Raw ISI values are plotted. The plots point out a regular firing pattern in the baseline and wash-out periods that is remarkably evidenced by the regular peaks in the ISI auto-correlograms. By contrast, a more irregular firing pattern of LC neurons during WAY application was clearly seen by the less defined peaks.