Fig 1.
Establishing lynx1 expression in brain regions associated with nociception.
(A) Evidence of lynx1 mRNA expression in the dorsal raphe nucleus via RT-PCR using lynx1-specific primers (expected band size of 62 bp). Expression of TPH2 (expected band size of 147 bp) validates that the isolation is in the correct region of interest. (B) Schematic of the brainstem at the level of the DRN, as a coronal plane of section (C) Expression of lynx1 protein (green) in the dorsal raphe nucleus, using anti-lynx1 pAb immunofluorescence staining [39, 64]and donkey anti-rabbit Cy2 secondary antibody, imaged at 4x magnification. (D and E) Side by side labeling of lynx1 (anti-lynx1 mAb, Alexa red), and TPH2 (anti-TPH2 pAb, Cy2, green), 10x magnification, scale bar = 200 μm. (F) Dual labeling immunofluorescence staining using anti-lynx1 mAb (red) and anti-TPH2 pAb (green), merge (yellow), 20x magnification, scale bar = 100 μm. (G) Dual labeling immunofluorescence staining using anti-lynx1 mAb (red) and anti-TPH2 pAb (green), merge (yellow), 40x magnification, scale bar = 50 μm.
Fig 2.
Modulatory effect of lynx1 on nicotine responses in the dorsal raphe nucleus.
(A) Photomicrograph of a live brain slice containing the dorsal raphe nucleus (B) Representative trace of an action potential from a GABAergic-like (without AP shoulder, left) and serotonergic-like neuron (with AP shoulder, right) recorded in current-clamp mode. Left upper panel is a single action potential at a faster time scale and lower panel is a spike train at high frequency of the GABAergic neuron. Right upper panel is a single AP at a faster time scale, and lower panel is a spike train at low frequency of the serotonergic neuron. Arrow points to the AP shoulder which is a hallmark of serotonergic neurons. (C) Representative original traces and average nicotine-evoked current amplitude of neuronal cell bodies recorded in voltage-clamp mode held at -70mV. Nicotine induced stronger responses both in GABAergic and serotonergic neurons of Lynx1 KO (white) neurons than wild-type (black) in dorsal raphe nucleus (GABAergic neurons: wt 47.8 ± 16.1 pA, n = 9 (mice N = 3) vs. lynx1KO 170 ± 38.2 pA, n = 17 (mice N = 6); p = 0.008, student t test. Serotonergic neurons: wt 61.0 ± 13.2 pA, n = 12 (mice N = 4) vs. lynx1KO 164 ± 34.6 pA, n = 19 (mice N = 6); p = 0.01, student t test).
Fig 3.
The effect of nicotine on antinociception assessed on a hot-plate assay.
(A) Antinociceptive responses in wt and lynx1KO mice after I.P. injections of saline (n = 8 wt, 8 KO. p = 0.899, two-way ANOVA, cohen’s D 0.13) or nicotine concentrations of 0.5 mg·kg-1 (n = 8 wt, 18 KO. p = 0.122, two-way ANOVA, cohen’s D 1.36), 1.0mg·kg-1 (n = 8 wt, 14 KO. p = 0.032, two-way ANOVA, cohen’s D 1.09) and 1.5mg·kg-1 (8 wt, 8 KO. p = 0.657, two-way ANOVA, cohen’s D 0.13) using the hot-plate assay. ED50 was 1.05 mg·kg-1 for wt and 0.44 mg·kg-1 for the lynx1KO group.
Fig 4.
lynx1 does not influence nicotine-mediated locomotor performance or body temperature.
(A) Effect of nicotine on locomotion in wt and lynx1KO mice after I.P. injections of nicotine concentrations 0.5 mg·kg-1 (n = 7 wt, 8 KO), 1.0mg·kg-1 (n = 6 wt, 6 KO), 1.5mg·kg-1 (6 wt, 6 KO). Locomotion were examined by scoring leg movements (seconds) in the time period 15–20 minutes post injection. Injection of nicotine induce the same amount of hypolocomotion in both genotypes. Each data point presented as mean ± SEM. wt: wild type, KO: lynx1 knockout. (B)The locomotor performance after nicotine injection (0.5 mg·kg-1) was binned into 5 minute time windows and showed no significant effect of genotype at any time window. (C) Effect of nicotine on body temperature in wt and lynx1KO mice after I.P. injections of either saline (n = 11 wt, 11 KO), or nicotine concentrations 1.0 mg·kg-1 (n = 9 wt, n = 11 KO) and 2.5 mg·kg-1 (n = 7 wt, 7 KO). Each bar presented as mean ± SEM.
Fig 5.
Mediation of lynx1 through α4β2 nAChRs.
(A) Antinociceptive responses in wt and lynx1KO mice after I.P. injection of the non-selective α4β2* nAChR agonist, epibatidine (5 μg·kg-1) (n = 24 wt, 21 KO, p = 0.029, Student’s T-test). Mice were tested on the hot-plate 15 minutes after injection. Epibatidine-mediated antinociception is augmented in lynx1KO mice compared to wt mice. Data presented as mean ± SEM time. *P<0.05 compared to wt controls. wt: wild type, KO: lynx1 knockout. (B) Antinociceptive responses in wt and lynx1KO mice after I.P. injection of the α4β2 nAChRs inhibitor dihydro-β-erythroidine hydrobromide (DHβE) (3.0 mg·kg-1) and nicotine (0.5 mg·kg-1) (nicotine treated lynx1KO mice (n = 8) vs. nicotine+DHβE treated lynx1KO mice (n = 6) using the hot-plate assay. Mice were injected with DHβE 25 minutes and nicotine 15 minutes prior to hot-plate testing. Injections of DHβE blocks the antinociceptive effect of nicotine in lynx1KO mice. Data indicates that lynx1 operates through the α4β2 nAChR to modulate antinociception. Data presented as mean ± SEM time. wt: wild type, KO: lynx1 knockout. (C) Schematic of lynx1 binding to the LS stoichiometry of α4β2 nAChRs preferentially over the HS stoichiometry. α4β2 nAChR pentamers shown in the high sensitivity (HS) and low sensitivity (LS) stoichiometry, made up of (α4)2(β2)3 vs. (α4)2(β2)3 nAChRs respectively. In our model, lynx1 preferentially binds and stabilizes the LS stoichiometry.
Fig 6.
Overall architecture of lynx1 at α4:α4 nAChR interface.
Molecular dynamic simulations of lynx1 binding to α4 nAChR subunits. The cell membrane is represented as a dashed line.
Fig 7.
Structural comparison between α4:α4/lynx1 and β2:β2/lynx1 complex model.
(A) α4:α4/lynx1 complex model. (B) β2:β2/lynx1 complex model (C) Structural details of boxed in A. (D) Structural details of boxed in B.