Article Figures & Data
Figures
- Figure 1.
A, Schematic of target MHb region and DIC image of target MHb neurons. Scale bar, 20 µm. B, Firing frequency mMHb neurons from PGVG-treated male and female mice in cell-attached mode. C, Whole-cell firing frequency with representative traces for male and female mMHb neurons (n = 5 females (15 neurons cell-attached, 14 neurons whole-cell), n = 5 males (13 neurons cell-attached, 11 neurons whole-cell)). D, Action potential duration, (E) threshold potential of mMHb neurons, (F) input resistance of control, and (G) maximal spiking ability of control mMHb neurons. H, Rheobase (minimal current necessary to elicit first action potential) of control mMHb neurons found from −20 to +70 (5 pA steps) current step protocol with representative traces from females and males. Red traces represent the first elicited action potentials. I, Current/voltage relationship plot of control mMHb neurons. X-axis represents currents injected from −20 to +70 pA and Y-axis is elicited action potentials per given current step. All results are mean ± SEM. Data represented as mean ± SEM were analyzed with an unpaired t-test (B–H) or mixed effects two-way ANOVA (I).
- Figure 2.
A, Timeline of EVSA paradigm. B–E, Active nose pokes for male and female mice in our EVSA paradigm assigned to 6 mg/ml nicotine (B), 6 mg/ml nicotine plus menthol (C), 60 mg/ml nicotine (D), and 60 mg/ml nicotine plus menthol (E). F, Mean FR3 active nose pokes for male and female mice. G, Mean breakpoint for male and female mice. Data are presented as mean ± SEM and analyzed via two-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.
- Figure 3.
A–D, Representative current clamp baseline firing traces for 6 mg/ml nicotine (A), 6 mg/ml nicotine plus menthol (B), 60 mg/ml nicotine (C), and 60 mg/ml nicotine plus menthol (D). E–H, Linear regression analysis of MHb baseline firing frequency to FR3 score for female mice assigned 6 mg/ml nicotine (E), 6 mg/ml nicotine plus menthol (F), 60 mg/ml nicotine (G), and 60 mg/ml nicotine plus menthol (H). Analysis was done through simple linear regression. Values for all animals are presented as mean per animal of two to five cells.
- Figure 4.
A–D, Representative current clamp traces of voltage steps for 6 mg/ml nicotine (A), 6 mg/ml nicotine plus menthol (B), 60 mg/ml nicotine (C), and 60 mg/ml nicotine plus menthol (D). E–H, Linear regression analysis of rheobase to FR3 score for female mice assigned 6 mg/ml nicotine (E), 6 mg/ml nicotine plus menthol (F), 60 mg/ml nicotine (G), and 60 mg/ml nicotine plus menthol (H). I–L, Linear regression analysis of maximum action potential spikes during current steps to FR3 score for female mice assigned 6 mg/ml nicotine (I), 6 mg/ml nicotine plus menthol (J), 60 mg/ml nicotine (K), and 60 mg/ml nicotine plus menthol (L). Values for all animals are presented as mean per animal. Blue R2 and p values with an asterisk(s) and red error bars represent significant correlations (p < 0.05). N = 6 mg/ml Nicotine, seven mice; 6 mg/ml nicotine plus menthol, nine mice; 60 mg/ml nicotine, eight mice; 60 mg/ml nicotine plus menthol: eight mice with two to five cells per animal.
- Figure 5.
A–D, Representative current-clamp traces baseline firing traces for male mice assigned to 6 mg/ml nicotine (A), 6 mg/ml nicotine plus menthol (B), 60 mg/ml nicotine (C), and 60 mg/ml nicotine plus menthol (D). E–H, Linear regression analysis of MHb baseline firing frequency to FR3 score for male mice assigned 6 mg/ml nicotine (E), 6 mg/ml nicotine plus menthol (F), 60 mg/ml nicotine (G), and 60 mg/ml nicotine plus menthol (H). Analysis was done through simple linear regression. Values for all animals are presented as mean per animal of 2–7 cells per animal. Blue R2 and p values with an asterisk(s) and red error bars represent significant correlations (p < 0.05).
- Figure 6.
A–D, Representative current clamp traces of voltage steps for 6 mg/ml nicotine (A), 6 mg/ml nicotine plus menthol (B), 60 mg/ml nicotine (C), and 60 mg/ml nicotine plus menthol (D). E–H, Linear regression analysis of rheobase to FR3 score for male mice assigned 6 mg/ml nicotine (E), 6 mg/ml nicotine plus menthol (F), 60 mg/ml nicotine (G), and 60 mg/ml nicotine plus menthol (H). I–L, Linear regression analysis of maximum action potentials spikes during current steps to FR3 score for male mice assigned 6 mg/ml nicotine (I), 6 mg/ml nicotine plus menthol (J), 60 mg/ml nicotine (K), and 60 mg/ml nicotine plus menthol (L). Analysis was done through simple linear regression. Values for all animals are presented as mean. Blue R2 and p values with an asterisk(s) and red error bars represent significant correlations (p < 0.05). N = 6 mg/ml Nicotine, eight mice; 6 mg/ml nicotine plus menthol, nine mice; 60 mg/ml nicotine, six mice; 60 mg/ml nicotine plus menthol, nine mice with two to seven cells per animal.
- Figure 7.
A, Comparison of rheobase between controls and tested nicotine dosages in male mice. B, Comparison of rheobase between controls and tested nicotine dosages in female mice. C, Comparison of maximal spiking ability between controls and tested nicotine dosages in male mice. D, Comparison of maximal spiking ability between controls and tested nicotine dosages in female mice. Data points represent mean rheobase or maximal spiking ability per animal. Bar graphs represent mean ± SEM and were analyzed via one-way ANOVA. *p < 0.05; **p < 0.01.
- Figure 8.
A,B, Representative UV and DIC images of α6-GFP neurons in the VTA. C–F, Representative current clamp recordings of individual voltage steps for rheobase and maximum spikes from two individual VTA dopamine neurons. G–I, Linear correlation of FR3 score with maximum action potential spike number (G), rheobase (H), and A:I ratio correlated to rheobase (I) for male mice. J–L, Linear correlation of FR3 score with maximum action potential spike number (J), rheobase (K), and A:I ratio correlated to rheobase (L) for female mice. Data are means of two to three cells for eight male and six female mice (each dot is the mean of individual mice).
- Figure 9.
A, Schematic of recording electrode and stimulator placement for brain slice FSCV assays in the NAc core. B, Representative voltammogram (insert) and color plot for a 60 Hz, 5-pulse stimulation from the NAc of a male mouse treated with nicotine plus menthol. E–H, Linear regression of FR3 score (mean FR3 active nose pokes) to tonic and phasic DA release (area under the curve, AUC) for male (E,F) and female (G,H) mice. I–L, Linear regression of PR score (mean breakpoint) to tonic and phasic DA release (area under the curve, AUC) for male (I,J) and female (K,L) mice. M–P, Linear regression of FR3 score (mean FR3 active nose pokes) to phasic/tonic ratio for male (M,N) and female (O,P) mice. Each dot represents mean data from individual mice.
Tables
6 mg/ml nic 6 mg/ml + menthol 60 mg/ml nic 60 mg/ml + menthol Whole-cell Hz R2 = 0.101
p = 0.121
R2 = 0.008
p = 0.747
R2 = 0.011
p = 0.75
R2 = 0.019
p = 0.546
Rheobase R2 = 0.002
p = 0.845
R2 = 0.000
p = 0.954
R2 = 0.048
p = 0.397
R2 = 0.065
p = 0.251
Max. spikes R2 = 0.149
p = 0.063
R2 = 0.088
p = 0.191
R2 = 0.009
p = 0.722
R2 = 0.061
p = 0.269
Table of correlations for female mice with varying nicotine dosages (6 mg/ml nicotine, 6 mg/ml nicotine plus menthol, 60 mg/ml nicotine, 60 mg/ml nicotine plus menthol) between PR score and metrics of mMHb intrinsic excitability (whole-cell firing frequency, rheobase, maximum spikes). Represented are R2 and p values from the linear regressions.
6 mg/ml nic 6 mg/ml + menthol 60 mg/ml nic 60 mg/ml + menthol Whole-cell Hz R2 = 0.182
p = 0.042
R2 = 0.169
p = 0.027
R2 = 0.072
p = 0.398
R2 = 0.349
p = 0.094
Rheobase R2 = 0.01
p = 0.653
R2 = 0.087
p = 0.113
R2 = 0.137
p = 0.144
R2 = 0.345
p = 0.097
Max. Spikes R2 = 0.000
p = 0.964
R2 = 0.107
p = 0.083
R2 = 0.092
p = 0.236
R2 = 0.051
p = 0.559
Table of correlations for male mice with varying nicotine dosages (6 mg/ml nicotine, 6 mg/ml nicotine plus menthol, 60 mg/ml nicotine, 60 mg/ml nicotine plus menthol) between PR score and metrics of mMHb intrinsic excitability (whole-cell firing frequency, rheobase, maximum spikes). Represented are R2 and p values from the linear regressions. Green values represent significant correlations with p < 0.05.
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