Article Figures & Data
Figures
- Figure 1.
mVTA to latVTA glutamate transmission is monosynaptic. a, AAV-DIO-ChR2-EYFP vectors were unilaterally microinjected into mVTA of VGluT2-Cre mice to permit optical stimulation of mVTA terminals in latVTA. b, TH/EYFP stain shows ChR2 expression in mVTA somata. c, TH/EYFP stain in latVTA shows ChR2 expression from mVTA in terminals surrounding TH+ cells. d, oEPSCs recorded in latVTA are sensitive to CNQX (10 μm)/d-AP5 (30 μm) application. e, An oEPSC trace family from a representative latVTA neuron is shown (gray, individual stimulation trials; blue, averaged trace). oEPSCs were measured at baseline (i) and following superfusion of the indicated drugs (ii–iv). f, Summary before/after scatter plot for all cells studied as described in e. n = 5 neurons from 4 mice (1 male, 3 female).
- Figure 2.
Nicotine modulation of monosynaptic mVTA-to-latVTA glutamate transmission. VGluT2+, ChR2-bearing fibers from mVTA were stimulated during recordings from latVTA cells at baseline, after superfusion with nicotine (0.3 μm), and following superfusion with nicotine + DhβE + MLA. a, Before/after plot summarizing all tested cells. b, Baseline versus nicotine data from a are plotted on an x–y plot where the blue dashed line indicates the unity line of no change. Open red circles indicate cells exhibiting a nicotine-mediated reduction, whereas open green circles indicate cells exhibiting a nicotine-mediated increase. c, An oEPSC trace family is shown from a representative latVTA neuron exhibiting a nicotine-mediated decrease in oEPSC amplitude (gray, individual stimulation trials; blue, averaged trace). d, Summary before/after scatter plot for all red data points indicated in b. n = 5 neurons from 4 mice (2 male, 2 female). e, An oEPSC trace family is shown from a representative latVTA neuron exhibiting a nicotine-mediated increase in oEPSC amplitude (gray, individual stimulation trials; blue, averaged trace). f, Summary before/after scatter plot for all green data points indicated in b. n = 6 neurons from 5 mice (2 male, 3 female).
- Figure 3.
Nicotine-mediated suppression of oEPSCs occurs through GABA release. a, An oEPSC trace family from a representative latVTA neuron, which exhibited nicotine-mediated suppression of the oEPSC, is shown (gray, individual stimulation trials; blue, averaged trace). oEPSCs were measured at baseline (i), after superfusion of nicotine (0.3 μm; ii), after nicotine plus PTX (50 μm; iii), and after nicotine/PTX plus CNQX (10 μm) and d-AP5 (30 μm; iv). b, Summary before/after scatter plot for all cells studied as described in a. n = 6 neurons from 4 mice (2 male, 2 female). c, An oEPSC trace family from a representative latVTA neuron, which exhibited nicotine-mediated enhancement of the oEPSC, is shown (gray, individual stimulation trials; blue, averaged trace). oEPSCs were measured at baseline (i), after superfusion of nicotine (0.3 μm; ii), after nicotine plus PTX (50 μm; iii), and after nicotine/PTX plus CNQX (10 μm) and d-AP5 (30 μm; iv). d, Summary before/after scatter plot for all cells studied as described in c. n = 8 neurons from 6 mice (4 male, 2 female).
- Figure 4.
Coexpression of VGluT2 and Gad2 in mVTA. a, FISH in mVTA neurons for probes: VGluT2, Gad2. Individual, representative VGluT2+/Gad2− (1), VGluT2−/Gad2+ (2), and VGluT2+/Gad2+ (3) cells are shown (images at bottom right). b, Scatter plot of VGluT2 (abscissa) versus Gad2 (ordinate) normalized percentage of coverage for all nuclei in mVTA FISH images [n = 3 mice (1 male, 2 female)]. Chrnb2 normalized percentage of coverage for each nucleus is represented via the indicated dot color, as defined by the scale at right. c, Top, Pie graph of VGluT2+ nuclei showing the fraction of Gad2+ and Gad2− nuclei. Bottom, Pie graph of VGluT2+/Gad2+ nuclei showing fraction of Chrnb2+ and Chrnb2− nuclei.
- Figure 5.
mVTA to latVTA GABA transmission is largely monosynaptic. a, AAV-DIO-ChR2-EYFP vectors were unilaterally microinjected into mVTA of Gad2-Cre mice to permit optical stimulation of mVTA terminals in latVTA. b, TH/EYFP stain shows ChR2 expression in mVTA somata. c, TH/EYFP stain in latVTA shows ChR2 expression from mVTA in terminals surrounding TH+ cells. d, Optical IPSCs recorded in latVTA are sensitive to PTX (50 μm) application. e, An oIPSC trace family from a representative latVTA neuron is shown (gray, individual stimulation trials; blue, averaged trace). oIPSCs were measured at baseline (i) and following superfusion of the indicated drugs (ii–iv). f, Summary before/after scatter plot for all cells studied as described in e. n = 12 neurons from 7 mice (3 male, 4 female). g, Data from f are shown normalized to their baseline responses. Data points in red indicate cells whose oIPSC was not recoverable with 4-AP following TTX treatment.
- Figure 6.
Nicotine modulation of monosynaptic mVTA to latVTA GABA transmission. a, An oIPSC trace family from a representative latVTA neuron is shown (gray, individual stimulation trials; blue, averaged trace). oIPSCs were measured at baseline and following superfusion of nicotine, then nicotine + DhβE + MLA. b, Summary before/after scatter plot for all cells studied, as described in a. The cell indicated by gray circles was excluded as an outlier from statistical analysis. n = 8 neurons (1 of 8 excluded) from 6 mice (3 male, 3 female).
Tables
Fig. Data structure Type of test p Value Effect size** 95% CI (pA) a 1f Normal One-way RM ANOVA 0.0017 0.8521 n/a b 1f Normal Sidak post hoc 0.0083* −92.3 pA −37.8 to −146.8 c 1f Normal Sidak post hoc 0.0028* 82.5 pA 119.o to 46.0 d 2a Normal One-way RM ANOVA 0.5454 0.0395 n/a e 2d Normal Paired t test (one tailed) 0.0019 14.5 pA 7.8 to 21.2 f 2f Normal Paired t test (one tailed) 0.0032 −20.47 pA −32.2 to −8.7 g 3b Normal Paired t test (one tailed) 0.0065 14.55 pA 4.2 to 24.9 h 3d Normal Paired t test (one tailed) 0.2721 −1.45 pA −7.2 to 4.3 i 5f Normal One-way RM ANOVA 0.0010 0.5563 n/a j 5f Normal Sidak post hoc 0.0000026* −60.45 pA −43.1 to −77.9 k 5f Normal Sidak post hoc 0.0051* 64.45 pA 107.8 to 21.1 l 6b Normal One-way RM ANOVA 0.00000031 0.9384 n/a m 6b Normal Sidak post hoc 0.000070* −20.67 pA −15.1 to −26.3 n 6b Normal Sidak post hoc 0.000069* 16.79 pA 21.3 to 12.3 Effect sizes and power were calculated with G*power 3.1. n/a, Not applicable; RM, repeated measures.
↵*Multiplicity-adjusted p value; **For RM ANOVA, effect size reported is R 2 = SStreatment/(SStreatment + SSresidual); for Sidak test and paired t test, effect size reported is the mean difference in pA.
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