Fig 1.
Conditional KO of α4 nAChR subunits.
a) α4-flox mice have loxP sites flanking exon 5 of the α4 nAChR gene. Infusion of viral vectors expressing Cre recombinase into the VTA results in conditional deletion of α4 in infected cells. b) The three main heteromeric nAChR subtypes found in neurons of the midbrain DA system, α4α6β2*, α6 β2(non-α4)*, and α4β2(non-α6)*, are shown. Deletion of α4 eliminates α4α6β2* and α4β2(non-α6)* subtypes, while isolating the activity of α6β2(non-α4)* nAChRs. c) Viral spread of AAV-GFP vectors. An AAV-GFP virus was infused bilaterally into the VTA of a C57BL/6 WT mouse, followed 3 weeks later by perfusion and anti-GFP staining of brain sections.
Fig 2.
Enhanced nicotine oral SA in mice with vMB α4 deletion.
a) Mean (± SEM) nicotine intake (mg/kg) over 4 days is shown for each nicotine concentration, in male and female α4-flox GFP(+) (n = 26) and Cre(+) (n = 24) mice. *p<0.05. b) Mean (± SEM) nicotine intake ratio (fraction of nicotine-containing fluid consumed relative to total fluid consumed) over 4 days is shown for each nicotine concentration in α4-flox GFP(+) and Cre(+) mice. c) Mean (± SEM) intake of nicotine solution (mL/kg), used to calculate mass of nicotine intake shown in (a), is shown for each nicotine concentration in α4-flox GFP(+) and Cre(+) mice. d) Mean (± SEM) water intake (mL/kg) over 4 days is shown for each nicotine concentration in α4-flox GFP(+) and Cre(+) mice. e) Mean (± SEM) total fluid intake (mL/kg) over 4 days is shown for each nicotine concentration in GFP(+) and Cre(+) mice. f) Mean (± SEM) body weight (g) for α4-flox GFP(+) and Cre(+) mice is shown for each nicotine concentration. S2, S3 and S4 Figs show intake data separated by sex.
Fig 3.
Nicotine CPP is reduced in mice with vMB α4 deletion.
a) CPP schematic. Prior to a 5-day, biased CPP procedure to establish nicotine CPP, mice were mildly food-restricted and handled. Drug-free pre-test and post-test days flanked 3 consecutive conditioning days that consisted of morning and afternoon saline (SAL) and nicotine (NIC) conditioning sessions. b) Nicotine CPP in C57Bl/6 WT mice. Groups of WT mice were conditioned with saline (n = 12), 0.25 mg/kg NIC (n = 12), or 0.5 mg/kg NIC (n = 12) to validate our CPP assay and identify a dose to be used subsequently in α4-flox mice. Mean (± SEM) place preference score is shown for the three drug doses. p values are for Dunnett’s multiple comparisons test. c) Nicotine CPP in α4-flox mice. AAV-GFP or AAV-Cre-GFP vectors were infused into vMB of α4-flox mice (GFP(+), n = 10; Cre(+), n = 10), and mice were subsequently conditioned with 0.25 mg/kg NIC. Mean (± SEM) place preference score is shown for both groups. p value is for unpaired t-test. d) Mean (± SEM) locomotor activity during the pre-test and post-test is shown for α4-flox mice conditioned with NIC. e) Mean (± SEM) locomotor activity during conditioning sessions 1, 2, and 3 is shown for α4-flox mice conditioned with NIC.
Fig 4.
Functional deletion of α4 subunits in VTA.
a) Recordings were made from α4-flox;Cre(-) and Cre(+) VTA DA neurons. ACh (1 mM) was applied by pressure ejection and inward currents were recorded. All recorded traces are shown in gray, and an averaged trace is shown in blue. Scale bar: 40 pA, 400 ms. b) Quantification of ACh-evoked currents in AAV-infected mice. Mean inward ACh-evoked currents from α4-flox;Cre(-) (n = 19 cells from n = 9 animals), α4-flox;Cre(+) (n = 14 cells from n = 5 animals), WT;Cre(-) (n = 7 cells from n = 2 animals), and WT;Cre(+) (n = 8 cells from n = 2 animals) cells ± SEM. ****p<0.0001 (unpaired t-test, t = 8.788). c) Residual ACh-evoked currents in α4-flox;Cre(+) neurons are αCtxMII-sensitive. For Cre(+) responses in (B), αCtxMII (100 nM) was applied by superfusion and ACh was re-applied after 12–15 min. Before-after responses for n = 4 αCtxMII-treated neurons (n = 2 animals) are shown. **p = 0.0052 (paired t-test, t = 7.348).
Fig 5.
Reduced AMPA/NMDA ratios in α4-flox;Cre(+) VTA DA neurons.
a) AMPA/NMDA ratios were measured as described in Materials and Methods. A representative evoked synaptic current in a neuron voltage clamped at -70 mV and +40 mV is shown. b) Quantification of AMPA/NMDA ratios. In each VTA DA neuron from α4-flox;Cre(+) and GFP(+) mice injected with saline (SAL) or nicotine (NIC; 0.17 mg/kg), mean (± SEM) AMPA/NMDA ratio is shown. Replicate cells and number of animals: GFP(+) SAL, n = 8 cells, n = 3 animals; GFP(+) NIC, n = 16 cells, n = 5 animals; Cre(+) SAL, n = 8 cells, n = 3 animals; Cre(+) NIC, n = 7 cells, n = 2 animals. ****p<0.0001 (Bonferroni’s multiple comparison test following two-way ANOVA).
Fig 6.
Cre virus infection of VTA DA and GABA neurons.
a) Targeting the VTA with AAV vectors. Mice were infused into the VTA with AAV-Cre-GFP vectors. After 2–3 weeks, mice were perfused and brains were sectioned for immunohistochemistry. Anti-GFP staining identifies infected neurons, while anti-TH staining identifies DA neurons. High magnification (60X) images show Cre-GFP expression in TH(+) and TH(-) neurons in VTA, indicated by one or two arrows, respectively. IPN: interpeduncular nucleus, ML: medial lemniscus. b) Cre recombinase expression in VTA GABA neurons. Slices from the infection shown in (a) were stained with anti-GAD65/67 antibodies. Native GFP signal was imaged without anti-GFP staining.