Article Figures & Data
Figures
- Figure 1.
Generation of α4 Lox-only mice. In the targeting construct used to generate α4 Lox-only mice, 34 bp loxP sites (black triangles) were inserted into the intronic regions on either side of exon V of the α4 gene. The neomysin resistance gene (neor) was included in the targeting construct to facilitate selection of ES cells that had undergone homologous recombination. The diphtheria toxin fragment A (DT-A) gene was inserted beyond the region of homologous DNA as a negative selection marker. In ES cells, the neomycin resistance gene was removed by Cre-mediated recombination. As recombination can occur between any two of the three loxP sites, several recombination products were obtained. ES cells containing product I with the floxed exon V but no neomycin resistance gene were injected into C57BL/6 mouse blastocysts and implanted into pseudopregnant females. PGK, Phosphoglucose kinase.
- Figure 2.
Deletion of α4 in dopaminergic regions in α4-DA mice. A–C, Expression of α4 mRNA (A), Cre recombinase mRNA (B), and high-affinity 125I-epibatidine binding sites (C) at the level of the dopaminergic cell bodies (VTA/SN) are shown in adjacent brain slices for wild-type, Cre-only control, Lox-only control, α4-DA, and α4-null mice. D, 125I-Epibatidine binding is also shown at the level of the ST that contains the terminal ends of mesolimbic dopaminergic neurons.
- Figure 3.
Selective deletion of α4 in dopaminergic neurons. A–H, Brain slices from wild-type controls (A–D) and α4-DA (E–H) mice were labeled for α4 mRNA (red, B, F), a marker for dopaminergic neurons (TH; green, C, G), and a marker of GABAergic neurons (GAD67; blue, D, H). Dopaminergic neurons are indicated by arrows, and GABAergic neurons by underlined arrows. Scale bars: 15 μm.
- Figure 4.
Functional confirmation of α4 deletion from dopaminergic neurons. A, B, α4β2*-nAChR-mediated (α-CtxMII-resistant; A) and α6β2*-nAChR-mediated (α-CtxMII-sensitive; B) dopamine release from two dopaminergic projection regions, OT and ST. C, α4β2*-nAChR-mediated GABA release from ST and CX. For both dopamine and GABA release, full dose–response curves were measured; representative concentrations are reported here (units normalized to baseline). K+-stimulated neurotransmitter release did not differ across genotypes (data not shown). D–F, 125I-Epibatidine binding (femtomoles per milligram of protein) in OT, ST, and CX. D, Cytistine-sensitive component (representing α4β2*-nAChRs). E, α-CtxMII-sensitive component (α6β2*-nAChRs). F, Total (representing all high-affinity heteromeric nAChRs). *p < 0.05.
- Figure 5.
α4-containing nAChRs on dopaminergic neurons mediate nicotine reward but not cocaine reward. A, Nicotine elicited significant place preference at 0.065 mg/kg (i.p.) in all three control groups (wild type, Cre-only control, and Lox-only control). Neither the α4-null mice nor the α4-DA mice developed a preference for the nicotine-paired side over a wide range of doses (0.04, 0.065, and 0.09 mg/kg). **p = 0.01. B, Cocaine conditioned place preference. There was no difference between mice at any dose studied.
- Figure 6.
α4-containing nAChRs on dopaminergic neurons partially mediate the anxiolytic effects of nicotine. A, B, Nicotine at 0.01 mg/kg significantly decreased anxiety in all three control groups (wild type, Cre-only control, and Lox-only control) as measured by increased time (A) and entries (B) into the open arm. α4-null mice did not show a change in anxiety as measured by time and entries into the open arm (*p < 0.05). α4-DA mice showed a decreased sensitivity to nicotine, with significantly less time spent in the open arm (#p < 0.05) than control mice. C, There were no differences between wild-type control, Lox-only control, α4-null, and α4-DA mice in activity (total entries into open and closed arms); however, Cre-only control mice showed increased activity (**p < 0.01). Data are scaled as the square root of dose.
- Figure 7.
α4-containing nAChRs on dopaminergic neurons are involved in locomotor suppression but not thermoregulatory effects of nicotine. A, B, Activity [crosses (A) and rears (B)] were recorded over 3 min in the Y-maze. All groups of mice showed locomotor suppression; however, α4-null mice showed decreased sensitivity (***p < 0.001, wild type by α4-null), whereas α4-DA mice showed increased sensitivity as measured by crosses (**p < 0.05, wild type by α4-DA). α4-null mice also showed decrease sensitivity to nicotine as measure by rears (*p < 0.05, wild type by α4-null). Although α4-DA mice did not show increased sensitivity relative to control mice in rears, they did show a significant difference from α4-null mice (#p < 0.05, α4-DA by α4-null). C, Similarly, all mice showed decrease in body temperature in response to increasing doses of nicotine. α4-null mice showed decreased sensitivity (***p < 0.0001, wild type by α4-null), whereas α4-DA mice did not differ from control groups. Data are scaled as the square root of dose.
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