Article Figures & Data
Figures
- Fig. 1.
Gene targeting of the neuronal nAChR α6 subunit. a, Construction of the targeting vector. The homologous recombination event generated a 4 kb genomic deletion that removes exons 1 and 2. The targeting vector was designed to obtain a replacement mutation and contains a neomycin resistance gene (Neor) as a positive selection marker and the diphtheria toxin gene (DTA) as a negative selection marker. The expected wild-type and mutant restriction fragments after PstI digest and hybridization with the Southern blot probe are shown as dotted lines. Restriction enzymes are as follows: E,EcoRI; H, HindIII;P, PstI; S,SalI. b, Southern blot analysis of wild-type (Wt), heterozygous (α6+/−), and homozygous null mutant mice (α6−/−). Genomic tail DNA was digested withPstI and hybridized with anSalI–PstI probe, external to the targeting vector. The 4 kb band corresponds to the mutated allele.c, Detection of α6 mRNA in the brain of Wt and α6−/− animals, by in situ hybridization with an oligonucleotide located in the sixth exon of the α6 gene. In Wt animals, α6 mRNA is detected in SN–VTA neurons, LC and RGC. No signal above background levels is detected in α6−/− animals.
- Fig. 2.
Semiquantitative analysis of α3, α4, α5, α7, β2, and β4 nAChR subunit mRNAs in Wt and Mt mice usingin situ hybridization. For each subunit, optical density value of the signal was measured and corrected for background and film linearity. This value was then normalized to 100 in the region where the signal was maximal. Results are expressed as means ± SEM of at least three animals. Statistical analysis was performed using an unpaired Student's t test (p < 0.05). Regions quantified were medial habenula (MHb), interpeduncular nucleus (IPN), thalamus (Th), cortex (Cx), VTA, and hippocampus (Hp). Note the absence of significant differences in the relative abundance of the mRNA of these subunits, in any brain region.
- Fig. 3.
Quantitative autoradiography for nicotinic agonists in the mouse brain. Quantitative analysis of [3H]cytisine (a), [3H]nicotine (b), [3H]epibatidine (c), and [3H]epibatidine in the presence of 50 nm unlabeled cytisine (d), in Wt and α6−/− mice. The results are expressed as the mean specific optical density ± SEM, normalized to 100 in the dLGN of Wt animals. AU, Arbitrary unit. Statistical analysis was performed using an unpaired Student's t test (*p < 0.05; **p < 0.01) versus Wt controls. Regions quantified were SC, dLGN, retina (Ret), VTA, Cpu, IPN, MHb, and Cx.
- Fig. 4.
Autoradiograms of cytisine-resistant [3H]epibatidine binding in the mouse brain. Sections were incubated with 400 pm[3H]epibatidine in the presence of 50 nm unlabeled cytisine. See Figure 3 for quantification.
- Fig. 5.
Autoradiograms of [125I]αCtxMII binding in the mouse brain. Sections were incubated with 0.5 nm[125I]αCtxMII, as described in Materials and Methods. Residual signal seen on the eye section of α6−/− animals corresponds to nonspecific binding to pigmented epithelium. MT, Medial terminal nucleus of the accessory tract; MVN, medial vestibular nucleus; VLGN, ventrolateral geniculate nucleus.
- Fig. 6.
Displacement of [3H]epibatidine binding by αCtxMII in mouse striatal membranes. a, Comparison of total (gray bars) and αCtxMII-resistant (10 μm, white bars) [3H]epibatidine binding sites in Wt and α6−/− animals. Each point represents the mean ± SEM of six separate determinations, expressed in femtomoles per milligram of protein. Statistical analysis was performed using an unpaired Student'st test. A statistically significant fraction (21%; *p < 0.01) of [3H]epibatidine binding sites is sensitive to αCtxMII displacement in Wt but not in α6−/− animals. αCtxMII-resistant [3H]epibatidine binding sites are significantly (°p < 0.01) upregulated in α6−/− animals compared with their Wt controls, whereas total [3H]epibatidine binding is unaffected by the genotype. b, Inhibition curve of [3H]epibatidine (500 pm) binding by αCtxMII (30 pm to 3 μm) in striatal membrane preparations of α6−/− (filled symbols) and Wt (open symbols) animals. Nonspecific binding, determined in the presence of 500 nmunlabeled epibatidine, was subtracted from each measurement. Results were then expressed as a percentage of specific binding in the absence of αCtxMII. Each value is the mean ± SEM of six separate determinations. See also Table 3.
- Fig. 7.
Displacement of [3H]epibatidine binding by cytisine in mouse striatal membranes. a, Inhibition curve of [3H]epibatidine binding by cytisine: membrane samples were incubated with 500 pm[3H]epibatidine in the presence of (30 pm to 3 μm) unlabeled cytisine. Nonspecific binding, determined in the presence of 500 nmunlabeled epibatidine, was subtracted from each measurement. Results were then expressed as a percentage of specific binding in the absence of cytisine. Each point represents the mean ± SEM of five separate determinations. Data obtained from α6−/− (filled symbols) or Wt (open symbols) striatal membranes preparations were fitted to a one- (dotted line) or two-site inhibition model (see Material and Methods), respectively, depending on the value of the Hill number (see below). Statistical analysis was performed using an unpaired Student's t test (*p < 0.05).b, Hill plot representation. Log(B/(Bo −B)) is plotted as a function of log([cytisine]), whereB is the amount of specific [3H]epibatidine binding andBo is the specific binding in the absence of cytisine. In these conditions, the slope of the curve represents the Hill number, nH. In Wt animals,nH is significantly <1 (p < 0.05), suggesting the existence of an heterogeneous population of binding sites. Conversely, in α6−/− animals, nH = 0.94 ± 0.05, which suggests a homogeneous population of binding sites. See also Table4.
Tables
Target Species Probe position Mismatch Sequence of the oligonucleotide Tm (°C) α3 Rat 138–182 ? 5′-GGACACCTCAAACTGGATGATGACTGGATGGGACACATTAGCCAC-3′ 65 α4 Mouse 570–614 0 5′-GAAGTCCAGTTGGTCCACACGGCTGTGCATGCTCACCAAGTCAAT-3′ 71 α5 Rat 1077–1121 5 5′-CCGAGATTTAGGTCCAgCccCacTCTCGGCTTCTTCTCTCTGAGT-3′ 73 α6 Mouse 1451–1497 0 5′-GGGACAATACCAATTAAGAGTTTCCTGTGTTCCCAAGCAGTGGCTGC-3′ 66 α7 Rat 1206–1250 2 5′-ATCATGTGTTGGGGAGCAGGCCAAACGgCCACAtACGACCCCAGA-3′ 71 β2 Mouse 1269–1313 0 5′-TCGCATATGGTCCGCAATGAAGCGTACACCGTCCACAGCTTCCCG-3′ 71 β4 Rat 1261–1305 ? 5′-AGCTGACACCCTCTAATGCTTCCTGTAGATCTTCCCGGAACCTCC-3′ 71 TH Rat 1429–1468 2 5′-AGGTGTGCAGCTCATCCTGGACCCCCTCcAAGGAGCGCt-3′ 79 D1 Mouse 592–638 0 5′-GCTGACGATCATAATGGCTACGGGGATGTAAAAGCTTATGAGGGAGG-3′ 63 D2 Mouse 928–972 0 5′-GTTTGGCAGGACTGTCAGGGTTGCTATGTAGACCGTGGTGGGATG-3′ 70 CCK Rat 264–308 3 5′-gAAATCCATCCAGCCCATGTAGTCCCGGTCACTTATcCTgTGGCT-3′ 74 PPE Rat 386–430 3 5′-GCATCCTTCTTCATGAAagCGCCATACCTCTTGGCAAGGATCTCg-3′ 67 PPT Rat 123–167 1 5′-GGCGATTCTCTGaAGAAGATGCTCAAAGGGCTCCGGCATTGCCTC-3′ 68 The melting temperature (Tm) is calculated for the most stringent wash condition (0.01 m NaCl, 0% formamide) as described byLe Novère et al. (1996). The indicated position is in reference to the A of the ATG. When the oligonucleotide was designed according to the rat cDNA sequence, and when corresponding mouse cDNA sequence was available, we indicated the number and position (lowercase characters) of the mismatches. (“?” means that the mouse cDNA sequence was not available).
- Table 2.
Comparison of the expression levels of dopaminergic markers in Cpu and SNc of α6−/− and Wt mice
Wt (Cpu) α6−/− (Cpu) Wt (SNc) α6−/− (SNc) Cell count 82.6 ± 19.4 89.3 ± 14.4 163.7 ± 5.5 158.0 ± 5.0 TH (ICC) 0.039 ± 0.002 0.037 ± 0.002 0.26 ± 0.03 0.25 ± 0.03 DAT (ICC) 0.098 ± 0.002 0.100 ± 0.002 0.31 ± 0.02 0.32 ± 0.02 TH (ISH) ND ND 0.83 ± 0.02 0.82 ± 0.01 D1 (ISH) 0.57 ± 0.05 0.56 ± 0.02 ND ND D2 (ISH) 0.70 ± 0.01 0.68 ± 0.01 0.53 ± 0.01 0.53 ± 0.02 PPE (ISH) 0.62 ± 0.01 0.58 ± 0.02 ND ND PPT (ISH) 0.70 ± 0.02 0.70 ± 0.01 ND ND CCK (ISH) ND ND 0.33 ± 0.01 0.33 ± 0.01 D1(LBA) 1.06 ± 0.03 1.02 ± 0.04 ND ND D2 (LBA) 0.60 ± 0.02 0.58 ± 0.01 0.041 ± 0.001 0.044 ± 0.001 In situ hybridization (ISH), immunocytochemistry (ICC), and ligand binding autoradiography (LBA) were used to compare the levels of expression of various markers in Wt and α6−/− mice at the striatal and mesencephalic levels. Only the results of semiquantitative analysis in Cpu and SNc are presented here. Except for the cell number, each value represents the mean specific optical density ± SEM, obtained from eight animals of each genotype. Cell count was performed on TH-immunostained (SNc) or Nissl-stained (Cpu) sections. [3]SCH23390 and [3H]raclopride were used for the determination of D1 and D2 dopamine receptor levels (respectively). ND, Not detected.
- Table 3.
αCtxMII inhibition of [3H]epibatidine binding on superior colliculus and striatal membranes
Total [3H]epibatidine binding (fmol/mg protein) αCtxMII sensitive population (fmol/mg protein) Ki (nm) Wt (SC) 173.4 ± 9.6 59.7 ± 3.8 ND α6−/−(SC) 102.0 ± 6.8 9.7 ± 1.8 ND Wt(Str) 57.9 ± 2.0 12.1 ± 1.0 1.4 ± 0.2 α6−/−(Str) 53.8 ± 1.7 −1.2 ± 0.8 NA Membranes were prepared from SC and striatum (Str) of Wt and α6−/− mice. αCtxMII-sensitive sites were measured using 10 μm unlabeled αCtxMII with 500 pm[3H]epibatidine. Each value represents the mean ± SEM of six separate experiments (expressed in femtomoles per milligram of protein). For Ki determination, data were fitted to a one site model (see Material and Methods). NA, Not applicable. ND, Not determined.
B1 (fmol/mg protein) Ki1(nM) B2 (fmol/mg protein) Ki2 (nM) Wt 45.9 ± 3.4 0.23 ± 0.03 12.8 ± 2.8 4.0 ± 1.3 α6−/− 56.5 ± 1.8 0.32 ± 0.03 — — Membranes were prepared from striatum of Wt and α6−/− mice. Membrane samples were incubated in the presence of 500 pm[3H]epibatidine with 30 pm to 3 μm unlabeled cytisine. Results were fitted to a one (α6−/−) or two (Wt) site model (see Material and Methods). Each value represents the mean ± SEM of five separate experiments.
