Reduction of DAT and TH immunostaining in striatum of DAT knockout mice. (A) DAT immunoreactivity in sections through striatum and nucleus accumbens from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) DAT knockout mice, as indicated. (B) TH immunoreactivity in striata and nucleus accumbens from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) DAT knockout mice, as indicated.

Spontaneous hyperlocomotion and attenuated cocaine-induced locomotion in DAT knockout mice. Locomotor activities were recorded for 1-hr periods in wild type (+/+, open bars) and heterozygous (+/−, gray bars) and homozygous (−/−, black bars) DAT knockout mice placed into an activity monitor cage to which they had not been previously exposed at the time 3 hr before injection with cocaine (10 mg/kg s.c.) at the arrow (time 0). Homozygous knockout mice show greater locomotor activity when placed in a novel environment, but less locomotor response to cocaine. Values represent mean ± SEM; ∗, P < 0.05 versus wild-type group; #, P < 0.05 versus the locomotor activity before injection; n = 7–14 mice per genotype.

Cocaine-conditioned place preferences in 5-HTT knockout mice. Conditioned place preference induced by cocaine in wild type (+/+, open bars) and heterozygous (+/−, gray bars) and homozygous (−/−, black bars) 5-HTT knockout mice. Time scores shown represent differences between post-conditioning (Post) and pre-conditioning (Pre) time spent in the cocaine-paired environment. Homozygous mice showed significantly more place preference than wild type (P < 0.05, ANOVA; n = 8–17 mice per genotype). 5-HTT knockout mice displayed significant place preference associated with 10 mg/kg cocaine in gene-dose-related fashion (∗, P < 0.01; ∗∗, P < 0.001 vs. saline- injected group by Student’s t test; n = 8–17 mice per genotype). 5-HTT knockout mice were constructed as in ref. 20. The following data show the real mean time scores ± SEM (sec) of pre-conditioning (Pre) and post-conditioning (Post) sessions on initially nonpreferred compartment in each genotype. Wild type [Pre/Post], heterozygote [Pre/Post], homozygote [Pre/Post]: saline ([324.7 ± 36.0/326.2 ± 51.7], [364.5 ± 52.5/426.3 ± 62.0], [296.5 ± 60.0/315.9 ± 76.7]), cocaine 10 mg/kg ([329.1 ± 33.6/575.3 ± 74.8], [221.5 ± 23.3/620.3 ± 106.0], [290.7 ± 44.1/848.1 ± 76.0].

Cocaine- and methylphenidate-conditioned place preferences in DAT knockout mice. (A) Conditioned place preference induced by cocaine in wild type (+/+, open bars) and heterozygous (+/−, gray bars) and homozygous (−/−, black bars) DAT knockout mice. Time scores shown represent differences between post-conditioning (Post) and pre-conditioning (Pre) time spent in the cocaine-paired environment. Wild-type mice displayed significant place preference associated with 5 and 10 mg/kg cocaine, whereas heterozygous and homozygous animals showed significant place preference associated with 10 mg/kg cocaine. ∗ P < 0.05 vs. saline-injected group by ANOVA; n = 8–23 mice per genotype. The following data show the real mean time scores ± SEM (sec) of pre-conditioning (Pre) and post-conditioning (Post) sessions on initially nonpreferred compartment in each genotype. Wild-type [Pre/Post], heterozygote [Pre/Post], homozygote [Pre/Post]: saline ([411.4 ± 30.0/382.8 ± 50.4], [387.0 ± 36.0/392.6 ± 47.2], [408.6 ± 29.1/364.6 ± 65.0]), cocaine 5 mg/kg ([304.1 ± 35.0/489.0 ± 67.8], [354.4 ± 37.7/495.9 ± 70.7], [348.2 ± 29.1/455.0 ± 114.8]), cocaine 10 mg/kg ([336.7 ± 25.6/526.0 ± 59.2], [327.9 ± 26.0/655.9 ± 64.9], [328.7 ± 26.9/597.5 ± 50.5]). (B) Conditioned place preference induced by methylphenidate in wild type (+/+, open bars) and heterozygous (+/−, gray bars) and homozygous (−/−, black bars) DAT knockout mice. Time scores shown represent differences between post-conditioning (Post) and pre-conditioning (Pre) time spent in the cocaine-paired environment. All three genotypes displayed significant place preference associated with 5 mg/kg methylphenidate. ∗∗, P < 0.01; ∗, P < 0.05 vs. saline-injected group by ANOVA; n = 12–21 mice per genotype. The following data show the real mean time scores ± SEM (sec) of pre-conditioning (Pre) and post-conditioning (Post) sessions on initially nonpreferred compartment in each genotype. Wild-type [Pre/Post], heterozygote [Pre/Post], homozygote [Pre/Post]: saline ([411.4 ± 30.0/382.8 ± 50.4], [387.0 ± 36.0/392.6 ± 47.2], [408.6 ± 29.1/364.6 ± 65.0]), methylphenidate 5 mg/kg ([356.6 ± 34.6/562.5 ± 68.3], [367.4 ± 22.1/552.6 ± 56.0], [376.6 ± 38.7/533.0 ± 77.2]).

Disruption of the DAT gene. The DAT gene was inactivated by replacement of exon I and II with a neomycin-resistance (neo^r) cassette. (A) Schematic representation of 5′ portions of the murine DAT gene, with positions of exons I–IV (filled boxes), the start codon (ATG), and BclI (Bcl), EcoRI (RI), BamHI (B), ApaI (Ap), SmaI (Sm), and SpeI (Sp) restriction endonuclease sites noted. Scale bar = 1 kb. (B) The pDATKO targeting vector, indicating neomycin resistance gene (neo^r) and MC1 thymidine kinase (TK) sequences. The direction of gene transcription is marked by the horizontal arrows. Abbreviations and scale as in A. (C) Predicted mutant allele resulting in the disrupted DAT gene. The locations of the 5′ probe used in the Southern blot are indicated. The first and second exons are missing from the mutant allele. Abbreviations and scale as in A and B. The sizes of the EcoRI fragment from wild-type (12 kb) and mutated alleles (5 kb) are indicated. (D) Genomic Southern blot analysis of hybridization of the 5′ DAT genomic probe to EcoRI-digested DNA extracted from wild-type (+/+), heterozygote (+/−), and homozygote (−/−) mouse tails. The presence of a 5-kb fragment indicates a homozygous mutant genotype, whereas wild-type fragments are 12 kb. (E) Scatchard analyses of saturation radioligand binding of [³H]CFT (WIN35,428) to striatal membranes from DAT knockout mice. Mean (± SEM, n = 3) values for B_max were 2.62 ± 0.5 pmol/mg of protein, 1.45 ± 0.18, and undetectable for wild-type (+/+, ○), heterozygous (+/−, •), and homozygous (−/−, ▴) DAT knockout mice, respectively. K_d values were 20.1 ± 1.6 nM, 27.0 ± 4.3 nM, and undetectable, respectively.