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. 2023 Jul 6;12:e87902. doi: 10.7554/eLife.87902

Figure 1. DAT::NrxnsKO mice exhibit impaired amphetamine-induced motor activity.

(A) Schematic representation of a mouse on a rotarod and the diagram of the rotarod testing protocol for the speed 1. (B) Performance on the accelerating rotarod during nine sessions over 3 consecutive days. Latency to fall was quantified at rotation speeds from 4 to 40 rpm over 10 min. (C) Performance of DAT::NrxnsKO and WT littermate mice on the rotarod was evaluated comparing the last session and the first session for each mouse. The results show a significant improvement in performance irrespective of genotype. (D) Quantification of the terminal speed over all the sessions shows no difference between the DAT::NrxnsKO and WT littermate mice. (E) Basal horizontal activity in a novel environment before and after a saline injection (10 mL/kg) over a total of 60 min. (F) Horizontal activity before and after a cocaine injection (20 mg/kg; 10 mL/kg) over a total of 60 min. (G) Horizontal activity before and after an amphetamine injection (5 mg/kg; 10 mL/kg) over 60 min shows reduced locomotion in the DAT::NrxnsKO compared to the control mice. For rotarod and locomotor activity experiments, 7–10 animals per group were used. For all analyses, the plots represent the mean ± SEM. Statistical analyses were carried out by two-way ANOVAs followed by Tukey’s multiple comparison tests or Sidak’s multiple comparisons test. The stars in panel D represent the level of significance of the post hoc tests (*p<0.05; **p<0.01).

Figure 1—source data 1. Contains the primary data for Figure 1 and Figure 1—figure supplement 2.

Figure 1.

Figure 1—figure supplement 1. Breeding scheme for generation of DAT::NrxnsKO; DAT::NrxnsWT, and DAT::NrxnsHET.

Figure 1—figure supplement 1.

Homozygous Nrxn 123flox mice were initially crossed with homozygous DATIRES-CRE mouse line. Heterogygous mice for Nrxn 123flox and DATIRES-CRE mice were then crossed with homozygous DATIRES-CRE mice. Heterozygous Nrxn 123flox mice; homozygous DATIRES-CRE mice were finally crossed with heterogygous mice for Nrxn 123flox to obtain the final generation of DAT::NrxnsKO; DAT::NrxnsWT and DAT::NrxnsHET. All animals were genotyped prior to weaning at P21 for both, Nrxn flox and DAT Cre genes.
Figure 1—figure supplement 2. Lack of notable changes in the behavioral performance of DAT::NrxnsKO mice.

Figure 1—figure supplement 2.

(A) Schematic representation of a mouse on a rotarod and the diagram of the rotarod testing protocol for speeds between 4 and 4 rpm over 2 min (speed 2). (B) Performance on the second accelerating rotarod task during nine sessions over 3 consecutive days. Latency to fall was quantified at rotation speeds from 4 to 40 rpm over 2 min. (C) Performance of DAT::NrxnsWT and KO littermate mice on the rotarod was evaluated comparing the last and first sessions for each mouse. No significant improvement in performance was detected, irrespective of genotype. (D) Quantification of the final speed over all sessions shows no difference between the DAT::NrxnsWT and KO littermate mice. (E) Summary graph of the time to turn in the pole test shows no genotype effect (WT = 7.10 ± 1.50 s and KO = 4.57 ± 0.75 s). (F) Summary graph showing that the time required to climb down the pole was significantly higher for the DAT::NrxnsKO mice; (unpaired t-test, p=0.034) (WT = 5.80 ± 0.53 s and KO = 8.14 ± 0.93 s). (G) Schematic representation of the sucrose preference testing protocol. (H) Quantification of sucrose preference in comparison to water consumption represented as a percentage. Initial 2 days: DAT::NrxnsKO CD1: 51.47±1.63% vs 48.52 ± 1.63% and CD2: 54.01 ± 3.17%, vs 45.99 ± 3.17%; DAT::NrxnsWT CD1: 50.58±1.47% vs 49.41 ± 1.47% and CD2: 54.73 ± 4.27%, vs 45.26 ± 4.27%. Results are presented as percentage of choice water/water. Following 3 test days: DAT::NrxnsKO TD1: 81.24±2.44% vs 18.75 ± 2.44%; TD2: 74.65 ± 1.39%, vs 25.34 ± 1.39% and TD3: 78.74 ± 1.37%, vs 21.25 ± 1.37%; DAT::NrxnsWT TD1: 80.52±1.74% vs 19.47 ± 1.74%; TD2: 76.21 ± 1.75%, vs 23.78 ± 1.75% and TD3: 78.58 ± 2.00%, vs 21.41 ± 2.00%. Results are presented as percentage of choice sucrose/water. For rotarod and locomotor activity experiments, 7–14 animals per group were used. For sucrose experiment, 7–8 mice per group were used. For the pole test experiment, 7–10 animals per group were used. For all analyses, the plots represent the mean ± SEM. Statistical analyses were carried out by two-way ANOVAs followed by Tukey’s multiple comparison tests or Student’s t-test (*p<0.05; ****p<0.0001).