Animals
Male mice (8–11 weeks old) from the following lines were used: C57BL/6N mice; NT-KO mice lacking the region encoding the proteolytic domain28; and its control NT-WT mice. All animals were housed in groups on standard a 12 h light: dark cycle with ad libitum access to food and water. The C57BL/6N strain was used for ATPase assay and Golgi-Cox staining. NT-KO and NT-WT mice were used for behavioral testing. All behavioral experiments were started at Zeitgeber time 4. All behavioral tests and intraperitoneal administration of digoxin were performed without anesthesia. Cervical dislocation was performed by an expert as a method of euthanasia before ATPase activity assay and spine analysis, and after behavioral tests. All experimental protocols were approved by animal care committee at Osaka Metropolitan University. All methods were carried out in accordance with relevant guidelines and regulations, and are reported below in accordance with ARRIVE guidelines.
Administration of digoxin
Digoxin (nacalai tesque, Kyoto, Japan) was dissolved in pyridine at a concentration of 0.02 mg/kg, 0.08 mg/kg, 1.3 mg/kg or 13 mg/kg, then diluted with saline up to a final concentration of 1 µg/kg, 4 µg/kg, 65 µg/kg, and 650 µg/kg of digoxin per 100 µl. Digoxin at the above doses was administered intraperitoneally without anesthesia. Na/K ATPase activity in rat hippocampus was reportedly increased by intraperitoneal administrations of digoxin at low concentration (65 µg/kg), but decreased at high concentration (650 µg/kg)14. Intraperitoneal administration of 65 or 650 µg/kg of digoxin corresponded to a dose below or above its IC50 for brain Na/K ATPase inhibition (25 nM for the α2 and α3 subunits; 130 µg/kg is equivalent to IC50), respectively, which was calculated from a previous study29. Concentrations of 1 µg/kg and 4 µg/kg were additionally adopted as doses approximately corresponding to clinical doses for humans. The effects of 1 and 4 µg/kg digoxin were analyzed in anticipation of drug repurposing as treatment for brain disorders in addition to cardiac failure.
ATPase activity assay
To assess the Na/K ATPase activity in brain, we calculated the amount of free phosphate ion released from ATP by ATPase using an ATPase Activity Assay Kit (BioVision, Waltham, MA, USA), because approximately 50% of ATP is consumed by Na/K ATPase in the brain30,31. Briefly, Following cervical dislocation and decapitation 1 h after intraperitoneal administration of vehicle only or digoxin at 1, 4, 65, or 650 µg/kg, we isolated and homogenized 40 mg of cerebral cortex from the parietal lobe of 10-week-old male C57BL/6N mice. After removing endogenous phosphate using the ammonium sulfate method, the amount of free phosphate ion released by ATPase was measured using Malachite Green Reagent according to the procedure recommended by the manufacturer. The absorbance of samples was measured at 650 nm using a microplate reader (MPR-A100; AS ONE Corp., Osaka, Japan).
Dendritic spine analysis
To visualize the dendritic spine, we performed Golgi-Cox staining using the FD Rapid Golgistain™ Kit (FD NeuroTechnologies, Columbia, MD, USA). Although the two-dimensional analysis using Golgi-Cox staining has limitations in acquiring 3D information, this method is still useful for comparing the spine morphology under various conditions. We used 10-week-old male C57BL/6N mice and 8-week-old male NT-KO mice for dendritic spine analysis. Following cervical dislocation and decapitation 1 h after intraperitoneal administration of vehicle only or digoxin at 1, 4, 65, or 650 µg/kg, we rapidly removed the brain. Removed brains were divided into equal thirds along the rostrocaudal axis, then the rostral and middle parts of the brain including cerebral cortex and hippocampus were stained by the Golgi-Cox method according to the instructions from the manufacturer and a previous study32. The border between rostral and middle parts was approximately located at the bregma level. Briefly, fresh brain blocks were immersed in a mixture of equal volumes of kit Solution A and B, then stored for 3 weeks under dark conditions. This solution was replaced with fresh solution after the first 24 h of immersion. Samples were subsequently immersed in Solution C for 1 week. Solution C was also replaced with fresh solution after the first 24 h of immersion. After immersion in Solution C, brain blocks were rapidly frozen in powdered dry ice, then tissue sections of 200 µm thickness along the rostrocaudal axis were sliced at -22 to -24°C using a cryostat microtome (HM525NX; PHC Corp., Tokyo, Japan). Sections were mounted with Solution C on glass slides precoated with 0.5% gelatin, then dried naturally at room temperature. Subsequently, the sections were stained using a solution containing 1 part Solution D, 1 part Solution E, and 2 parts distilled water (DW) for 5 min. After staining, slides were rinsed in DW twice for 4 min each, then dehydrated in a serial dilution of ethanol and cleared in xylene. Finally, slides were coverslipped and sealed using Entellan (Merck, Darmstadt, Germany). We performed two times of independent staining using 2 mice under each condition.
Bright field images of stained sections were acquired using a microscope (BX50; Olympus Corp., Tokyo, Japan) with the NY-X9 digital photographic device system (Microscope Network Corp, Saitama, Japan). The morphological characterization of newly formed dendritic spines (filopodia and thin-type spines) is that the spine is longer than a matured spine (stubby- and mushroom-type spines; <1 µm in length)4,17,18. Because the average length of dendritic spines is 0.5-2 µm in the CNS spiny neurons17, we counted protrusions with a length ≥2 µm from dendritic shafts as an index of spine turnover, that is, spine formation or inhibition of recycling of young spines, along the basal dendrites of pyramidal neurons in layer 3 of the cerebral cortex, and along primary branches of the apical dendrites of CA1 pyramidal neurons. All researchers who counted dendritic spines were blinded to the experimental condition of each image.
Open field test
To evaluate the locomotor activity of mice, the open field test was performed using the video tracking software (Smart 3.0; Panlab Harvard Apparatus, Barcelona, Spain). Male NT-KO and NT-WT mice at 11 weeks old were intraperitoneally injected with vehicle only or digoxin (1, 4, 65, or 650 µg/kg) 1 h before the test. We measured the total travel distance by freely moving mice in a gray acryl box (30 × 30 × 30 cm) for 20 min.
Rotarod test
We performed the rotarod test to assess motor learning in mice. The rotarod test is a widely used primary assay for the study of motor learning33,34, because coordinate sensorimotor responses must be learned in order to remain on the accelerating rotarod without falling. We adopted the accelerating rotation condition (from 4 to 40 rpm in 5 min) because a steep learning curve till 15 trials was obtained35 and morphological change of spines was detected in striatum under this condition5. We used a rotarod machine with automatic timers, falling sensors and a 3-cm-diameter rod (LE8200; Panlab Harvard Apparatus, Barcelona, Spain). Male NT-KO and NT-WT mice at 10 weeks old were subjected to the rotarod test 1 h after intraperitoneal administrations of vehicle only or digoxin at 1, 4, 65, or 650 µg/kg. Mice performed 4 trials on the accelerating rotarod (from 4 to 40 rpm in 5 min) for 3 consecutive days (Days 1, 2, and 3). The time to fall from the accelerating rotarod was recorded. Seven days after Day 3 (Day 10), retention of motor memory was assessed by two additional trials on the accelerating rotarod at the same rate without injecting digoxin to exclude a transient influence of changes in cardiopulmonary endurance by digoxin on test performance. The trial interval on each day was set to approximately 5 min. The timeline of rotarod test was shown in Fig. 5a.
Statistical analysis
R software (version R-2.8.1 for Windows) was used for statistical data analysis. To evaluate differences between wild-type and NT-KO, unpaired t testing, Welch’s t test, or Mann-Whitney U test was performed according to the results of the Shapiro-Wilk normality test and Levene homoscedasticity test. Differences among four or five conditions of digoxin concentrations (vehicle only, 1, 4, 65, or 650 µg/kg) were examined by one-way ANOVA, Welch’s ANOVA, or the Kruskal-Wallis test and their corresponding post-hoc test (Tukey test, Games-Howell test, or Steel-Dwass test), respectively, according to the results of the Shapiro-Wilk normality test and Levene homoscedasticity test. Statistical significance was set at the level of P < 0.05.