Intracerebroventricular injection of ouabain causes mania-like behavior in mice through D2 receptor activation

Intracerebroventricular (ICV) administration of ouabain, an inhibitor of the Na, K-ATPase, is an approach used to study the physiological functions of the Na, K-ATPase and cardiotonic steroids in the central nervous system, known to cause mania-like hyperactivity in rats. We describe a mouse model of ouabain-induced mania-like behavior. ICV administration of 0.5 µl of 50 µM (25 pmol, 14.6 ng) ouabain into each lateral brain ventricle results in increased locomotor activity, stereotypical behavior, and decreased anxiety level an hour at minimum. Fast-scan cyclic voltammetry showed that administration of 50 µM ouabain causes a drastic drop in dopamine uptake rate, confirmed by elevated concentrations of dopamine metabolites detected in the striatum 1 h after administration. Ouabain administration also caused activation of Akt, deactivation of GSK3β and activation of ERK1/2 in the striatum of ouabain-treated mice. All of the abovementioned effects are attenuated by haloperidol (70 µg/kg intraperitoneally). Observed effects were not associated with neurotoxicity, since no dystrophic neuron changes in brain structures were demonstrated by histological analysis. This newly developed mouse model of ouabain-induced mania-like behavior could provide a perspective tool for studying the interactions between the Na,K-ATPase and the dopaminergic system.


Supplementary materials and methods
Guide cannulas for intracerebroventricular (ICV) injections were made as small as possible in order to avoid using screws for the fixation of cannulas with dental cement because mouse skull is thin (0,1-0,2 mm) making additional brain damage by screws unavoidable. As seen in Fig. S1, guide cannulas on the mouse head were small enough to prevent animals from knocking them off the head and any discomfort during movements.
Guide cannulas were placed at coordinates AP = -0.5, L = 1.0, depth 2.0 (coordinates are given in millimeters relative to the bregma 1 ) to inject ouabain into the lateral ventricles directly above the striatum (Fig. S2).
Supplementary Figure S2. Schematic image of guide cannulas position in the mouse brain (frontal slice 1 ).
Supplementary Figure S3. Histological analysis of a frontal slice of an operated mouse on the level of ICV cannula implantation. The tissues were stained with hematoxylin and eosin.
It is visible that the guide cannula have reached the targeted lateral ventricles (Fig. S3). It is also visible on the histological microphotographs of the tissues that the guide cannula were placed in such a way that only the motor cortex in the area of the operation was damaged, and no significant damage was done to the nearby areas. No inflammation or edema was caused by the manipulations.

Locomotor activity of mice after injection of different concentrations of ouabain
Preliminary experiments with different concentrations of ouabain were conducted to find the dose of ouabain that causes hyperactivity in mice. The groups of mice treated with other ouabain concentrations (10 μM and 200 μM) were small (N=3) to prevent unnecessary waste of animals. Bilateral injection of 0,5 μl of 10 μM ouabain did not cause any effect on the locomotor activity of mice. Bilateral injection of 0,5 μl of 200 μM ouabain caused decreased locomotor activity and "freezing" of the animals (Fig. S4).
Supplementary Figure S5. Locomotor activity of mice in the open field test. Distance travelled by mice, calculated separately for every 10 min of a 1h open field test during habituation (a), immediately after the injection (b), 3 h after the injection (c), and 24 h after the injection (d). Data is presented as mean ± SEM, N=10 per group.
During the 20 minutes post administration in the control group 33 ± 6 rearings were observed, while in the ouabain-treated group 65 ± 15 rearings were observed.
1h track visualization of a control mouse and 50 μM ouabain-treated mouse illustrates stereotypical movements of ouabain-treated mice in the open field test (Fig. S6).

Catecholamine content in the striatum after ICV ouabain administration
As seen in Fig. S7, ouabain administration caused 3,4-dihydroxyphenylacetic acid and homovanillic acid levels in the striatum of mice to increase without any effect on the level of dopamine.

Akt, GSK3β and ERK1/2 activation in the striatal tissue of the experimental animals
To determine the influence of D2 dopamine receptor activation on ouabain-induced intracellular signal cascades in the striatum of mice, Akt, GSK3β and ERK1/2 activation was evaluated 30 min after the bilateral ICV injection of 0.5 μl of 50 μM ouabain and 0.5 μl of 200 μM ouabain (i. e. 1 h after intraperitoneal administration of haloperidol, 70 µg/kg).
Since the 200 μM ouabain group was not included in the data set in the main article, the full set of representative bands is presented in the supplementary materials. As you can see on