Mania-like behavior induced by genetic dysfunction of the neuron-specific Na+,K+-ATPase α3 sodium pump

Abstract

Bipolar disorder is a debilitating psychopathology with unknown etiology. Accumulating evidence suggests the possible involvement of Na(+),K(+)-ATPase dysfunction in the pathophysiology of bipolar disorder. Here we show that Myshkin mice carrying an inactivating mutation in the neuron-specific Na(+),K(+)-ATPase α3 subunit display a behavioral profile remarkably similar to bipolar patients in the manic state. Myshkin mice show increased Ca(2+) signaling in cultured cortical neurons and phospho-activation of extracellular signal regulated kinase (ERK) and Akt in the hippocampus. The mood-stabilizing drugs lithium and valproic acid, specific ERK inhibitor SL327, rostafuroxin, and transgenic expression of a functional Na(+),K(+)-ATPase α3 protein rescue the mania-like phenotype of Myshkin mice. These findings establish Myshkin mice as a unique model of mania, reveal an important role for Na(+),K(+)-ATPase α3 in the control of mania-like behavior, and identify Na(+),K(+)-ATPase α3, its physiological regulators and downstream signal transduction pathways as putative targets for the design of new antimanic therapies.

Fig. 1.

Exploration and ambulation in Myk/⁺ mice. Myk/⁺ show an increased number of explorations of (A) novel objects (+/+ mice n = 10, Myk/⁺ n = 11) and (B) nosepokes compared with +/+ mice and do not habituate (+/+ mice n = 8, Myk/⁺ n = 7). (C) In an open field Myk/⁺ travel a further distance and (D) spend more time in the center (+/+ mice n = 27, Myk/⁺ n = 22) in 5-min bins compared with +/+ mice. (E) Myk/⁺ exhibit unusual walking patterns compared with +/+ mice. (F) In 60 min in the open field, Myk/⁺ increase locomotor activity in response to d-amph, but +/+ mice do not (+/+ vehicle n = 9, +/+ d-amph n = 9; Myk/⁺ vehicle n = 8, Myk/⁺ d-amph n = 9). All data are presented as mean SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with +/+ mice, ^###P < 0.001 compared with Myk/⁺ vehicle mice.

Fig. 2.

Sleep and endogenous circadian period in Myk/⁺ mice. (A) Myk/⁺ (n = 6) experience more wake time than +/+ mice (n = 6) across 24 h with a reduction of both non-REM and REM sleep, as assessed by EEG and EMG. (B and C) Myk/⁺ show deficits in sleep duration only during the light phase; (D) Myk/⁺ have fewer REM sleep bouts but no change in non-REM bouts. (E) Non-REM bout length is reduced and REM bout length was unchanged in Myk/⁺. (F) REM sleep latency is reduced in Myk/⁺. (G) Wheel running actograms from +/+ and Myk/+ mice. Animals were held on a light-dark (LD) cycle for 14+ d, released into constant dark for 7 d to assess free running period, and reentrained to a LD12:12 cycle. Shaded area represents dark portion of LD cycle. Vertical arrows indicate continuation of nocturnal activity into light. (H) Endogenous period is extended in Myk/⁺ because of longer periods of (I) activity (α). All data are presented as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with +/+ mice.

Fig. 3.

Mania-like behavior in Myk/⁺ mice. (A) Myk/⁺ prefer to explore the open arm of the EPM (+/+ mice n = 29, Myk/⁺ n = 19) and (B) the light side of the LDB (+/+ mice n = 27, Myk/⁺ n = 18) for longer durations than +/+ mice. (C) Myk/⁺ (n = 8) show a higher preference for 0.1% sucrose than +/+ mice (n = 16) over 4 d and (D) consume more sucrose on the first presentation of sucrose. W, water; S, sucrose. (E) Myk/⁺ (n = 14) are active for a longer duration than +/+ mice (n = 15) in the Porsolt forced swim test. (F) PPI scores are impaired in Myk/⁺ (n = 20) compared with +/+ mice (n = 33) at all prepulse intensities tested. (G and H) Myk/⁺ (n = 16) had a startle habituation deficit compared with +/+ when presented with a repeated auditory stimulus (n = 12). All data are presented as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with +/+ mice.

Fig. 4.

Attenuation of mania-like behavior by lithium and VPA in Myk/⁺ mice. (A) Chronic treatment with lithium reduces total distance traveled by Myk/⁺ (n = 20) compared to untreated Myk/⁺ (n = 22) over 30 min in the open field and had no effect in +/+ mice (n = 28 control, n = 27 lithium). (B) VPA reduced total distance traveled by Myk/⁺ (n = 20) compared to vehicle-treated Myk/⁺ (n = 22), and had no effect on +/+ mice (n = 28 vehicle, n = 27 VPA) over 30 min. (C) Lithium reduces open-arm duration in Myk/⁺ (n = 18) compared with control Myk/⁺ (n = 19), but had no effect on +/+ mice (n = 29 control, n = 29 lithium) in the EPM. (D) VPA reduces open-arm duration in Myk/⁺ (n = 12) compared with vehicle-treated Myk/⁺ (n = 10) but had no effect on +/+ mice (n = 12 control, n = 12 VPA) in the EPM. (E) ERK inhibitor SL327 decreased distance traveled in Myk/⁺ (n = 12) compared with vehicle-treated Myk/⁺ (n = 9), and had no effect in +/+ mice (n = 12 vehicle, n = 12 SL327) in the open field. (F) SL327 reduced open-arm duration in Myk/⁺ (n = 6) compared with Myk/⁺ vehicle (n = 6) but had no effect on +/+ mice (n = 6 control, n = 6 SL327) in the EPM. (G) Rostafuroxin decreased distance traveled in Myk/⁺ (n = 12) compared with vehicle treatment (n = 12) and had no effect in +/+ mice (n = 13 vehicle, n = 15 rostafuroxin) in the open field. (H) In the EPM rostafuroxin reduced open arm duration in Myk/⁺ (n = 12) compared with vehicle treated Myk/⁺ (n = 12) and had no effect in +/+ mice (n = 12 vehicle, n = 15 rostafuroxin). All data are presented as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 compared with +/+ mice, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with Myk/⁺ vehicle mice.

Fig. 5.

Free intracellular Ca²⁺ and Ca²⁺-dependent signaling in Myk/⁺ mice. (A) Mean resting intracellular ([Ca²⁺]_i) is stably elevated in cortical cells cultured from Myk/⁺ (n = 47) than +/+ mice (n = 19), as measured by the ratio of fura-2 fluorescence emission upon 340-nm and 380-nm excitation (P < 0.01). (B) Myk/⁺ cortical neurons show a prolonged peak in [Ca²⁺]_i compared with +/+ in response to bath superfusion of 10 μM glutamate (Glu). (C) When normalized to baseline [Ca²⁺]_i, glutamate-evoked [Ca²⁺]_i transients were prolonged in neurons from Myk/⁺ compared with neurons from +/+ (D) Immunoreactivity of p-Akt1/2/3 and p-ERK1/2 was elevated in Myk/⁺ compared with +/+ hippocampus. Transgenic overexpression of NKA α3 in Myk/⁺/Tg mice did not alter hippocampal levels of p-ERK1/2 but reduced p-Akt1/2/3. +/+, n = 8 (Akt, ERK); Myk/+, n = 11 (Akt), n = 10 (ERK); Myk/+/Tg, n = 5 (Akt, ERK). (E) Model of NKA α3 signaling at the synapse in +/+ and Myk/⁺ mice. Myk/⁺ mice have reduced NKA activity that augments [Ca²⁺]_i and activation of p-ERK and p-Akt. These intracellular signals may independently, additively or synergistically contribute to behavioral phenotypes of mania. All data are presented as means ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with +/+ mice, ⁺⁺P < 0.01 compared with Myk/⁺/Tg mice.