Construction of Atp1a3-deficient mice and behavioural analyses
We constructed and prepared the knockout mouse line Atp1a3 (Fig. 1). Mice homozygous for the Atp1a3 deletion mutation (Atp1a3−/−) died just after birth due to complete lack of breathing movements (K. Ikeda & H. Onimaru, unpubl. data). Because haploinsufficiency of ATP1A3 (ATP1A3+/−) is thought to contribute to RDP, we compared heterozygous mice (Atp1a3+/−) with their wild-type littermates (WT). Atp1a3+/− showed no gross morphological defects, no apparent histological anomalies in the brain and survived as WT. Adult Atp1a3+/− were hyperactive (increased locomotor activity) in both the home cage and the open field, compared with WT (Table 1 and Fig. 2A; Moseley et al. 2007; DeAndrade et al. 2011). The time spent in the centre area of the open field was not different between Atp1a3+/− and WT (Fig. 2B), suggesting that there was no difference in anxiety behaviour between the two. The Atp1a3+/− also showed enhanced performances on the rotarod test (Fig. 2C) and balanced beam paradigm (Fig. 2D) compared with WT. Although relevance to the clinical symptoms is not clear, the observation of a better sense of motor balance in Atp1a3+/− motivated us to analyse altered cerebellar function.
Table 1. Spontaneous motor activity in the home cage of the adult wild-type and Atp1a3+/− littermates for 48 h
| Atp1a3 genotype||Spontaneous activity (counts day−1)|
|Wild-type||16413 ± 1304||14729 ± 1125||1548 ± 215|
| Atp1a3 +/− ||24614 ± 3326||21038 ± 2811||3577 ± 551|
Figure 2. Altered behaviours in Atp1a3 heterozygous mice (Atp1a3+/−) A and B, open field test (10 week-old mice); n= 12 for each male genotype mouse. A, total path length in the open-field test. Atp1a3+/− showed significantly increased activities compared with wild-type littermates (WT). B, percentage of time spent in the centre area. C, rotarod test (3 week-old female mice); n= 18 for WT and n= 19 for Atp1a3+/−. Latency to fall from the rod was measured. Atp1a3+/− showed significantly better performance than WT at days 1 and 2. No significant difference was observed on day 3. D, balanced beam test (11–14 week-old mice); n= 12 for each male genotype mouse. Time to reach an escape box was measured. Performance on days 1–3, but not day 4, was significantly better in Atp1a3+/− than WT. E, duration of sustained dystonic response (score D4/D5, Pizoli et al. 2002). Atp1a3+/− showed significantly increased time during which dystonic responses were observed. F, susceptible time of dystonia induction to disturbance. Atp1a3+/− showed significantly longer sensitivity to induced dystonic response; n= 16 (WT) and n= 15 (Atp1a3+/−). Data are mean ± SEM. Open bars: WT, filled bars: Atp1a3+/−. *P < 0.05.
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Induction of dystonia by intracerebellar injection of kainate
Atp1a3 +/− did not develop dystonia either spontaneously under normal environment or after various mild behavioural stimuli, such as suspension by the tail (each mouse was suspended by its tail for 15 min) and forced swimming, or restraint stress (data not shown). We hypothesized that one of the reasons for the lack of manifestation of symptoms was inadequate developmental changes in the synaptic circuits by the above mild and transient stresses in Atp1a3+/−. Therefore, dystonia was induced by pharmacological disturbance of synaptic circuit. To specify the primary focal region of dystonia, we used microinjection of low doses of kainate, an excitatory glutamate agonist, directly into the cerebellar vermis. This experimental approach was based on a previous well-described refined procedure that induces abrupt disruption of synaptic transmission in the cerebellum with resultant dystonia phenotype (Pizoli et al. 2002). Following injection of kainate into the cerebellum, the duration of sustained dystonic response was measured over 1 min every 10 min until 2 h after injection. The first sign of dystonia appeared 10–20 min after the injection in both WT and Atp1a3+/−. A typical dystonic response encompassed persistent hold up of the hindlimb against the trunk. Furthermore, intentional movements often induced dystonic posture in both WT and Atp1a3+/−. The total time of sustained dystonic response (score D4/D5 in Pizoli et al. 2002) was longer in Atp1a3+/− than in WT (Fig. 2E). Furthermore, recovery from the effect of kainate injection (i.e. disappearance of dystonia) was significantly later in Atp1a3+/− than in WT (Fig. 2F). In addition, the mean D4/D5 response (number of events) was significantly larger in Atp1a3+/− than in WT (Fig. S1A). However, the mean duration of individual events was not different between Atp1a3+/− and WT (Fig. S1B). The position of the microinjection site was not different between the two groups (data not shown), indicating that the increased response in Atp1a3+/− was not due to differences in the site of kainate injection in the cerebellum.
Expression of Atp1a3 in the cerebellum
The distribution of Na pump α3 protein in the brain of 3-month-old (adult) mice was investigated recently by immunohistochemistry (Bøttger et al. 2011). In the present study, we confirmed the mRNA expression of Atp1a1, Atp1a2 and Atp1a3 in the cerebella at younger ages [postnatal days (P) 35–40]. The expression of each α subunit gene showed a distinct pattern (Fig. 3). Atp1a1 transcript was observed in the granular layer (GL), while Atp1a2 was noted mainly in the Purkinje cell layer (PCL), with scattered signal in the GL and white matter (WM) of the cerebellum, and in the pia mater (arrowhead in Fig. 3D), as reported previously (Moseley et al. 2003). Atp1a3 showed unique expression patterns: it was observed in the cell bodies in PCL, throughout the ML, especially in interneurons, basket and stellate cells, and also in Golgi cells of the GL, but not in granule cells, indicating that Atp1a3 is expressed in GABAergic neurons. High levels of Atp1a3 transcripts were also observed in neurons of the cerebellar nuclei (CN), which are projected by GABAergic inhibitory synapses from PCs. Our results add support to previous findings of Atp1a3 mRNA expression in GABAergic neurons, namely PCs, basket and stellate cells in ML, as well as Golgi cells in GL of juvenile rodents (Hieber et al. 1989, 1991; Chauhan & Siegel, 1997).
Figure 3. mRNA expression of Na pump α subunits in the cerebellum of juvenile mouse (postnatal day 40) Alternate sections were examined by in situ hybridization using different probes. A–C, Atp1a1 mRNA expression. D–F, Atp1a2 mRNA expression. Atp1a2 mRNA expression in the pia matter (arrowhead). G–I, Atp1a3 mRNA expression. Boxes in B, E and H show areas that are pictured beneath at higher magnifications (C, F and I, respectively). In each panel, the top is the dorsal and the right is the medial side. CN, cerebellar nuclei; ML, molecular layer; GL, granular layer; PCL, Purkinje cell layer; WM, white matter.
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We further examined the expression of Atp1a3 in the developing pup (P14), because dystonia symptoms in RDP patients most commonly appear in young age (Brashear et al. 2008). In the cerebellum, the expression level and pattern of Atp1a3 were essentially similar to those at P40 (data not shown). Atp1a3 was highly expressed in the subthalamic nucleus and substantia nigra, moderately expressed in the globus pallidus and less abundantly expressed in the caudate putamen, which are considered primary causal regions of dystonia.
Localization of the Na pump α3 subunit in the cerebellum
Immunostaining of the α3 subunit was observed as dots in the ML and on the surface of PC soma (Fig. 4B and F). The presynaptic marker of GABAergic neurons, VGAT, was positive, showing a dotted appearance on the membrane of PC bodies and dendrites, and those signals mostly overlapped with that of the α3 subunit (Fig. 4E–H), indicating accumulation of the α3 subunit in the inhibitory synapses. Moreover, excess staining for the α3 subunit was noted in the cerebellar pinceaux, together with VGAT (Fig. 4D and H). The pinceaux is a thick cup-like structure inferior to the PC somata and dense terminal plexus around the axon hillock and initial segment of PC. It is formed by convergence of axonal collaterals descending from ML basket cells and stellate cells, and is highly immunopositive for VGAT (Takayama & Inoue, 2004; Sotelo, 2008). The α3 subunit was also co-immunostained with GAD65/67, which were expressed in most GABA-containing neurons (Fig. 4I). The α3 subunit was co-localized with excitatory presynaptic markers, VGLUT1 and VGLUT2, in the mossy fibre-granule cell synapses in GL (Fig. 4J and K), but not in PCs (Fig. 4J and K). These results indicate the expression of Atp1a3 in inhibitory neurons and high accumulation of its products in inhibitory terminals and pinceaux.
Figure 4. Expression of Na pump α3 subunits, VGAT, GAD65/67, VGLUT1 and VGLUT2 in the cerebellum of young wild-type mice Immunofluorescence using antibodies to α3 (green, B and F) and VGAT (red, C and G). Nuclei are stained with DAPI (blue, A and E). E–H, higher magnifications of A–D. Merged figures of α3 and VGAT are shown in D and H. A merged figure of immunofluorescence using antibodies to α3 (red) and GAD65/67 (green) is shown in I. Merged figures using antibodies to α3 (green) and VGLUT1 (red, J) or VGLUT2 (red, K) are shown. A–I, P26 mice; J and K, P39 mice.
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Characterization of neurotransmission from ML interneurons to PCs
The above results showed increased response to dystonia induction by kainate injection into the cerebellum of Atp1a3+/− and strong expression of Atp1a3 in inhibitory neurons in the developing cerebellum. The inhibitory microcircuitry of the cerebellar cortex has been thought to play a key role in orchestrating synaptic integration and precise timing of action potentials in PCs, the sole output neuron from the cerebellum (for a review, see Jörntell et al. 2010). Taken together, it is likely that disturbance of the inhibitory synaptic transmission onto PCs could occur in Atp1a3+/−. Accordingly, we compared the properties of GABAergic neurotransmission from ML interneurons to PCs between WT and Atp1a3+/− using the whole-cell voltage-clamp technique.
IPSCs were recorded from visually identified PCs of cerebellar slices prepared from young pup littermates (P12–15, VH=−30 mV), and stimulation electrodes were placed in the inner fourth of the ML to activate axons of ML interneurons (thus, they were mostly basket cells). When stimulation intensity was increased from 10 to 100 μA, the amplitude of IPSCs increased almost linearly in both Atp1a3+/− and WT (Fig. 5A and B). The effect of stimulation in eliciting IPSCs appeared to be consistently stronger in PCs of Atp1a3+/− (solid triangles) than WT (open circles), although the difference was not statistically significant (F1,231= 1.65, P= 0.20, two-way ANOVA). A further increase in the stimulation intensity to 300–500 μA increased the amplitude of IPSCs to a similar plateau level in both WT and Atp1a3+/−; the mean IPSC amplitude at 500 μA was not different between WT and Atp1a3+/− (1026 ± 223 pA, n= 12 and 970 ± 226 pA, n= 11, respectively, P= 0.86, Fig. 5A and B). This suggests that the majority of ML interneuron axons present in the activation field were stimulated by the saturated intensity (namely, 300–500 μA). Comparison of the relationship between IPSC amplitude and stimulation intensity after normalization of the amplitude by the amplitude of IPSCs evoked at a stimulation intensity of 500 μA (Fig. 5C) showed a significant leftward shift in the relationship in Atp1a3+/− (F1,219= 15.6, P < 0.001). This suggests a decreased threshold of ML interneuron response to electrical stimulation in Atp1a3+/−. To get insight into the mechanism underlying the enhancement of cerebellar inhibitory neurotransmission in Atp1a3+/−, we applied a pair of stimulation pulses at varying intervals, and compared the paired-pulse ratio (PPR) of the amplitude, i.e. the second IPSC amplitude/the first IPSC amplitude (Fig. 5D; Satake et al. 2000; Zucker & Regehr, 2002). As shown in Fig. 5D and E, the PPR of IPSCs was not significantly different between WT and Atp1a3+/− at any tested intervals (F1,175= 0.061, P= 0.81).
Figure 5. Comparison of ML interneuron-mediated inhibitory neurotransmission onto Purkinje cells (PCs) between WT and Atp1a3+/− A, representative traces of IPSCs recorded from a single PC of WT and Atp1a3+/−. The IPSCs were evoked by electrical stimulation with a series of different intensity (from 10 to 500 μA for 100 μs), and superimposed for stimulations at 10 (pale grey lines), 60 (dark grey lines) and 500 μA (black lines). Each trace is derived from averaging the IPSCs of several successive traces recorded every 15 s. Stimulation artifacts were truncated for clarity. B, relationship between IPSC amplitude and stimulation intensity for WT (open circles, n= 12) and Atp1a3+/− (filled triangles, n= 11). Data are mean ± SEM. C, relationship between IPSC amplitude and stimulation intensity of WT (open circles, n= 12) and Atp1a3+/− (filled triangles, n= 11). Data are relative to the amplitude examined at stimulation intensity of 500 μA. Note the significantly higher response to the weak stimulation intensity in Atp1a3+/−. D, representative averaged traces of IPSCs examined by the paired-pulse protocol in PCs of WT and Atp1a3+/−. The traces were normalized relative to the peaks of the first IPSC. E, relationship between the paired-pulse ratio (PPR) and the inter-stimulus intervals (ISI) for IPSCs examined in WT (open circles, n= 11) and Atp1a3+/− (filled triangles, n= 12).
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At young age, the mouse cerebellar inhibitory neurotransmission between ML interneurons and PCs shows remarkable paired-pulse depression (Fig. 5D and E; for further details, see Pouzat & Hestrin 1997). Because the PPR appears to be less sensitive to presynaptic potentiation at the high release probability synapse, for example, compared with the frequency of miniature IPSC (mIPSC) (Mitoma & Konishi, 1999; Saitow et al. 2000), we next recorded mIPSCs in PCs in the presence of 1 μm TTX, and compared their frequency and amplitude between WT and Atp1a3+/−. The frequency of mIPSCs was much higher in Atp1a3+/− than in WT (Fig. 6A, B and D, P= 0.034). In contrast, the amplitude distribution of mIPSCs was similar (Fig. 6C) and the mean amplitude was not different between WT and Atp1a3+/− (P= 0.84, Fig. 6E). These results suggest that the properties of the postsynaptic GABA receptor do not seem to be significantly different between WT and Atp1a3+/−, but rather that enhancement of GABAergic transmission in Atp1a3+/− originates in a presynaptic mechanism. The latter agrees with the presynaptic expression of Atp1a3 in ML interneurons (Fig. 3). We further examined the resting membrane potentials (Em) of ML interneurons in the current-clamp mode, assuming that reduced expression of α3 subunits leads to membrane depolarization and thereby increases the excitability of ML interneurons. The Em was examined in the presence of 1 μm TTX; Em was −41.3 ± 2.5 mV in WT (n= 12) and −40.1 ± 2.1 mV in Atp1a3+/− (n= 11), and the difference was not statistically significant (P= 0.72, Fig. 6F). We also compared the excitability of ML interneurons between WT and Atp1a3+/− by injecting square wave currents from the recording pipette, but did not find any remarkable difference in the response (Fig. 6G). Furthermore, comparison of the spontaneous firing rate of interneurons by loose cell-attached recordings showed no significant difference between WT (4.6 ± 1.6 Hz, n= 15) and Atp1a3+/− (5.2 ± 1.2 Hz, n= 10) (P= 0.77, Fig. S2). These results rule out the possibility that the enhancement of cerebellar GABAergic neurotransmission in Atp1a3+/− is due to increased somatic excitability of ML interneurons. Together with the presynaptic accumulation of α3 subunit immunoreactivity in ML interneurons (Fig. 4), it is likely that the increase in inhibitory neurotransmission in Atp1a3+/− originates in axonal, but not somatic, depolarization of the interneuron.
Figure 6. Characterization of miniature IPSCs (mIPSCs) in Atp1a3+/− compared with WT A, successive traces of mIPSCs recorded from single PCs of WT and Atp1a3+/− in the continuous presence of TTX (1 μm) and CNQX (20 μm). B and C, cumulative distribution of the inter-event intervals (IEI in B) and amplitude of mIPSCs (C) of WT (grey lines) and Atp1a3+/− (black lines). Data were calculated from the trace in A. Note the significant leftward shift in the IEI distribution in the Atp1a3+/− (P < 0.001, Kolmogorov–Smirnov test in B) without a change in the amplitude distribution (P= 0.33, Kolmogorov–Smirnov test in C). D and E, comparison of the frequency (D) and mean amplitude (E) between mIPSCs recorded from PCs of WT (open bar, n= 13) and Atp1a3+/− (filled bar, n= 12). F, comparison of the resting membrane potential of ML interneurons between WT (open bar and circles, n= 12) and Atp1a3+/− (filled bar and triangles, n= 11) (P= 0.46, unpaired t test). G, representative traces of voltage responses recorded from single ML interneurons from WT and Atp1a3+/−. Each trace is derived from several successive traces recorded by the current-clamp technique. Current injection protocol is shown under the trace. Data are mean ± SEM. *P < 0.05.
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Properties of PF- and CF-PC synapses
Next, we compared the properties of the excitatory synaptic transmission from PFs or CFs to PCs between WT and Atp1a3+/− at P12–15 (VH=−60 mV or −30 mV, respectively). The amplitudes of PF-mediated EPSCs increased almost linearly depending on the increment in stimulation intensity in WT and Atp1a3+/− (Fig. 7A). Stimulation with a higher intensity (300–500 μA) increased the amplitude of EPSCs to a plateau level and the mean amplitude at 500 μA was not significantly different between WT and Atp1a3+/− (1.35 ± 0.26 nA, n= 11 and 1.54 ± 0.34 nA, n= 11, respectively; P= 0.66). There was also no significant difference between WT and Atp1a3+/− even when the relationship between stimulation intensity and EPSC amplitude was examined after normalization by the amplitude of EPSCs evoked at 500 μA (F1,259= 0.087, P= 0.77, Fig. 7B). Application of a mixture of 2 mmγ-DGG, a low-affinity competitive antagonist of AMPA receptors (Wadiche & Jahr, 2001; Kodama et al. 2006), and 50 μm cyclothiazide, a blocker of AMPA receptor desensitization (Partin et al. 1993), reduced the amplitude of PF-EPSCs to almost the same level in WT and Atp1a3+/− (51.4 ± 6.0%, n= 10 and 57.8 ± 3.5%, n= 7, respectively; P= 0.66, data not shown). These results suggest that the concentration of PF transmitter in the synaptic clefts is similar between WT and Atp1a3+/− during the single EPSC. PF-EPSCs are known to exhibit a prominent paired-pulse facilitation (Fig. 7C). The magnitudes of the PPR of PF-EPSCs were not significantly different between WT and Atp1a3+/− at any intervals tested in either the absence (F1,202= 0.24, P= 0.62) or the presence (F1,128= 0.49, P= 0.49) of γ-DGG and cyclothiazide (Fig. 7D and E).
Figure 7. Comparison of parallel fibre (PF)- and climbing fibre (CF)-mediated excitatory neurotransmission onto PCs between WT and Atp1a3+/− A, representative averaged traces of PF-mediated EPSCs recorded from a single PC of WT and Atp1a3+/−. The EPSCs were evoked by electrical stimulation with a series of different intensity (from 10 to 500 μA for 100 μs), and superimposed for stimulations at 10 (pale grey lines), 60 (dark grey lines) and 500 μA (black lines). Each trace is derived from averaging the EPSCs of several successive traces recorded every 15 s. Stimulation artifacts were truncated for clarity. B, relationship between the amplitude of PF-PC EPSCs and stimulation intensity for WT (open circles, n= 14) and Atp1a3+/− (filled triangles, n= 13). Data are mean ± SEM and are relative to the amplitude examined at a stimulation intensity of 500 μA. C, representative averaged traces of PF-PC EPSCs examined by the paired-pulse protocol in PCs of WT and Atp1a3+/−. The traces were normalized relative to the peaks of the first EPSC. D and E, relationship between the PPR and ISI for PF-PC EPSCs examined in WT (D, circles) and Atp1a3+/− (E, triangles). The magnitudes of PPR of the EPSC were not significantly different between WT and Atp1a3+/− at any interval tested in the absence (open symbols) and presence (filled symbols) of 2 mmγ-DGG and 50 μm cyclothiazide. F, representative averaged traces of mGluR1/TRPC1-mediated inward currents examined in PCs of WT and Atp1a3+/−. The currents were evoked by trains of PF stimuli (100 Hz for 100 ms) in the presence of 20 μm CNQX and 100 μm picrotoxin. G, representative averaged traces of CF-EPSCs examined by the paired-pulse protocol in PCs of WT and Atp1a3+/−. The traces were normalized relative to the peaks of the first EPSC. H and I, relationship between the PPR and ISI for CF-PC EPSCs examined in WT (H, circles) and Atp1a3+/− (I, triangles). The PPR of CF-EPSCs was not different between WT and Atp1a3+/−, when they were compared in the absence (open symbols) and presence (filled symbols) of γ-DGG and cyclothiazide.
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The metabotropic glutamate receptor mGluR1 in PCs plays an important role in mediating synapse formation, synaptic plasticity (long-term depression) and motor coordination (Ichise et al. 2000). Repetitive activation of PFs elicits mGluR1-mediated slow inward currents in PCs (Batchelor et al. 1994; Hirono et al. 1998), and a recent study indicates that this inward current is mediated by the cation channel TRPC1 (Kim et al. 2003). The amplitude of the mGluR1/TRPC1-mediated currents was closely dependent on the expression level of neuronal glutamate transporter EAAT4 (Wadiche & Jahr, 2005), which is specifically expressed in PCs, and their glutamate uptake capacity is highly coupled with Na pump activity of the PC. Assuming that deficient Na pump activity causes a reduction of glutamate uptake, thereby leading to enhanced activation of the perisynaptic mGluR1 in PCs, we compared the mGluR1/TRPC1-mediated currents between WT and Atp1a3+/−. The mGluR1/TRPC1 currents were evoked by trains of PF stimulation (100 Hz for 100 ms) and recorded from PCs in lobule III, where the EAAT4 expression level in PCs is low (Wadiche & Jahr, 2005; Satake et al. 2010), in the presence of 20 μm CNQX and 100 μm picrotoxin. The amplitude of the mGluR1/TRPC1 current of Atp1a3+/− (195.9 ± 32.1 pA, n= 19) appeared to be slightly smaller than that of WT (220.6 ± 57.9 pA, n= 16), although the difference was not different statistically (P= 0.71, Fig. 7F). The currents were similarly suppressed by the mGluR1 antagonist AIDA (0.3 mm); the amplitude reached 68.9 ± 7.0% of control in WT (n= 8) and 60.7 ± 7.3% in Atp1a3+/− (n= 10) (P= 0.44, data not shown). These results indicate that the properties of PF-PC excitatory neurotransmission, which includes not only ionotropic receptor-mediated fast synaptic transmission but also metabotropic receptor-mediated slow perisynaptic transmission, are similar between WT and Atp1a3+/−.
At early postnatal stages, most PCs are innervated by multiple numbers of olivocerebellar CFs. Then, by the end of the third postnatal week, the supernumerary CFs are eliminated until each PC is innervated by a single CF (for a review, see Watanabe & Kano, 2011). Therefore, more than one discrete CF-EPSC can be evoked in some PCs in young animals if the intensity of CF stimulation increases. We next compared properties of the single CF-mediated (namely, mature CF-mediated) EPSCs, all of which were corroborated by observing whether the EPSC is evoked in an all-or-none fashion (Kodama et al. 2006). As shown in Fig. 7G, we did not find any significant differences in their amplitudes between WT and Atp1a3+/− (3.82 ± 0.53 nA, n= 9 and 3.58 ± 0.64 nA, n= 7, respectively; P= 0.78, Fig. 7H and I). The amplitudes of CF-EPSCs observed in WT and Atp1a3+/− decreased similarly after application of 2 mmγ-DGG and 50 μm cyclothiazide (51.0 ± 4.9%, n= 9 and 51.7 ± 5.8%, n= 6, respectively; P= 0.93, data not shown), suggesting that the concentration of CF transmitter in the synaptic clefts is similar during the single EPSC. Furthermore, the PPR of CF-EPSCs was not statistically different between WT and Atp1a3+/−, both in the absence (F1,143= 1.60, P= 0.21) and in the presence (F1,134= 0.062, P= 0.80) of γ-DGG and cyclothiazide (Fig. 7G–I).
The above results suggest that excitatory synaptic transmission converging onto PCs is not disturbed in Atp1a3+/−, which is in sharp contrast to the enhanced inhibitory synaptic transmission.