Article Figures & Data
Figures
- Figure 1.
Generation of Slc26a11 knock-out mice. A, A Slc26a11 conditional targeting vector containing the Slc26a11 genomic region was constructed to flox exons 9 and 10, flanked by long and short homology arms, respectively. The linearized targeting vector was electroporated into ES cells (for details see Materials and Methods). B, DNA was digested with StuI, and was hybridized with a probe targeted against the 5′ external region. The expected sizes are indicated on the schematic and positive clones were further confirmed by Southern blot analysis (left). C, DNA from the same clones was digested with NsiI, and was hybridized with a probe targeted against the 3′ internal region. The expected sizes are indicated on the schematic and positive clones were confirmed by Southern blotting (left).
- Figure 2.
KBAT is specifically deleted from PCs. A, Northern blot analysis of cerebellar RNA showing a significant reduction in KBAT mRNA expression levels in KBAT knock-out mice. B, Western blot analysis of cerebellar proteins showing a significant reduction in KBAT protein abundance in KBAT knock-out mice. C, Immunofluorescent staining of KBAT in cerebellar tissues of wild-type mice shows a strong labeling of KBAT in cell body and dendrites of PCs (top), whereas KBAT is deleted from PCs of L7-KBAT KO mice. KBAT localization is shown in green. Blue represents DAPI nuclear staining. Yellow arrows indicate PC cell bodies and red arrows their dendrites.
- Figure 3.
KBAT regulates EGABA. A, Left, The schematic representation of the protocol used for calculation of EGABA by perforated patch-clamp recording to avoid disruption of the [Cl−]i. Right, Raw traces of a L7-KBAT KO PC and a control PC. B, I/V curve of the same representative PCs in A. Each data point indicates the maximum current amplitude evoked by puff application of muscimol to a L7-KBAT KO PC and a control PC when clamped at various holding potentials (ranging from −120 to −60 mV with 10 mV intercept). C, Comparing the reversal potential of GABA obtained from PCs of L7-KBAT KO mice shows a significant negative shift compared to littermate controls (p = 0.03, n = 6 per group). D, The estimated intracellular concentration of Cl− is significantly reduced in L7-KBAT KO mice (p = 0.03, n = 6 per group). E, The membrane potential of PCs is not significantly different between L7-KBAT mice and controls (n = 8 for control and n = 9 for L7-KBAT KO PCs; p = 0.8).
- Figure 4.
sIPSCs in PCs. A, Representative traces of sIPSCs of a L7-KBAT KO PC (right) and a control PC (left). B, Frequency of sIPSCs is significantly higher in PCs of L7-KBAT KO mice (n = 22 control PC and 18 L7-KBAT KO PC; p = 0.01). C, Comparing the maximum amplitude of sIPSCs did not show a significant difference between the two groups (p = 0.51). D, E, No significant difference was detected in neither the rise time of sIPSCs (P = 0.12) nor the decay time (p = 0.95). F, In vitro cell-attached recordings of MLIs did not show a significant deference in the spiking activity of presynaptic interneurons. Each panel shows individual data points with mean values ± SEM.
- Figure 5.
Spontaneous and evoked action potential firing in PCs. A, Representative traces of intrinsic firing activity of a L7-KBAT KO PC (left) and a control PC (right). B, Spontaneous firing frequency was significantly higher in KO PCs compared to the controls when the inhibitory input was present (n = 15 control PC and 17 L7-KBAT KO PC; P = 0.004). C, Firing frequency of PCs remained significantly higher when both inhibitory and excitatory inputs were blocked (n = 23 control PC and 20 L7-KBAT KO PC; p = 0.01). D, The regularity of firing between the two groups did not show a significant difference (p = 0.9). E, The excitability of PCs was compared by injecting depolarizing currents to PC in steps of 100 pA and did not show a significant difference between the groups (repeated-measures ANOVA, p = 0.2, n = 12 per group). F–H, Action potential amplitude, threshold and half-width were not significantly different (n = 12 per group; p > 0.05). I, The amplitude of AHP was significantly smaller in KO PCs compared with controls (n = 12 per group; p = 0.04).
- Figure 6.
In vivo PC firing. A, Single-unit traces of a L7-KBAT KO PC (left) and a control PC (right). B, The frequency of simple spikes (SS) was significantly higher in PCs lacking KBAT (n = 19 control PC and 15 L7-KBAT KO PC; p = 0.02). C, The regularity of simple spike firing (CV) did not show a significant difference between the groups (p = 0.09). D, The regularity of interspike intervals (CV2) of simple spikes was not affected in L7-KBAT KO mice (p = 0.4). E, The frequency of complex spikes was not changed in the knock-out cells (p = 0.3). F, The regularity of complex spike firing (CV) was not significantly different between the groups (p = 0.2). G, The minimal CF pause of the recorded cells was not significantly different (p = 0.1).
- Figure 7.
L7-KBAT KO mice show smaller step size compared with littermate controls. A, Comparison of average number of steps during baseline (non-perturbed) sessions shows significant higher number of steps per session in L7-KBAT knock-out mice compared to the controls (p = 0.02, n = 10 per group). B, Average number of steps per session during both perturbed and non-perturbed sessions shows that the difference in the number of steps remains significantly different during all sessions (p = 0.02). C, L7-KBAT knock-out mice show higher percentage of small steps in all sessions compared to the wild-type control mice (p = 0.02). D, Percentage of large steps is significantly lower in L7-KBAT KO mice (p = 0.01). Non-perturbed (np) and perturbed (p) sessions are indicated in each panel.
- Figure 8.
Eye movement tests were not affected in L7-KBAT mice. A–C, Compensatory eye movements in adult control (n = 12) and L7-KBAT KO (n = 12) were compared. Mice were subjected to visual (OKR) and/or vestibular stimulation (in dark, VOR; in light, VVOR) and gain (ratio of eye to stimulus velocity) and phase (difference in degrees between eye and stimulus) were calculated. None of these parameters were detected to be different between knock-out and control mice (p values > 0.05). D, VOR gain-decrease learning was not impaired in L7-KBAT KO mice compared with controls (p = 0.1). E, Likewise, VOR phase reversal training did not show a difference in motor learning and consolidation between the L7-KBAT KO mice and the control mice (p = 0.5).
Tables
RMP, mV Iholding, pA Rs, MΩ Rin, MΩ Cm, pF τm, ms L7-KBAT KO −56.3 ± 1.6 −262 ± 42 10.5 ± 0.7 59.4 ± 4.5 180 ± 10 10.6 ± 0.9 Controls −55.5 ± 1.6 -308 ± 90 9.89 ± 0.39 60.0 ± 6.9 196 ± 19 11.3 ± 1.0 p value 0.72 0.26 0.44 0.94 0.47 0.66 RMP: Resting membrane potential; Iholding: holding current at −60 mV, Rs: series resistance; Rin: input resistance; Cm: membrane capacitance; τm: membrane time constant. N = 6 per group.
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