Protein kinase Cγ in cerebellar Purkinje cells regulates Ca2+-activated large-conductance K+ channels and motor coordination

Significance The cerebellum, the site where protein kinase C (PKC) was discovered, contains the highest amount of PKCγ in the central nervous system. PKCγ in the cerebellum is exclusively confined to Purkinje cells (PCs), sole outputs from the cerebellar cortex. Systemic PKCγ-knockout mice show impaired motor coordination; however, the cause of motor defects remains unknown. Here we show that activation of PKCγ suppresses the Ca2+-activated large-conductance K+ (BK) channels located along the PC dendrites. A consequential increase in the membrane resistance attenuates electrical signal decay during propagation, resulting in an altered complex spike waveform. Our results suggest that synaptically activated PKCγ in PCs plays a critical role in motor coordination by negative modulation of BK currents.

To evaluate motor performance, the mice were subjected to a beam-walking test. The beam consisted of a 100-cm horizontal steel round bar (11 mm in diameter) placed 50 cm above the floor. After the habituation trials, the mice were placed at the starting point of the bar. The slip number of the fore or hind limb from the bar and the time to cross the 80-cm distance on the bar were measured accordingly. Sessions consisting of three trials per day with a 10-minutes intertrial interval were conducted for these mice.

Footprint test
Footprints were made using waterproof black ink and white paper. Ink was applied to the hind paws of the mice. The mice were allowed to walk forward in a narrow alley. For each mouse, the stride length and width were measured and accordingly averaged.

Voltage-clamp recordings
Input resistance and membrane capacitance were estimated from passive currents induced by applying hyperpolarizing pulses (from -70 to -80 mV or -10 to -20 mV, 200 ms duration). PF-EPSCs, CF-EPSCs, and mIPSCs were recorded as described previously [6]. CF-EPSCs were recorded at -70 mV under some conditions. Multiple CF innervation was estimated as described previously [7]. In other experiments recording CF-EPSCs, maximal stimulation was applied to activate all the CFs innervating the individual PCs. CF-LTDs were recorded from P14-17 mouse PCs as described previously [8]. Some BK currents were measured from P10-14 mouse PCs. The PCs were first held at -100 mV for 500 ms, and the currents were then evoked by moving the holding potentials to various potentials. BK currents were calculated by subtracting the traces before and after the application of 100 nM iberiotoxin (4235-s, Peptide Institute Inc., Osaka, Japan). The leak subtraction method was used to record the BK currents to compensate for the leak current. The extracellular solution contained 0.1 mM picrotoxin, except for the solution used to record mIPSCs, which contained 1 μM TTX. The extracellular solution used to record the BK currents contained 1 μM TTX and 5 mM 4-AP and the solution that was used to record the CF-EPSCs at -70 mV contained 0.5 µM NBQX. In some experiments, the extracellular medium contained 500 µM TEA. Data were discarded when the resistance values changed by >20% of the basal value during the experiment.

Current-clamp recordings
Before starting the experiments, the baseline membrane potentials of the recording PCs were adjusted to approximately -70 mV. CF-mediated depolarization and complex spikes were evoked by the CF stimulation. To evaluate the complex spike waveform, we counted the number of Na + spikes superimposed on the sustained depolarization of the complex spike, whose amplitudes were over 5 mV. Some recordings were made in the presence of 10 nM apamin (4257v, Peptide Institute Inc., Osaka, Japan) and/or 100 nM iberiotoxin.

Confocal Live Calcium imaging
CF-evoked Ca 2+ signals in the cerebellar PCs were examined by confocal microscopy using an upright microscope (BX51WI, Olympus) equipped with a 40× water immersion objective (LUMPLFLN 40XW, Olympus, Tokyo, Japan), a water-cooled CCD camera (iXon3 DU-897E-CS0-#BV-500, Andor, UK), and a high-speed spinning-disc confocal unit (CSU-X1, Yokogawa Electric, Tokyo, Japan), as described previously [9], but with some modifications. The pipette solution for the Ca imaging contained the following (in mM): 135 potassium gluconate, 10 HEPES, 5 KCl, 5 NaCl, 5 Mg-ATP, 0.5 Na-GTP, 0.1 EGTA, 5 phosphocreatine, and 0.1 Oregon Green 488 BAPTA-1; pH 7.3. To optimize CF stimulation, the amplitude of CF-evoked EPSCs was maximized by adjusting the stimulus intensity and its location, as described above, in a voltage-clamp mode at -70 mV. Subsequently, the amplifier recording mode was switched to the current-clamp mode, which enabled the measurement of physiological Ca 2+ responses. CFevoked Ca 2+ signals were acquired after setting the resting membrane potential of the recorded PCs to approximately -65 mV by current injection. Confocal fluorescence Ca 2+ images were obtained at ~30 frames/s (33 ms exposure time, 512 × 512 pixels, no binning) with Andor iQ3 software (Andor, UK), and background-subtracted images were used for further analysis. Fb is the average basal fluorescence value during pre-stimulus frames (> 30 frames), and the relative Ca 2+ signal change (∆F/Fb) was calculated pixel-by-pixel, where Ft is the fluorescence intensity at time t and ∆F = Ft -Fb. CF-evoked dendritic Ca 2+ responses in PCs can vary depending on their dendritic location [10,11]. Thus, to minimize the chance of missing or underestimating the Ca 2+ signals, dozens of regions of interest (ROIs) (usually >20 ROIs) were set on the whole area of the active PC dendritic processes, and each ROI was smaller than the entire size of the dendritic Ca 2+ signal spatial spread ( Supplementary Fig. 5A) (9). The validity of the ROI position was routinely confirmed on averaged images of more than 20 frames ( Supplementary Fig. 5, insets). The dendritic Ca 2+ trace from each ROI was obtained as the time course of the spatially averaged values of ∆F/Fb within each ROI. The peak values, integrals, and half-decay times of the Ca 2+ traces were measured from all the ROIs in each PC, and their maximum values were used to represent each PC [9]. Image processing and analysis were performed with Andor iQ3 (Andor, UK), and a set of custom-made macros or programs of ImageJ, Python, and Igor Pro 8 (WaveMetrics, USA) written by NH.

Supplementary Fig. 5. Faster decay time constants of CF-EPSCs in PKCγ-cKO mouse PCs were not restored by re-expression of PKCγ
Decay time constants were analyzed from the CF-EPSCs recorded in Fig. 4 and Supplementary  Purkinje cells, BK -/-; BK channel-knockout, KO; knockout, CF; climbing fiber; CF-LTD; climbing fiber long-term depression of synaptic transmission.

Supplementary Movie 1. Significant restoration of beam-walking performance in PKCγ-
KO mice by PC-specific expression of PKCγ.
The wild-type mouse (WT) walked smoothly to the shelter with almost no slipping, whereas the PKCγ-knockout mouse (KO) wobbled on the bar with frequent slips, resulting in a significantly longer duration to reach the shelter. AAV vector-mediated PC-specific rescue of PKCγ in PKCγknockout mouse (KO + PKCγ) significantly improved their performance (fewer slips and shorter time to reach the shelter).

Supplementary Movie 2. Poor beam-walking performance in the Prkcg fl/fl mouse that lost
PKCγ expression specifically from PCs after maturation.
The Prkcg fl/fl mouse received injection of AAV vectors expressing Cre by the PC-specific L7-6 promoter, resulting in PC-specific elimination of PKCγ expression (conditional knockout; cKO).
In contrast to good performance of the wild-type mouse (WT), the cKO mouse showed significantly poorer performance, indicating that PKCγ expressed in mature mice PCs plays a critical role in motor coordination.