Inferior Olivary TMEM16B Mediates Cerebellar Motor Learning

Abstract

Ca²⁺-activated ion channels shape membrane excitability and Ca²⁺ dynamics in response to cytoplasmic Ca²⁺ elevation. Compared to the Ca²⁺-activated K⁺ channels, known as BK and SK channels, the physiological importance of Ca²⁺-activated Cl^- channels (CaCCs) in neurons has been largely overlooked. Here we report that CaCCs coexist with BK and SK channels in inferior olivary (IO) neurons that send climbing fibers to innervate cerebellar Purkinje cells for the control of motor learning and timing. Ca²⁺ influx through the dendritic high-threshold voltage-gated Ca²⁺ channels activates CaCCs, which contribute to membrane repolarization of IO neurons. Loss of TMEM16B expression resulted in the absence of CaCCs in IO neurons, leading to markedly diminished action potential firing of IO neurons in TMEM16B knockout mice. Moreover, these mutant mice exhibited severe cerebellar motor learning deficits. Our findings thus advance the understanding of the neurophysiology of CaCCs and the ionic basis of IO neuron excitability.

Keywords: ANO2; CaCC; TMEM16B; anoctamin-2; calcium-activated chloride channel; calcium-activated ion channels; eyeblink conditioning; inferior olive; motor learning; olivo-cerebellum.

Figure 1. A large Ca²⁺-activated Cl⁻ current exists in inferior olivary (IO) neurons

(A) Depolarizing an IO neuron to +10 mV elicited a large Ca²⁺ sensitive, time-dependent, outward rectifying current (I_tdo) with a slow decaying tail current (I_tail) at the end of depolarization. This Ca²⁺-sensitive current (CTL) can be inhibited by 100 μM niflumic acid (NFA) and it is completely abolished by 400 μM Cd²⁺, a potent voltage-gated Ca²⁺ channel blocker. (B) Summary of NFA inhibitory effects on I_tdo and I_tail (paired t-test: n = 6. p = 0.014 for I_tdo and p = 0.005 for I_tail). Error bar represents SEM. (C) Measurements of reversal potential in different extracellular Cl⁻ concentrations indicate that the Ca²⁺-sensitive current in IO neurons arises from Ca²⁺-activated Cl⁻ channels (CaCCs). The voltage protocol is shown at the top. (D) The I–V relation in the presence of high and low extracellular Cl⁻ ([Cl⁻]_ex) concentrations. n = 11 and 6 for high (136.8 mM) and low (28 mM) [Cl⁻]_ex concentrations, respectively. Error bar represents SEM. Dotted lines mark I = 0.

Figure 2. TMEM16B but not TMEM16A is highly expressed in IO neurons

(A) TMEM16A is not expressed in IO neurons. (B) TMEM16B is expressed in IO neurons, which express neuronal marker NeuN. (C) TMEM16B expression is eliminated and farnesylated mCherry expression is introduced in the IO neurons from TMEM16B knockout (KO) mice. (D) Depolarization to +10 mV induced large Ca²⁺-sensitive I_tdo and I_tail in WT IO neurons. (E) Genetic ablation of TMEM16B completely eliminated I_tdo and I_tail in TMEM16B KO IO neurons, indicating that TMEM16B CaCC mediates the Ca²⁺-sensitive I_tdo and I_tail in IO neurons. The small inward current was carried by the high-threshold voltage-gated Ca²⁺ channels in IO neurons. (F) Comparison of I_tail amplitudes of the IO neurons from TMEM16B WT and KO mice. To avoid membrane capacitive currents, tail current amplitudes 50 ms after the repolarization (−80 mV) were measured. Unpaired t-test: n = 24 for WT and n = 17 for TMEM16B KO mice, *** p =1.3 × 10⁻⁸. Error bar represents SEM. Dotted lines mark I = 0.

Figure 3. TMEM16B-CaCC can be activated by dendritic high-threshold voltage gated Ca²⁺ (Ca_V) channels

(A, B) Current traces elicited from TMEM16B knockout (KO, A) and wildtype (WT, B) IO neurons. Membrane potential was depolarized from −20 to +40 mV to open the dendritic high-threshold Ca_V channels and then repolarized to −80 mV. (c) The I-V relationship of peak I_tdo and I_tail current from WT and KO IO neurons. Peak current amplitudes were measured at the end of the depolarization pulses and tail current amplitudes were measured at 50 ms after the repolarization pulse. Unpaired t-test: n = 24 for WT and n = 17 for KO. * p = 0.0006 for I_tdo and p = 2.7 × 10⁻⁸ for I_tail. Error bars represent SEM. (D) Current changes in response to a 50 ms depolarization to 0 mV right after membrane break-in to establish whole-cell patch clamp (grey) and 4 minutes later (black), after allowing 5 mM EGTA to diffuse into the WT IO neuron. (E) Time course of the tail current while 5 mM EGTA diffuses into the cell, leading to total elimination of the CaCC-mediated CaCC tail current. The tail currents were measured 50 ms after the repolarization pulse and normalized to the amplitudes of the initial tail current. n = 4. Error bars represent SEM. (F) Prolongation of depolarization enabled opening of TMEM16B-CaCC, resulting in a gradual increase of the outward I_tdo and I_tail current in the presence of 5 mM EGTA. Current was recorded after EGTA had entered the cell under whole-cell patch recording. Inset, enlarged view of currents at the beginning of the test pulses. Dotted lines mark I = 0.

Figure 4. IO neurons from TMEM16B KO mice exhibited reduced membrane excitability

(A, B) Representative firing of IO neurons from wildtype (WT, A) and TMEM16B knockout (KO, B) mice in response to a 400 pA excitatory stimulus for 1 second. Insets show expanded view of the spikes. The black, red and blue dotted lines mark the resting potential, the maximum repolarization potential, and the steady state potential in response to continuous 400 pA excitatory stimulus, respectively. Definitions of spike parameters are labeled with blue, cyan and red color. (C) The average number of spikes for WT and TMEM16B KO IO neurons in response to the excitatory stimulus. p = 0.001. Error bar represents SEM. (D) The averaged action potential duration of 90% repolarization (APD90%) of the first high-threshold Ca²⁺ spike from WT and TMEM16B KO IO neurons. APD90% was used to define after depolarization. p = 0.004. Error bar represents SEM. (E) The averaged after-hyperpolarization (AHP) duration of WT and TMEM16B KO IO neurons. p = 0.0001. Error bar represents SEM. (F) The averaged AHP slope of WT and TMEM16B KO IO neurons. p = 0.0002. Error bar represents SEM. Unpaired t-test: 37 IO neurons from 12 TMEM16B WT mice and 34 IO neuron from 10 TMEM16B KO mice, ** p < 0.01, *** p < 0.001.

Figure 5. TMEM16B-CaCC is specifically expressed in inferior olivary neurons in the olivo-cerebellar system

(A) No TMEM16B expression was observed in the Purkinjie cell (PC) layer. Anti-calbindin labels PCs. The farnesylated mCherry signal from TMEM16B KO mice was used to label TMEM16B expressing cells. mCherry was only detected in climbing fibers from IO neurons. (B) No TMEM16B expression was observed in the granule cell (GC) layer. Anti-NeuN labels GCs. mCherry was only detected in climbing fibers. (C) No TMEM16B expression was observed in the deep cerebellar nucleus (DCN). Anti-calbindin labels DCN neurons. mCherry was only detected in climbing fibers.

Figure 6. TMEM16B knockout (KO) mice exhibited impaired motor learning assessed via eyeblink classical conditioning

(A) Acquisition of conditioned responses (CR) of a wildtype (WT) and a TMEM16B KO mouse during a seven-day training section. The eyeblink conditioning training paradigm is shown on top, with the conditioned stimulus (CS, 350 ms, 2.7 Hz tone and white light LED) and the 100 ms unconditioned stimulus (US, airpuff, 4~5psi) starting at 250 ms after the initiation of CS. CS and US are co-terminated. Each animal was trained for 90 paired CS-US trials followed by 10 CS-only trials in seven consecutive days. The eyelid closure traces during the last 10 CS-US trials were averaged and shown in the figure. The initial responses shown in the dashed lines refer to the average of the first 10 CS-US trials for the naïve mice on Day 1. (B) TMEM16B KO exhibited impaired CR acquisition. CR acquisition was defined by eyelid closures that are greater than 15% of complete eye closure during the last 10 CS-only trials at the end of each training day. A repeated measure ANOVA was used for comparison of CR acquisition for TMEM16B KO and WT mice in all the training sessions. There were highly significant differences of the factors genotype (F(1,16) = 56.93, p < 0.0001) and time (F(6,96) = 40.63, p < 0.0001). The interaction was also significant (F(6,96) = 4.215, p = 0.0008).