Fig 1.
Ano2 is expressed in cerebellar Purkinje cells.
(A) Overview of in situ hybridization of a sagittal cerebellar slice reveals a continuous expression of Ano2 in the Purkinje cell layer (PCL). No staining is visible in the molecular layer (ML) and only few cells are labelled in the granule layer (GL) and the area where the deep cerebellar nuclei (DCN) are located. Control experiments using the sense probes revealed no staining in the cerebellum (inset). Scale bars = 1 mm (B) Detail of the Purkinje cell layer demonstrating the colocalization (lower) of the in situ hybridization (upper) with an immunohistochemical staining for calbindin (green) (middle) following the in situ hybridization procedure. A DAPI nuclear stain (blue) was added to visualize the GL. Scale bars = 25 μm.
Fig 2.
Neuroanatomy of the cerebellar cortex is comparable between wildtype and Ano2-/- mice.
(A) Representative immunohistochemical stainings for calbindin (green) and parvalbumin (red) on sagittal cerebellar slices of wildtype (left) and Ano2-/- mice (right). Stainings were used to compare Purkinje cell density (B) and size (C) as well as the density of molecular layer interneurons (D) between wildtype and Ano2-/- mice. DAPI (blue) was used to visualize cell nuclei. (B)–(D) No significant differences were detected in Purkinje cell density (B; wt 41.1 ± 6.4 per mm PCL, n = 61 images from N = 6 animals, Ano2-/- 38.5 ± 7.5 per mm PCL, n = 42 N = 6; Student’s t-test p = 0.0811) and diameter (C; wt 20.7 ± 3.4 μm, n = 342 cells in 61 images from N = 6 animals, Ano2-/- 21.1 ± 3.7 μm, n = 246 in 42 images N = 6; Student’s t-test p = 0.2091), as well as in the density of molecular layer interneurons (D; wt 15.9 per 105 μm3 ML IQR 13.3–20.7, n = 62 images from N = 6 animals; Ano2-/- 17.1 per 105 μm3 ML IQR 13.3–23.6, n = 45 N = 6; Wilcoxon-Mann-Whitney test p = 0.501) between wildtype and Ano2-/- mice. (E) Immunohistochemical staining for calbindin (blue), VGluT2 (green) and VGAT (red) on sagittal cerebellar slices. As expected, GABAergic synapses (red) cover the entire molecular layer, whereas climbing fiber synapses (green) only span the proximal two thirds of the molecular layer. (F) Detail of a Purkinje cell with primary and secondary dendrites (calbindin; blue) with climbing fiber synapses (VGluT2; green) and GABAergic synapses (VGAT; red). (G), (H) No significant differences were detected in density of GABAergic (G; wt 22.0 ± 6.8 per 102 μm2 ML, n = 42 images from N = 5 animals, Ano2-/- 21.8 ± 5.5 per 102 μm2 ML, n = 47 N = 5; Student’s t-test p = 0.8788) and climbing fiber synapses (H; wt 3.5 ± 1.0 per 102 μm2 ML, n = 42 images from N = 5 animals; Ano2-/- 3.2 ± 1.0 per 102 μm2 ML, n = 48 N = 5; Student’s t-test p = 0.1989) in the proximal half of the molecular layer between wildtype and Ano2-/- mice. (I) Histograms of the distances measured between adjacent climbing fiber synapses on a Purkinje cell dendrite reveal a comparable distribution of climbing fiber synapses between wildtype and Ano2-/- mice (bin size = 0.5 μm; wt n = 1698 distances in 35 images from N = 9 animals, Ano2-/- n = 1762 in 30 images N = 6). ML = molecular layer; PCL = Purkinje cell layer; GL = granule layer. Scale bars = 20 μm.
Fig 3.
Spontaneous simple spike firing of Purkinje cells is unaltered in Ano2-/- mice.
Representative traces of spontaneous simple spike activity in wildtype (black) and Ano2-/- mice (red) before (A) and after (B) addition of 20 μM gabazine to the bath solution. The arrows in (A) mark spontaneous inhibitory postsynaptic currents (IPSCs) that were then blocked by addition of gabazine to the bath solution. Comparison of spontaneous simple spike firing rates reveals no significant difference between wildtype and Ano2-/- mice without (A; wt 43.3 ± 12.9 Hz, n = 10 cells of N = 3 animals; Ano2-/- 53.2 ± 21.4 Hz, n = 15 N = 3; Student’s t-test p = 0.1631) and with 20 μM gabazine (B; wt 62.3 ± 21.5 Hz, n = 12 cells of N = 3 animals; Ano2-/- 59.8 ± 20.4 Hz, n = 23 N = 5; Student’s t-test p = 0.7448).
Fig 4.
ANO2 reduces excitability during strong and threshold activation.
Analysis of simple spike counts upon threshold activation (A), moderate activation (B) and strong activation (C). Representative traces (left) of simple spike activity of Purkinje cells from wildtype (black) and Ano2-/- mice (red) at physiological ECl. Comparison of simple spike counts during the first 500 ms of the respective current injection at physiological (middle) and elevated ECl (right) (A middle: wt 1.0 IQR 0.0–7.0, n = 39 cells of N = 8 animals, Ano2-/- 6.0 IQR 0.0–13.0, n = 43 cells of N = 11 animals, Wilcoxon-Mann-Whitney test p = 0.040; A right: wt 5.0 IQR 0.0–14.3, n = 32 cells of N = 6 animals, Ano2-/- 9.0 IQR 1.0–14.75, n = 36 cells of N = 8 animals, Wilcoxon-Mann-Whitney test p = 0.368; B middle: wt 33.1 ± 11.0, n = 35 cells of N = 8 animals, Ano2-/- 35.9 ± 11.8, n = 31 N = 9, Student’s t-test p = 0.3255; B right: wt 38.9 ± 11.1, n = 29 cells of N = 6 animals, Ano2-/- 38.5 ± 10.6, n = 31 N = 8, Student’s t-test p = 0.9116; C middle: wt 46.1 ± 11.0, n = 21 cells of N = 6 animals, Ano2-/- 54.1 ± 10.6, n = 19 N = 7, Student’s t-test p = 0.0255; C right: wt 57.6 ± 11.4, n = 27 cells of N = 6 animals, Ano2-/- 60.0 ± 13.1, n = 29 N = 7, Student’s t-test p = 0.4788). * p < 0.05.
Fig 5.
ANO2-mediated chloride currents gradually increase interspike intervals.
Progression of interspike intervals (ISIs) during the first sixteen simple spikes upon moderate (A) and strong activation (B) of Purkinje cells in wildtype (black) and Ano2-/- mice (red). At physiological ECl (left), the interspike intervals in wildtype mice grow longer from the second ISI on, causing a divergence of the ISIs from wildtype and Ano2-/- mice. In contrast, the progression of ISIs at elevated ECl (right) develops without divergence (A left: Genotype effect: F(1, 930) = 22.65, p < 0.001; ISI number x genotype interaction: F(14, 930) = 0.09, p = 1; A right: Genotype effect: F(1, 900) = 0.18, p = 0.6712; ISI number x genotype interaction: F(14, 930) = 0.01, p = 1; B left: Genotype effect: F(1, 570) = 29.37, p < 0.001; ISI number x genotype interaction: F(14, 570) = 0.14, p = 1; B right: Genotype effect: F(1, 840) = 4.2, p = 0.0430; ISI number x genotype interaction: F(14, 840) = 3.7, p = 1). * p < 0.05; *** p < 0.001.
Fig 6.
ANO2 increases the hyperpolarization following strong activation of Purkinje cells.
(A) Representative traces of the afterhyperpolarization (AHP) following strong activation of wildtype (black/grey) and Ano2-/- (red/dark red) Purkinje cells at physiological and elevated ECl. The holding current was set at -500 pA or -550 pA for all cells in this example. (B) AHP amplitude after strong activation is significantly larger in wildtype compared to and Ano2-/- mice at physiological ECl, but not at elevated ECl (ECl -87 mV: wt -11.2 ± 2.8 mV, n = 20 cells of N = 6 animals; Ano2-/- -8.9 ± 2.4 mV, n = 19 N = 7; Student’s t-test p = 0.0097; ECl -48 mV: wt -9.0 ± 3.0 mV, n = 27 cells of N = 6 animals; Ano2-/- -9.2 ± 1.9 mV, n = 31 N = 7; Student’s t-test p = 0.7983) (C) Time till the maximal AHP is reached after strong activation is unaltered by ANO2 or the elevation of ECl (ECl -87 mV: wt 187.8 ± 53.2 ms, n = 20 cells of N = 6 animals; Ano2-/- 178.1 ± 35.6 ms, n = 19 N = 7; Student’s t-test p = 0.5073; ECl -48 mV: wt 172.4 ± 50.7 mV, n = 27 cells of N = 6 animals; Ano2-/- 181.4 ± 49.8 ms, n = 31 N = 7; Student’s t-test p = 0.5035). ** p < 0.01.
Fig 7.
Stimulation of molecular layer interneurons and control experiments.
(A) Schematic illustration of the neuronal circuits in the cerebellar cortex and the protocol for the stimulation of molecular layer interneurons. Purkinje cells (PC) are the central element in the olivo-cerebellar circuit. Purkinje cells project to the deep cerebellar nuclei (DCN) and receive excitatory input from the mossy fiber (MF)-parallel fiber (PF) pathway and from climbing fibers (CF), which originate in the inferior olivary nucleus (IO) of the brainstem. Basket cells (BC) and stellate cells (SC) both inhibit Purkinje cells, whereas Golgi cells (GoC) inhibit the mossy fiber-granule cell (GrC) synapse. Evoked inhibitory postsynaptic currents (eIPSCs) are induced by extracellular stimulation of molecular layer interneurons (P2). eIPSCs are recorded at the Purkinje cell soma with the patch pipette (P1). Reduction of eIPSC amplitudes is induced by a train of five depolarizing pulses with an amplitude of 50 mV via the patch pipette (inset). GL = granule layer, PCL = Purkinje cell layer, ML = molecular layer (B) Representative trace of an eIPSC recording described in (A). At the beginning of each recording, a test pulse (a) was applied to monitor series resistance. The holding potential was increased to -60.5 mV (b) before inducing two eIPSCs (c) with an interstimulus interval of 50 ms (arrows in the command trace). The red arrow marks the amplitude of eIPSC that was analyzed in these experiments (C), (D) Representative traces of eIPSC recordings of one cell before (black) and after (grey) adding 0.5 μM TTX (C) or 20 μM gabazine (D) to the bath solution. eIPSCs are completely abolished by both TTX and gabazine.
Fig 8.
ANO2 promotes reduction of eIPSC amplitude.
(A) Representative traces of eIPSC recordings in wildtype (upper) and Ano2-/- mice (lower) before (black/dark red) and after depolarization (grey/light red) of Purkinje cells. The dotted line marks the holding current right before stimulation of interneurons from which the amplitude was calculated. (B) Time course of eIPSC amplitude in wildtype (upper) and Ano2-/- mice (lower). Reduction (blue) was defined as the difference between eIPSC amplitude of the last measurement before depolarization and eIPSC amplitude of the second measurement after depolarization. eIPSC amplitude was significantly reduced in both mouse lines after depolarization (wt before depol. 110.4 ± 16.3%, wt after depol. 59.6 ± 13.4%, paired Student’s t-test p = 0.0001; Ano2-/- before depol. 101.6 ± 14.5%, Ano2-/- after depol. 74.5 ± 18.4%, paired Student’s t-test p = 0.0004). (C) Recovery of eIPSC amplitude in wildtype Purkinje cells after depolarization (B, upper). An exponential fit (red) to the mean values of eIPSC amplitude was performed to obtain the time constant τ = 30.6 s of recovery. (D) Comparison of reduction of eIPSC amplitudes (blue in B) between wildtype and Ano2-/-mice. The reduction of eIPSC amplitude is significantly attenuated in Ano2-/- mice (wt 50.8 ± 20.9%, Ano2-/- 27.2 ± 21.2%, Student’s t-test p = 0.0173). wt n = 9 cells from N = 6 animals; Ano2-/- n = 14, N = 9; * p < 0.05, *** p < 0.001.
Fig 9.
ANO2 in the olivo-cerebellar circuit.
Schematic illustration of a cerebellar module summarizing the recent findings about the role of ANO2 in the olivocerebellar cortex. Glutamatergic neurons are depicted in green and GABAergic neurons in red. ANO2 has been shown to be expressed in inferior olivary neurons, where it accelerates repolarization after high-threshold calcium spikes and, thus, promotes the generation of these calcium spikes [9]. In Purkinje cells, ANO2 attenuates excitability upon strong activation. First indications of an expression of ANO2 in neurons of the deep cerebellar nuclei is presented in Fig 1A, but functional involvement in the cerebellar module awaits investigation.