Enhanced neuronal excitability in the absence of neurodegeneration induces cerebellar ataxia

Abstract

Cerebellar ataxia, a devastating neurological disease, may be initiated by hyperexcitability of deep cerebellar nuclei (DCN) secondary to loss of inhibitory input from Purkinje neurons that frequently degenerate in this disease. This mechanism predicts that intrinsic DCN hyperexcitability would cause ataxia in the absence of upstream Purkinje degeneration. We report the generation of a transgenic (Tg) model that supports this mechanism of disease initiation. Small-conductance calcium-activated potassium (SK) channels, regulators of firing frequency, were silenced in the CNS of Tg mice with the dominant-inhibitory construct SK3-1B-GFP. Transgene expression was restricted to the DCN within the cerebellum and was detectable beginning on postnatal day 10, concomitant with the onset of cerebellar ataxia. Neurodegeneration was not evident up to the sixth month of age. Recordings from Tg DCN neurons revealed loss of the apamin-sensitive after-hyperpolarization current (IAHP) and increased spontaneous firing through SK channel suppression, indicative of DCN hyperexcitability. Spike duration and other electrogenic conductance were unaffected. Thus, a purely electrical alteration is sufficient to cause cerebellar ataxia, and SK openers such as the neuroprotective agent riluzole may reduce neuronal hyperexcitability and have therapeutic value. This dominant-inhibitory strategy may help define the in vivo role of SK channels in other neuronal pathways.

Figure 1

Characterization of SK3-1B-GFP Tg mice. (a) Locations of Thy1.2 (gray) and SK3-1B-GFP in transgene cassette with BamHI restriction sites indicated by arrows (numbers above arrows represent kilobases from 5′ end of cassette). Initiation codon is indicated by inverted arrowhead. A 5.4-kb probe for Southern blot analysis is indicated by line below cassette. (b) BamHI digests of tail DNA in D11 and D2. Molecular weights (in kilobases: 23, 9.4, 6.6, 2.3). A 9.2-kb endogenous band (E) seen in all lanes. Tg-specific bands (Tg) seen in D11 lanes 1, 2, 4, 6, 7 and in D2 lanes 1 and 3. (c) Western blot analysis of brain extracts probed with anti-GFP and anti–β-actin Ab’s.

Figure 2

Performance on motor tasks. (a) Stationary rod; (b) rotarod; (c) hanging wire task; (d) hindfoot-forefoot discordance (n = 7–14 Tg; 6–12 non-Tg). D11 non-Tg (filled triangles), D2 non-Tg (open triangles), D11 Tg (closed squares), D2 Tg (open squares) (n = 10–19 Tg; 9–18 non-Tg). Body weights: 4 weeks old: Tg 18.5 ± 0.9 g, non-Tg 17.6 ± 0.8 g; 12 weeks old: Tg 26.9 ±1.3 g, non-Tg 25.7 ± 1.2 g. Error bars = SEM.

Figure 3

SK3-1B-GFP is expressed in DCN neurons that express SK1 and SK2 and does not cause neurodegeneration. (a) Immunoperoxidase staining for GFP with hematoxylin counterstaining revealed transgene expression in the large neurons in the DCN (brown cells indicated by arrowheads in middle and right panels) but not in the cerebellar cortex (indicated by arrowhead pointing to granule cell layer in cerebellar cortex) shown at 5 weeks of age. GC, granule cell layer. (b) In sections from 12-week-old mice stained with H&E, cerebellar architecture appears normal. (c) There is no evidence of reactive gliosis in Tg cerebellar sections immunostained for GFAP at 12 weeks of age. (d) Fluorojade shows no staining (blue DAPI counterstained cells with no green fluorojade-positive cells) in cerebellar sections from mice aged 12 weeks. Scale bars = 100 μm.

Figure 4

Transgene expression in different regions of the brain. H&E staining (left) and transgene expression (right) in the layer V neocortex, spinal cord anterior horn cells, red nucleus, and pontine nucleus (brown). In the midbrain section tyrosine hydroxylase–positive neurons appear blue. SN, substantia nigra, RN, red nucleus. Scale bars = 100 μm.

Figure 5

Grading scheme for transgene expression levels. Examples of sections immunostained for GFP showing relative expression levels and corresponding grade from – to +++. Scale bar = 100 μm.

Figure 6

Transgene-expressing DCN neurons are hyperexcitable. (a) DCN neurons express SK2 (indicated by arrowheads pointing to brown SK2 cells) and SK1 (arrowheads points to blue SK1 cells). SK3 was detected in the midbrain but not in the DCN (not shown). Scale bars = 100 μm. (b) I_AHP in non-Tg DCN. (c) I_AHP in non-Tg DCN is apamin-sensitive. (d) I_AHP is absent from Tg DCN. (e) I_AHP amplitude data summarized. (f) Non-Tg DCN neurons fire spontaneously at approximately 6.3 Hz and exhibit a prominent post-spike after-hyperpolarization. (g) Non-Tg DCN exposed to 100 nM apamin fire more rapidly at approximately 15.2 Hz and do not show the after-hyperpolarization. (h) Tg DCN fire spontaneously at approximately 16.6 Hz and lack the after-hyperpolarization. (i) Summary of mean firing frequency (left) and spike width measured at half amplitude (right). Error bars = SEM.

Figure 7

Other conductances in Tg neurons are unaffected. (a) Amplitude of apamin-resistant K⁺ currents in non-Tg and Tg DCN neurons (n = 6 non-Tg, 32 Tg). (b) Peak voltage-gated Ca²⁺ currents in Tg and non-Tg DCN neurons. (c) Ca²⁺ currents in Tg DCN neurons demonstrating Ca²⁺-CaM–dependent but not Ba²⁺-dependent current inactivation. The Ba²⁺ trace is scaled to the peak amplitude of the Ca²⁺ trace (d). Ca²⁺ current inactivation summarized (n = 5 non-Tg, 8 Tg; P > 0.05). Ca_V, voltage-gated CA²⁺. Error bars = SEM.