Axonal spines in Purkinje cells of ankyrin-G-deficient mice

Spiny membranous protrusions receiving excitatory synaptic input are a characteristic feature of dendrites. Here we demonstrate that spines can be induced in axons by interfering with the structural integrity of the axon initial segment (AIS). Axonal spines were identified in a mouse model with a cerebellum-specific knock-out for ankyrin-G (AnkG). AnkG is a membrane cytoskeleton adapter that is required for restriction of the voltage-gated Na channel, neurofascin, and beta-4 spectrin at the AIS. The morphology of Purkinje cells in AnkG -/- mice was examined using calbindin immunohistochemistry. Surprisingly, a fraction of AnkG -/- Purkinje cell axons traversing the granule cell layer was densely covered with spines. These spiny axons could be followed for up to 150 µm and eventually fasciculated with non-spiny axonal processes of Purkinje cells within the white matter. The size and shape of spines covering these processes was similar to that of spines normally associated with Purkinje cell dendrites. The axonal nature of the spine-bearing processes was verified by immunostaining against the axonal marker tau protein. Interestingly, spine-bearing axons received dense synaptic input as indicated by numerous synaptophysin-positive boutons contacting either the shaft or the spines of these axons. These aberrant axo-axonal terminals were characterized using markers against GABAergic (anti-GAD65) and glutamatergic terminals (anti-VGlut1). Spine-bearing axons were contacted by both GABAergic and glutamatergic boutons. These results indicate that aberrant axonal spines can be induced by interfering with the structural integrity of the AIS-specific membrane cytoskeleton. The associaton of such dendritic features with AnkG-deficient axons indicates that the AnkG-based membrane cytoskeleton of the AIS contributes to the maintenance of axo-dendritic polarity.