Characterisation of the axon initial segment and intrinsic excitability in the sub-acute phase post-ischaemic stroke

Stroke is a leading cause of disability, and stroke-induced changes in cortical excitability are thought to impede functional recovery. Identifying cellular targets that contribute to maladaptive excitability holds great potential for the development of therapeutic interventions to improve stroke outcomes. One potential target is the axon initial segment, the specialized neuronal domain where action potentials are initiated. In the acute phase post-stroke, neurons in the peri-infarct zone have previously been shown to have abnormal axon initial segment structural properties, which may contribute to altered neuronal excitability. However, whether this continues into the sub-acute phase post-stroke, a period with heightened plasticity and when physical rehabilitation typically begins, is unknown. We induced a photothrombotic unilateral ischaemic stroke to the right motor cortex of 13-week-old mice alongside adeno-associated virus labelling of layer 2/3 and layer 5 pyramidal neurons in the peri-infarct zone and contralesional motor cortex. Immunofluorescence staining for Ankyrin-G and whole-cell patch clamp electrophysiology measures were made at 28 days post-stroke to assess changes in axon initial segment structure and function. Additionally, we investigated potential hemispheric-, cortical layer-, and sex-dependent differences in axon initial segment and intrinsic excitability properties. Our results show that normal axon initial segment structure is preserved in the sub-acute phase post-stroke. However, we found that stroke increased action potential half-width and membrane capacitance across both hemispheres and sexes. Additionally, stroke-injured male mice showed hyperpolarized action potential thresholds but reduced maximum spike firing frequencies in the contralesional hemisphere and reduced evoked spike firing frequencies across both hemispheres, while stroke-injured females showed reduced action potential amplitudes and maximum spike firing frequencies in the peri-infarct zone but increased action potential amplitudes in the contralesional hemisphere, along with preserved maximum and evoked firing frequencies in this region. Our results show that despite the preservation of normal axon initial segment structure, changes to intrinsic excitability contribute to the abnormal cortical excitability observed in the sub-acute phase post-stroke. We also provide evidence that stroke induces sex-dependent differences in neuronal function. These findings suggest that intrinsic mechanisms should be considered as a cellular target for stroke therapies and emphasize the importance of considering sex as a biological variable in studies of post-stroke neuronal plasticity and in the development of targeted therapeutic interventions.