AIS Plasticity In Four Hippocampal Neuronal Cell Populations

In neurons, action potentials initiate at a specialized region of the axon called the axon initial segment (AIS). The AIS is a highly structured region consisting of a high density of K⁺ and Na⁺ channels that regulate the firing of action potentials. In hippocampal neurons, the AIS moves more distally down the axon in response to high activity conditions but the mechanisms of this plasticity are currently unknown. These mechanisms may further our understanding of conditions involving abnormal neuronal activity such as epilepsy. Until recently we have only known about AIS movement in hippocampal neurons in general, therefore this study aimed to further our understanding of the mechanisms of AIS plasticity in individual cell types. Immunohistochemical staining was used to label the four predominant populations of cells found in the hippocampus. Mature Dentate Granule (DG) neurons express the transcription factor Prox1 throughout all stages of their life, therefore we used this protein as a marker for DG cells. GABAergic cells were stained by targeting GABA which is found only in neurons that use it as a neurotransmitter. Cornu Ammonis (CA)3 neurons were identified by αPy staining for as yet unidentified filaments unique to these cells, and an absence of GABA staining. Cornu Ammonis (CA)1 neurons were identified by an absence of aforementioned protein stains. The AIS was labelled by staining for AnkyrinG (AnkG), the main scaffolding protein of the AIS. Preliminary data in the lab suggested that the calcineurin inhibitor FK506 halted movement of the AIS in high activity conditions. Therefore, cells were treated for 48 hours with or without the calcineurin inhibitor FK506 at 10 days in vitro (DIV) in the presence or absence of 15mM K⁺ to stimulate high electrical activity. In DG cells, the AIS moved distally by ~5.5μm in high activity conditions but in the presence of FK506 this movement was not observed. A large number of DG cells were observed at 12DIV, which may have been attributed to proliferation of this cell type in culture. BrdU incorporation at four time points, however, suggested no DG cells were born in vitro. In CA1 neurons, high K⁺ treatment produced AIS movement of ~10μm which was also arrested in high K⁺ + FK506 conditions. GABAergic neurons showed a small but significant, and calcineurin dependent distal movement of ~2μm at the start point of the AIS, but no significant movement was observed in the maximum and end points of the AIS. In CA3 neurons αPy staining was observed in control conditions but not high K⁺ conditions. This was not affected by the presence or absence of FK506. Although AIS movement could not be measured for this population of neurons, these surprising results warrant future investigation. Overall, data gathered from this study show that different hippocampal cell types have different AIS responses to elevated electrical activity, but all are blocked by the calcineurin inhibitor FK506.