The axon initial segment (AIS) is a structure at the proximal end of the axon with a high density of sodium and potassium channels that defines the site of action potential generation. It has recently been shown that this structure is plastic and can change its position along the axon as well as its length in a homeostatic manner (Grubb & Burrone 2010; Kuba et al. 2010). Here, we investigated AIS plasticity in CA1 neurons of rat hippocampal organotypic cultures, using optogenetics to excite individual pyramidal neurons expressing channelrhodopsin-2 (ChR2). 48 hour photostimulation (20 ms blue light flashes delivered in bursts of 5 at 20 Hz, mean of 1 Hz) led to an outward shift of the AIS by around 7 μm. This effect was cell-autonomous and independent of network activity. The structural plasticity of the AIS was accompanied by an increase in current threshold for eliciting an action potential and a shift in the input/output curve towards higher input currents resulting in an overall decrease in excitability. In parallel to this, we also mapped and characterised the axo-axonic synapses that Chandelier interneurons form exclusively onto the AIS of pyramidal cells. We find that these synapses, labelled with either presynaptic (vGAT) or postsynaptic (gephyrin, GABA-A alpha2 receptor) markers, did not translocate with the AIS after chronic stimulation. As a result, the synapses formed onto the proximal end of the AIS were left behind, creating a partial mismatch between axo-axonic synapses and the AIS. We are currently exploring the functional consequences of this intriguing form of plasticity.