A key component allowing a neuron to function properly within its dynamic environment is the axon initial segment (AIS), the site of action potential generation. In visual cortex, AIS of pyramidal neurons undergo periods of activity-dependent structural plasticity during development. However, it remains unknown how AIS morphology is organized during development for downstream cells in the visual pathway (retinal ganglion cells; RGCs) and whether AIS retain the ability to dynamically adjust to changes in network state. Here, we investigated the maturation of AIS in RGCs during mouse retinal development, and tested putative activity-dependent mechanisms by applying visual deprivation with a focus on the AIS-specific cisternal organelle (CO), a presumed Ca 2+ -store. Whole-mount retinae from wildtype and Thy1-GFP transgenic mice were processed for multi-channel immunofluorescence using antibodies against AIS scaffolding proteins ankyrin-G, βIV-spectrin and the CO marker synaptopodin (synpo). Confocal microscopy in combination with morphometrical analysis of AIS length and position as well as synpo cluster size was performed. Data indicated that a subset of RGC AIS contains synpo clusters and that these show significant dynamic regulation in size during development as well as after visual deprivation. Using super resolution microscopy, we addressed the subcellular localization of synpo in RGC axons. Similar to cortical neurons, RGCs show a periodic distribution of AIS scaffolding proteins. A previously reported scaffold-deficient nanodomain correlating with synpo localization is not evident in all RGC AIS. In summary, our work demonstrates a dynamic regulation of both the AIS and synpo in RGCs during retinal development and after visual deprivation, providing first evidence that the AIS and CO in RGCs can undergo structural plasticity in response to changes in network activity.

Rodent neocortical neurons undergo prominent postnatal development and maturation. The process is associated with structural and functional maturation of the axon initial segment (AIS), the site of action potential initiation. In this regard, cell size and optimal AIS length are interconnected. In sensory cortices, developmental onset of sensory input and consequent changes in network activity cause phasic AIS plasticity that can also control functional output. In non-sensory cortices, network input driving phasic events should be less prominent. We, therefore, explored the relationship between postnatal functional maturation and AIS maturation in principal neurons of the primary motor cortex layer V (M1LV), a non-sensory area of the rat brain. We hypothesized that a rather continuous process of AIS maturation and elongation would reflect cell growth, accompanied by progressive refinement of functional output properties. We found that, in the first two postnatal weeks, cell growth prompted substantial decline of neuronal input resistance, such that older neurons needed larger input current to reach rheobase and fire action potentials. In the same period, we observed the most prominent AIS elongation and significant maturation of functional output properties. Alternating phases of AIS plasticity did not occur, and changes in functional output properties were largely justified by AIS elongation. From the third postnatal week up to five months of age, cell growth, AIS elongation, and functional output maturation were marginal. Thus, AIS maturation in M1LV is a continuous process that attunes the functional output of pyramidal neurons and associates with early postnatal development to counterbalance increasing electrical leakage due to cell growth.