Our data suggest that the molecular mechanisms for stabilization of the AIS in adult neurons in vivo are distinct from the mechanisms used Z-VAD-FMK in vivo for assembly of the AIS in developing neurons. We propose

a dynamic model for maintenance of the mature AIS, whereby Nfasc186 is constitutively required for anchoring of new protein components to the AIS complex. To test whether proteins known to be constituents of the initial segment complex could cluster appropriately in the absence of the Neurofascins (the neuronal isoform Nfasc186 and the glial isoform Nfasc155), we examined the cerebella of wild-type and Neurofascin null mice at P6. NrCAM was the only component of the AIS complex found to be affected in mutant Purkinje cells (Figure 1A), Selleckchem LY2157299 and the number of Purkinje cells positive for NrCAM was reduced from 93.0% ± 1.3% to 7.4% ± 0.1% (mean values ± SEM, n = 3,

40 cells per animal, p < 0.0001, unpaired Student's t test). In order to establish if the presence of NrCAM at the AIS was dependent on the neuronal isoform of Neurofascin, Nfasc186, we generated transgenic mice expressing FLAG-tagged Nfasc186 on a Neurofascin null background. The transgenic Nfasc186 was targeted appropriately and rescued NrCAM at the AIS (Figure 1B). Interestingly, although the stable targeting of NrCAM to the AIS was dependent on Nfasc186, the converse Idoxuridine was not true (see Figure S1 available online); neither was NrCAM required for the long-term stability of the AIS (Figure S1). We concluded that although Nfasc186 is not required for in vivo assembly of voltage-gated sodium channels at the AIS, it recruits NrCAM to the AIS complex. Since the Neurofascins are not required for the clustering of sodium channels or the majority of their associated proteins in the AIS complex,

we asked if instead they have a role in maintaining the complex. Since Neurofascin null mice die at P7 (Sherman et al., 2005), it is not possible to study the long-term stability of their initial segments in vivo. Hence, we first examined organotypic slice cultures derived from Neurofascin null cerebella. Such cultures are known to maintain viability for months (Kessler et al., 2008). In the absence of the Neurofascins clustering of components of the AIS was complete after 9 days in vitro (DIV). The exception was NrCAM, as found in vivo (Figures 1A and 2). Further culture for up to 15 days resulted in the dispersal of sodium channels, AnkyrinG and βIV-Spectrin, whereas the wild-type AIS remained intact (Figure 2). This suggests that the Neurofascins are required for AIS stability, at least in vitro.