Active zone proteins Bassoon and Piccolo at the calyx of Held : age - dependent localization and targeted in vivo perturbation

Neurons communicate with each other via synaptic transmission. Chemical synapses transfer information through the release of neurotransmitter. In contrast to the very detailed knowledge of the functional capabilities of synapses and the fact that most presynaptic proteins have been identified, the molecular mechanisms underlying neurotransmitter release remain poorly understood. The active zone (AZ), the site of Ca2+-dependent neurotransmitter release in nerve terminals, is a morphological specialization of the presynaptic plasma membrane with a set of proteins necessary for the organization of exo- and endocytotic molecular machineries. Bassoon and Piccolo are structurally related, large multidomain proteins specifically and exclusively located in AZs of the mammalian nervous system, they are thought to organize AZs through their multidomain capability of interaction with many other proteins. Specific deletion of Bassoon in mice resulted in a significantly lower number of active synapses in hippocampal autaptic cultures. Bassoon deletion did not result in compensatory changes of AZ proteins but Piccolo, which was increased 1.4 times. Hence, the presence of Piccolo may prevent a loss of function in Bassoon knockout mice. To assess the role of Bassoon and Piccolo in neurotransmitter release, we examined their localization in the calyx of Held giant presynaptic terminal and attempted a simultaneous knockdown of both proteins using RNA interference. First, we examined the three-dimensional (3D) localization of Bassoon and Piccolo in the rat calyx of Held between postnatal days (P) 9 and 24, a period characterized by pronounced structural and functional changes. To unequivocally assign immunohistochemical (IHC) signals to the calyx, we expressed membrane-anchored GFP (mGFP) or synaptophysin-GFP in the calyx using targeted stereotaxic delivery of adeno-associated virus (AAV) vectors. We then examined the distribution of Bassoon and Piccolo using IHC in slices containing calyces with labeled plasma membrane or synaptic vesicles (SV) using confocal microscopy and 3D reconstructions. We found that both Bassoon and Piccolo were arranged in clusters resembling the size of AZs. These clusters were located in the presynaptic membrane facing the principal cell, close to and partially overlapping with SV clusters. Simultaneous application of both antibodies revealed a ~90% overlap, indicating that both proteins co-localize. We found about 200-400 clusters in both P9 and P24 calyces. The number and distribution of clusters did not differ, suggesting that these parameters do not contribute to postnatal functional maturation. Furthermore, we observed IHC-signals in the spaces between finger-like protrusions of the calyx, consistent with intermingled non-calyceal inputs located on the principal cell. As these signals mimic a calyx-like distribution, particularly in 2D images, pre-labeled calyces are essential for IHC studies of protein distribution in the calyx of Held. To understand the function of Bassoon and Piccolo in AZ organization and their contribution to neurotransmitter release, we attempted to down-regulate each of these proteins in vivo in the calyx of Held using RNA interference. Small hairpin RNAs (shRNA) directed against Bassoon and Piccolo were expressed through AAV vectors. Viral particles were stereotaxically delivered to the ventral cochlear nucleus, where the somata of neurons giving rise to calyx terminals are located. Using 3D fluorescence immunohistochemistry, we could demonstrate a down-regulation of Piccolo at its most relevant site - the nerve terminal. With this approach we were able to show a decreased amount of Piccolo in the calyces treated with shRNA as compared to control calyces. Preliminary results suggest a knockdown of Bassoon using the same approach. However, low titers of the virus preparations did not yield numbers of perturbed calyces sufficient for functional analyses in brain slices. This also precluded knocking down Bassoon and Piccolo simultaneously. Attempts of improving viral titers remained unsuccessful, posing a potential general limitation to AAV-mediated applications of shRNAs for targeted in vivo RNA interference