nserved, presynaptic SR-protein kinase, SRPK79D. LRP4 and SRPK79D interact genetically and epistatically, as SRPK79D overexpression can suppress lrp4-related phenotypes. Unexpectedly, this role for LRP4 occurs preferentially in excitatory neurons, as impairing lrp4 in inhibitory neurons has no effect. As little is known about the presynaptic determinants of excitatory versus inhibitory synapses, this may suggest a new mode for distinguishing such synapses from the presynaptic side. Thus, LRP4 may represent a conserved synaptic organizer that functions presynaptically, cell autonomously, and independently of agrin to coordinate synapse number and function. Mosca et al. eLife 2017;6:e27347. DOI: 10.7554/eLife.27347 2 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19827996 of 29 Research article Neuroscience Results LRP4 is a synaptic protein expressed in excitatory neurons We identified CG8909 as the fly LRP4 homologue, which is predicted to be a single-pass transmembrane protein whose domain organization resembles that of mammalian LRP4. Drosophila LRP4 shares 38% identity with human LRP4 overall, 61% identity within the LDL-repeat containing extracellular portion, and 28% identity in the intracellular tail. Consistent with previous expression data from whole-brain microarrays, we determined that LRP4 was expressed throughout the adult brain using antibodies against the endogenous protein or an lrp4-GAL4 transgene that expresses GAL4 under the lrp4 promoter and visualized with either Syt-HA or an HA epitope-tagged LRP4. All methods revealed similar patterns of expression in the antennal lobes, optic lobes, and higher olfactory centers including the mushroom body and the lateral horn. Antibody specificity was validated by the complete loss of signal in a deletion of the lrp4 coding region. We further investigated LRP4 in the antennal lobe, the first olfactory processing center in the Drosophila CNS, which has emerged as a model circuit for studying sensory processing and whose synaptic organization was recently mapped at high resolution. LRP4 was enriched in the synaptic neuropil of the antennal lobe. As this neuropil is made up of processes from multiple classes of olfactory neurons, 
all of which make presynaptic connections there, we used intersectional strategies with lrp4-GAL4 to identify which neurons expressed lrp4. These approaches revealed lrp4 expression in both olfactory receptor neurons and projection neurons. Because of the observed neuropil expression of LRP4, we sought to examine the localization of LRP4 with regards to a known synaptic protein, the active zone scaffolding component Bruchpilot. However, due to the density of CNS neuropil, colocalization analyses using light level microscopy have inherently low resolution. Therefore, we applied 2883-98-9 web expansion microscopy to the Drosophila CNS to improve the resolution of colocalization analysis. This technique uses isotropic expansion of immunolabeled tissue while maintaining the spatial relationship between protein targets and allowing for enhanced resolution with confocal microscopy. Using protein-retention expansion microscopy, we obtained reliable, ~4 fold isotropic expansion of Drosophila CNS tissue. To specifically examine the relationship between LRP4 and active zones only in ORNs, we expressed HA-tagged LRP4 and Brp-Short-mStraw using the pebbled-GAL4 driver. LRP4-HA expressed using lrp4-GAL4 localizes to similar regions as LRP4 antibody staining, suggesting the fidelity of this transgene. Within individual expanded glomeruli of
