Postsynaptic Effectors of Neuron Morphology and Function: Part I. Characterization of Postsynaptic Drosophila Syndapin. Part II. Chimeric Light-Activated Receptors for the Control of 5-ht_1a Signaling

PART I

Vertebrate syndapin (Synd) was identified as a dynamin interacting protein, which binds Wiskott-Aldrich Syndrome protein (WASp). WASp is a known activator of the Arp-2/3 complex (actin related protein) that promotes actin polymerization. In Drosophila, Synd has a postsynaptic distribution at neuromuscular junctions (NMJ). To determine the synaptic function of Synd, we have generated and characterized Drosophila Synd loss-of-function mutants. These mutants display a NMJ overgrowth phenotype manifested by increased synaptic span, bouton number, and extent of branching. The synd mutants also contain hyperbranching boutons that give rise to multiple “satellite” boutons. We can rescue this phenotype by expression of a synd transgene postsynaptically, but not presynaptically. Loss of synd resembles the NMJ morphological phenotype of wsp loss-of-function mutants, suggesting a common functional pathway. Furthermore, the NMJ overgrowth phenotype is most pronounced in synd and wsp double mutants and dependent on gene dosage – demonstrating that the two genes interact. Finally, relative postsynaptic staining of Wsp is decreased in a synd mutant, but Synd remains intact in a wsp mutant. Taken together, this suggests that Syndapin serves as an adaptor protein recruiting Wsp postsynaptically to the NMJ. Loss of synd is likely to modulate synapse growth by disrupting Wsp localization and function.

PART II

The 5-HT_1A receptor is a metabotropic G protein-coupled receptor (GPCR) linked to the Gi/o signaling pathway and has been specifically implicated in the pathogenesis of depression and anxiety. To understand and precisely control 5-HT_1A signaling, we created a light-activated GPCR, which targets into 5-HT_1A receptor domains and substitutes for endogenous 5-HT_1A receptors. To induce 5-HT_1A-like targeting, vertebrate rhodopsin (Rh) was tagged with the C-terminal domain (CT) of 5-HT_1A (Rh-CT_5-HT1A). Rh-CT_5-HT1A activates G protein-coupled inward rectifying K+ channels (GIRK) in response to light and causes membrane hyperpolarization in hippocampal neurons, similar to the agonist-induced responses of the 5-HT_1A receptor. The intracellular distribution of Rh-CT_5-HT1A resembles that of wild type 5-HT_1A receptor; Rh-CT_5-HT1A localizes somatodendritically and efficiently traffics to distal dendritic processes. Additionally, neuronal expression of Rh-CT_5-HT1A, but not Rh, inhibits responses to 5-HT_1A agonist, suggesting that Rh-CT_5-HT1A and endogenous 5-HT_1A receptors compete to interact with the same trafficking machinery. Finally, Rh-CT_5-HT1A is able to substitute for absent 5-HT_1A signaling in cultured neurons and dorsal raphe slices of KO mice, showing that Rh-CT_5-HT1A functionally compensates for native 5-HT_1A. Thus, as an optogenetic tool, Rh-CT_5-HT1A has the potential to directly correlate in vivo 5-HT_1A signaling with 5-HT neuron activity and behavior in both wild type animals and animal models of neuropsychiatric disease.