Biology subjects

Sanfilippo, P.

Publications and source records attributed to Sanfilippo, P..

3 recordsLinked to original sources

bioRxiv2024

Gradients of Recognition Molecules Shape Synaptic Specificity of a Visuomotor Transformation

Converting sensory information into motor commands is fundamental to most of our actions. In Drosophila, visuomotor transformations are mediated by Visual Projection Neurons (VPNs). These neurons convert object location and motion into directional behaviors downstream through a synaptic gradient mechanism. However, the molecular origins of such graded connectivity remain unknown. We addressed this question in a VPN cell type called LPLC2, which integrates looming motion and transforms it into an escape response through two parallel dorsoventral synaptic gradients at its inputs and outputs. We identified two corresponding dorsoventral expression gradients of cell recognition molecules within the LPLC2 population that regulate this synaptic connectivity. Dpr13 determines synaptic outputs of LPLC2 axons by interacting with its binding partner DIP-{varepsilon} expressed in the Giant Fiber, a neuron that mediates escape. Similarly, beat-VI regulates synaptic inputs onto LPLC2 dendrites by interacting with Side-II expressed in upstream motion-detecting neurons. Behavioral, physiological, and molecular experiments demonstrate that these coordinated molecular gradients control differential synaptic connectivity, enabling the accurate transformation of visual features into motor commands. As within-neuronal-type variation in gene expression is also observed in the mammalian brain, graded expression of cell recognition molecules may represent a common mechanism underlying synaptic specificity.

neuroscience↗

bioRxiv2023

Constitutive and conditional epitope-tagging of endogenous G protein coupled receptors in Drosophila

To visualize the cellular and subcellular localization of neuromodulatory G-protein coupled receptors (GPCRs) in Drosophila, we implement a molecular strategy recently used to add epitope tags to ionotropic receptors at their endogenous loci. Leveraging evolutionary conservation to identify sites more likely to permit insertion of a tag, we generated constitutive and conditional tagged alleles for Drosophila 5-HT1A, 5-HT2A, 5-HT2B, Oct{beta}1R, Oct{beta}2R, two isoforms of OAMB, and mGluR. The conditional alleles allow for the restricted expression of tagged receptor in specific cell types, an option not available for any previous reagents to label these proteins. We show that 5-HT1A and 5-HT2B localize to the mushroom bodies and central complex respectively, as predicted by their roles in sleep. By contrast, the unexpected enrichment of Oct{beta}1R in the central complex and of 5-HT1A and 5-HT2A to nerve terminals in lobular columnar cells in the visual system suggest new hypotheses about their function at these sites. Using an additional tagged allele of the serotonin transporter, a marker of serotonergic tracts, we demonstrate diverse spatial relationships between postsynaptic 5-HT receptors and presynaptic 5-HT neurons, consistent with the importance of both synaptic and volume transmission. Finally, we use the conditional allele of 5-HT1A to show that it localizes to distinct sites within the mushroom bodies as both a postsynaptic receptor in Kenyon cells and a presynaptic autoreceptor. Significance StatementIn Drosophila, despite remarkable advances in both connectomic and genomic studies, antibodies to many aminergic GPCRs are not available. We have overcome this obstacle using evolutionary conservation to identify loci in GPCRs amenable to epitope-tagging, and CRISPR/Cas9 genome editing to generated eight novel lines. This method also may be applied to other GPCRs and allows cell-specific expression of the tagged locus. We have used the tagged alleles we generated to address several questions that remain poorly understood. These include the relationship between pre- and post-synaptic sites that express the same receptor, and the use of relatively distant targets by pre-synaptic release sites that may employ volume transmission as well as standard synaptic signaling.

neuroscience↗

bioRxiv2023

Mapping of multiple neurotransmitter receptor subtypes and distinct protein complexes to the connectome

Neurons express different combinations of neurotransmitter receptor (NR) subunits and receive inputs from multiple neuron types expressing different neurotransmitters. Localizing NR subunits to specific synaptic inputs has been challenging. Here we use epitope tagged endogenous NR subunits, expansion light-sheet microscopy, and EM connectomics to molecularly characterize synapses in Drosophila. We show that in directionally selective motion sensitive neurons, different multiple NRs elaborated a highly stereotyped molecular topography with NR localized to specific domains receiving cell-type specific inputs. Developmental studies suggested that NRs or complexes of them with other membrane proteins determines patterns of synaptic inputs. In support of this model, we identify a transmembrane protein associated selectively with a subset of spatially restricted synapses and demonstrate through genetic analysis its requirement for synapse formation. We propose that mechanisms which regulate the precise spatial distribution of NRs provide a molecular cartography specifying the patterns of synaptic connections onto dendrites.

neuroscience↗