SUMMARY Mapping neural circuits across defined synapses is essential for understanding brain function. Here we describe trans-Tango, a technique for anterograde transsynaptic circuit tracing and manipulation. At the core of trans-Tango is a synthetic signaling pathway that is introduced into all neurons in the animal. This pathway converts receptor activation at the cell-surface into reporter expression through site-specific proteolysis. Specific labeling is achieved by presenting a tethered ligand at the synapses of genetically defined neurons, thereby activating the pathway in their postsynaptic partners and providing genetic access to these neurons. We first validated trans-Tango in the Drosophila olfactory system and then implemented it in the gustatory system, where projections beyond the first-order receptor neurons are not fully characterized. We identified putative second-order neurons within the sweet circuit that include projection neurons targeting known neuromodulation centers in the brain. These experiments establish trans-Tango as a flexible platform for transsynaptic circuit analysis.

22The COVID-19 pandemic has severely disrupted worldwide supplies of viral transport media 23 (VTM) due to widespread demand for SARS-CoV-2 RT-PCR testing. In response to this 24 ongoing shortage, we began production of VTM in-house in support of diagnostic testing in our 25 hospital network. As our diagnostic laboratory was not equipped for reagent production, we took 26 advantage of space and personnel that became available due to closure of the research division of 27 our medical center. We utilized a formulation of VTM described by the CDC that was simple to 28 produce, did not require filtration for sterilization, and used reagents that were available from 29 commercial suppliers. Performance of VTM was evaluated by several quality assurance 30 measures. Based on Ct values of spiking experiments, we found that our VTM supported highly 31 consistent amplification of the SARS-CoV-2 target (coefficient of variation = 2.95%) using the 32 Abbott RealTime SARS-CoV-2 EUA assay on the Abbott m2000 platform. VTM was also found 33 to be compatible with multiple swab types and, based on accelerated stability studies, able to 34 maintain functionality for at least four months at room temperature. We further discuss how we 35 met logistical challenges associated with large-scale VTM production in a crisis setting including 36 use of staged, assembly line for VTM transport tube production. 37 38 on June 9, 2020 by guest http://jcm.asm.org/ Downloaded from 39The COVID-19 pandemic has led to an unprecedented need for diagnostic testing. The ideal specimen type is currently believed to be a nasopharyngeal (NP) swab 41 specimen transported to a molecular microbiology laboratory in viral transport medium (VTM). 42Starting in March 2020, increasing demand for testing led to a national shortage of both NP 43 swabs and VTM that created significant bottlenecks in large-scale testing efforts. 44VTM exists in several formulations, all of which consist of a buffered salt solution, a 45 complex source of protein and/or amino acids, and antimicrobial agents. Its purpose is to 46 preserve virus for later amplification by NAAT technology and/or viral culture. Although 47 simpler formulations, for example, saline, are technically compatible with RT-PCR, most NAAT 48 assays for respiratory pathogens have been developed and FDA-cleared for use with more 49 complex transport media (i.e. VTM and universal transport medium, UTM). Overgrowth of 50 bacteria may also occur in media lacking antimicrobial agents. As such, we chose to reproduce a 51 standard of care transport medium to serve our healthcare network. 52In response to the COVID-19 pandemic, our molecular microbiology laboratory was able 53 to quickly scale up SARS-CoV-2 RT-PCR testing to approximately 1000 tests per day to support 54 the needs of our hospital network. However, national shortages of collection materials including 55 nasopharyngeal swabs and VTM were projected to limit our ability to continue testing at this 56 level. We discuss a nationwide collaborative effort to prod...

The mushroom body (MB) is a well-characterized associative memory structure within the Drosophila brain. Analyzing MB connectivity using multiple approaches is critical for understanding the functional implications of this structure. Using the genetic anterograde transsynaptic tracing tool, trans-Tango, we identified divergent projections across the brain and convergent downstream targets of the MB output neurons (MBONs). Our analysis revealed at least three separate targets that receive convergent input from MBONs: other MBONs, the fan-shaped body (FSB), and the lateral accessory lobe (LAL). We describe, both anatomically and functionally, a multilayer circuit in which inhibitory and excitatory MBONs converge on the same genetic subset of FSB and LAL neurons. This circuit architecture enables the brain to update and integrate information with previous experience before executing appropriate behavioral responses. Our use of trans-Tango provides a genetically accessible anatomical framework for investigating the functional relevance of components within these complex and interconnected circuits.

A powerful feature of adaptive memory is its inherent flexibility. Alcohol and other addictive substances can remold neural circuits important for memory to reduce this flexibility. However, the mechanism through which pertinent circuits are selected and shaped remains unclear. We show that circuits required for alcohol-associated preference shift from population level dopaminergic activation to select dopamine neurons that predict behavioral choice in Drosophila melanogaster. During memory expression, subsets of dopamine neurons directly and indirectly modulate the activity of interconnected glutamatergic and cholinergic mushroom body output neurons (MBON). Transsynaptic tracing of neurons important for memory expression revealed a convergent center of memory consolidation within the mushroom body (MB) implicated in arousal, and a structure outside the MB implicated in integration of naïve and learned responses. These findings provide a circuit framework through which dopamine neuronal activation shifts from reward delivery to cue onset, and provide insight into the maladaptive nature of memory.