BAcTrace a new tool for retrograde tracing of neuronal circuits
Posted on: 10 July 2020
Preprint posted on 25 January 2020
Categories: genetics, neuroscience
Background
Understanding the underlying connectivity of a neural circuit is critical for ultimately understanding its function. The gold standard technique for describing a neural circuit is electron microscopy, but it is extremely laborious, time intensive, and costly, preventing many labs from being able to use this technique to study neural circuits. To combat these difficulties, viral and genetic techniques have been developed to identify synaptic connectivity between neurons of interest in a wide variety of model organisms. Classical approaches using dye traces in non-human primates were some of the first neuroanatomical studies performed to identify connectivity (Goldman-Rakic et al. 1984).
Genetic approaches began to take off with the development of GFP reconstituted across synaptic partners (GRASP) in C. elegans (Feinberg et al. 2008). Modified GRASP approaches in Drosophila melanogaster, namely sybGRASP (Macpherson et al. 2015) and Tango-Trace (Jagadish S et al. 2014), took this framework and improved the synaptic specificity. More recently, true transsynaptic tracers have been developed, trans-TANGO and TRACT [(Talay et al. 2017; Huang et al. 2017) disclosure – I am an author on the TRACT paper Huang et al. 2017]. Yet, both of these tracers are anterograde, meaning they label neurons postsynaptic to a given neuronal population. There is a need for a retrograde tracer in Drosophila that this paper attempts to fill. This paper takes advantage of the specificity of Botulinum Neurotoxin A (BoNT/A) for human SNAP25 to design a reporter system for synaptic connectivity. The mechanism of action of the light chain of BoNT/A has been known for a long time (Schiavo et al. 1992), allowing the authors to design a fully transgenic system for transsynaptic tracing.
Key Findings
To develop their retrograde tracer, the authors use a transsynaptic system that expresses BoNT/A fused to a CD2 transmembrane protein in postsynaptic neurons. Synaptic specificity is achieved by expressing synaptobrevin-fused GFP specifically in receiver (presynaptic) cells. The transgenically expressed light chain of BoNT/A binds synaptobrevin-GFP through a GFP nanobody that replaces the receptor binding domain of the neurotoxin. It remains bound to the receiver cell, and is taken up by eventually being endocytosed. After being taken up by the receiver neuron, BoNT/A eventually cleaves human SNAP25 to release a QF transcription factor to alter transcription in the receiving neuron (throughout the paper this cleavage of the QF transcription factor drives tdTomato or Halo expression). The authors demonstrate the system’s effectiveness in vitro, then move in vivo to the fly olfactory system.
Impressively, they see robust labeling of projection neurons in vivo when BoNT/A is expressed in kenyon cells, a major postsynaptic partner of projection neurons. To get a better sense of the specificity, the authors move on to labeling projection neurons using feedback synapses onto olfactory receptor neurons. These synapses are weaker, and a corresponding delay in labeling of projection neurons is observed with labeling being stronger in 10 day old flies. The authors also examine the potential that BAcTrace is neurotoxic, and mostly rule out the possibility through electrophysiological recordings of labeled projection neurons.
Highlights
I was impressed by how effectively the authors were able to make their BoNT/A system work. Certainly many years of work went into making it as effective as it is in the paper, and there are benefits to using BoNT/A, including a reduced likelihood of off-target effects by BoNT/A because it is such a specific protease. In principle, having the QF driver fused to human SNAP25 as opposed to a different protein with a more general cleavage site should lead to lower background. The main source of background seemed to be independent expression of BoNT/A by UAS that is reduced by the B3 stop cassette. Of course, all components of the system have some background expression, but mostly at very low levels.
The authors’ also clearly observe activity dependence of their tracing system, which is likely an overall beneficial feature considering the density of synaptic connectivity in the fly olfactory system. The activity dependence almost certainly comes from the syb:GFP transgene, which is critical for uptake of BoNT/A. With some modifications, this system could be used to label highly active synapses during a specific window.
An unfortunate aspect of the author’s transgenic system is the sheer number of transgenes. The system fully saturates the Gal4/UAS, LexA/LexAop, and QF/QUAS systems. This minimizes the potential use of BAcTrace to examine how genetic manipulations alter neuronal connectivity. Also, by using the Gal4/UAS system and known strong insertion sites attP2 and attP40 to drive donor transgene expression, they potentially miss the opportunity to enhance the labeling by using these sites to drive the weak link in the BAcTrace labeling system. For BAcTrace to be used to label neurons for electrophysiology, ideally labeling would be strong at 2-4 days old because that is when it is easiest to record from fly neurons. Of course, the upside of using split-Gal4 lines inserted at attP2 and attP40 is that you get more interpretable results, so changing the system is not without tradeoffs.
Lastly, I am encouraged by the author’s reports of background driven by the V5 protein tag. This fits with my personal experience of it being a poor tag that is both difficult to detect and comes with many unwanted effects. I hope others in the field take note and reduce their use of this tag, and I thank the authors for reporting this in their preprint.
Questions
Part of the paper that stands out is the repeated use of the VT033006 line as the receiver driver. Have the authors’ tried other receiver driver lines with any success? Specifically, have the authors tried using a panneuronal driver for the receiver transgene expression? The ability of any tracer tool to generalize across lines is important for its ability to be adapted by the wider community.
Separately, have the flies been raised at different temperatures to see how that affects labeling? In both trans-TANGO and TRACT, non-standard temperatures are used to get the best effects (18ºC for trans-TANGO and one day at 29ºC for TRACT). This could help improve the signal that is weaker in figures 4 and 5.
References
Feinberg, E., Vanhoven, M., Bendesky, A., Wang, G., Fetter, R., Shen, K., Bargmann, C. (2008). GFP Reconstitution Across Synaptic Partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57(3), 353-63. https://dx.doi.org/10.1016/j.neuron.2007.11.030
Goldman-Rakic, P., Selemon, L., Schwartz, M. (1984). Dual pathways connecting the dorsolateral prefrontal cortex with the hippocampal formation and parahippocampal cortex in the rhesus monkey Neuroscience 12(3), 719-743. https://dx.doi.org/10.1016/0306-4522(84)90166-0
Huang, T., Niesman, P., Arasu, D., Lee, D., Cruz, A., Callejas, A., Hong, E., Lois, C. (2017). Tracing neuronal circuits in transgenic animals by transneuronal control of transcription (TRACT). eLife 6(), e32027. https://dx.doi.org/10.7554/elife.32027
Jagadish, S., Barnea, G., Clandinin, T., Axel, R. (2014). Identifying functional connections of the inner photoreceptors in Drosophila using Tango-Trace. Neuron 83(3), 630-44. https://dx.doi.org/10.1016/j.neuron.2014.06.025
Macpherson, L., Zaharieva, E., Kearney, P., Alpert, M., Lin, T., Turan, Z., Lee, C., Gallio, M. (2015). Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation Nature Communications 6(1), 10024. https://dx.doi.org/10.1038/ncomms10024
Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Laureto, P., DasGupta, B., Montecucco, C. (1992). Tetanus and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of synaptobrevin Nature 359(6398), 832-835. https://dx.doi.org/10.1038/359832a0
Talay, M., Richman, E., Snell, N., Hartmann, G., Fisher, J., Sorkaç, A., Santoyo, J., Chou-Freed, C., Nair, N., Johnson, M., Szymanski, J., Barnea, G. (2017). Transsynaptic Mapping of Second-Order Taste Neurons in Flies by trans-Tango. Neuron 96(4), 783-795.e4. https://dx.doi.org/10.1016/j.neuron.2017.10.011
doi: https://doi.org/10.1242/prelights.22764
Read preprintSign up to customise the site to your preferences and to receive alerts
Register hereAlso in the genetics category:
Intracellular diffusion in the cytoplasm increases with cell size in fission yeast
Leeba Ann Chacko, Sameer Thukral
HIF1A contributes to the survival of aneuploid and mosaic pre-implantation embryos
Anchel De Jaime Soguero
Significantly reduced, but balanced, rates of mitochondrial fission and fusion are sufficient to maintain the integrity of yeast mitochondrial DNA
Leeba Ann Chacko
Also in the neuroscience category:
Deciphering the nanoscale architecture of presynaptic actin using a micropatterned presynapse-on-glass model
Felipe Del Valle Batalla
The RNA binding protein HNRNPA2B1 regulates RNA abundance and motor protein activity in neurites
Felipe Del Valle Batalla
Pharyngeal neuronal mechanisms governing sour taste perception in Drosophila melanogaster
Matthew Davies
preListsgenetics category:
in theBSDB/GenSoc Spring Meeting 2024
A list of preprints highlighted at the British Society for Developmental Biology and Genetics Society joint Spring meeting 2024 at Warwick, UK.
List by | Joyce Yu, Katherine Brown |
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
List by | Reinier Prosee |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
List by | Sergio Menchero et al. |
Semmelweis Symposium 2022: 40th anniversary of international medical education at Semmelweis University
This preList contains preprints discussed during the 'Semmelweis Symposium 2022' (7-9 November), organised around the 40th anniversary of international medical education at Semmelweis University covering a wide range of topics.
List by | Nándor Lipták |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
List by | Nándor Lipták |
2nd Conference of the Visegrád Group Society for Developmental Biology
Preprints from the 2nd Conference of the Visegrád Group Society for Developmental Biology (2-5 September, 2021, Szeged, Hungary)
List by | Nándor Lipták |
EMBL Conference: From functional genomics to systems biology
Preprints presented at the virtual EMBL conference "from functional genomics and systems biology", 16-19 November 2020
List by | Jesus Victorino |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
List by | Maiko Kitaoka et al. |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
List by | Hiral Shah |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Zebrafish immunology
A compilation of cutting-edge research that uses the zebrafish as a model system to elucidate novel immunological mechanisms in health and disease.
List by | Shikha Nayar |
Also in the neuroscience category:
2024 Hypothalamus GRC
This 2024 Hypothalamus GRC (Gordon Research Conference) preList offers an overview of cutting-edge research focused on the hypothalamus, a critical brain region involved in regulating homeostasis, behavior, and neuroendocrine functions. The studies included cover a range of topics, including neural circuits, molecular mechanisms, and the role of the hypothalamus in health and disease. This collection highlights some of the latest advances in understanding hypothalamic function, with potential implications for treating disorders such as obesity, stress, and metabolic diseases.
List by | Nathalie Krauth |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
List by | Helen Zenner |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
List by | Ana Dorrego-Rivas |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
List by | Madhuja Samaddar et al. |
SDB 78th Annual Meeting 2019
A curation of the preprints presented at the SDB meeting in Boston, July 26-30 2019. The preList will be updated throughout the duration of the meeting.
List by | Alex Eve |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
List by | Sandra Malmgren Hill |
Young Embryologist Network Conference 2019
Preprints presented at the Young Embryologist Network 2019 conference, 13 May, The Francis Crick Institute, London
List by | Alex Eve |