BAcTrace a new tool for retrograde tracing of neuronal circuits

Abstract

11New tools and techniques have enabled many key advances in our understanding of the brain. To elucidate circuit function, it is necessary to identify, record from and manipulate networks of connected neurons. Here we present BAcTrace (Botulinum Activated Tracer), the first fully genetically encoded, retrograde, transsynaptic labelling system. BAcTrace is based on C. botulinum neurotoxin A, Botox, which we have engineered to act as a Trojan horse that jumps retrogradely between neurons to activate an otherwise silent transcription factor. We validate BAcTrace at three connections in the Drosophila olfactory system and show that it enables electrophysiological recordings of connected neurons. Finally, in a challenging circuit with highly divergent connections, we used Electron Microscopy connectomics to show that BAcTrace correctly identifies 12 out of 16 connections.The development of genetic tools to elucidate connectivity and manipulate neurons and circuits has 13 been key to advancing our understanding of how the brain works. Increasingly these tools are being 14 used to study diseases of the nervous system and develop effective treatments [31, 57]. 15In the context of circuit research, the ability to identify and manipulate pre-or post-synaptic cells to 16 neurons of interest is of crucial importance. For example, if genetic drivers are available for sensory 17 neurons in the skin then one might want to label downstream, post-synaptic cells in the nerve cord. 18Conversely, when studying motor circuits, genetic drivers for motor neurons might be available and 19 revealing upstream, pre-synaptic cells will be appropriate. Tools to label downstream neurons (e.g. for 20 "walking" from sensory input towards motor-outputs as in the first example) are called anterograde, 21 while retrograde tools reveal the input neurons to a given population. 22Drosophila melanogaster is a key model organisms to study the genetic and circuit basis of animal 23 behaviour (e.g. see [62, 15]). The fly has a rich behavioural repertoire encoded in a relatively small 24 nervous system. This numerical simplicity is paired with extensive collections of genetic reagents, 25 both to investigate gene function (e.g. mutant and RNAi collections) and to label and manipulate 26 most neuronal classes (using the orthogonal expression systems Gal4, LexA, QF and their split versions 27 [4, 45, 30, 58]). While these reagents offer excellent genetic access to neurons, until recently the 28 fly lacked tools to map synaptic connections between neurons. This has recently changed with the 29 development of electron microscopy methods to map connections in larval [38] and adult [63] brains. 30 Furthermore, two genetically encoded systems for anterograde tracing: trans-Tango [54] and TRACT 31 [22] have recently been developed. Despite these important additions to the experimental toolbox, a 32 retrograde labelling system is still missing. Rabies virus and its modifications constitute the most notable 33 examples of retrograde transsynaptic tools [31]. While...

BAcTrace, a tool for retrograde tracing of neuronal circuits in Drosophila

“…Once all antennal lobes were blindly annotated in relation to LHN type, we used the toxin labelling (CD2) channel to assign each specimen to the correct LHN cell type. Stacks are available online [ 47 ] for readers to examine.…”

“…All plasmids are available upon request from the authors and their sequences deposited in GenBank as described in Tables Supplementary Table 9 , Supplementary Table 12 , Supplementary Table 13 and Supplementary Table 14 . Brain confocal stacks used for making ORN and LHN plots are available from the Zenodo repository [ 47 ].…”

Information flow, cell types and stereotypy in a full olfactory connectome

“…Structural connectivity from the hemibrain does not necessarily capture functional connections assayed by physiology. Encouragingly, however, recent work with a retrograde genetic system for finding neurons that input onto genetically targetable cells found 6/7 glomerular connections to LHPD2a1/b1 neurons of above 10 synapses in FAFB, and 8/9 for LHAV1a1 (Cachero et al, 2020).…”

Leucokinin and Associated Neuropeptides Regulate Multiple Aspects of Physiology and Behavior in Drosophila

“…Intersectional genetic techniques have made it possible to target subpopulations of neurons expressing specific genes, and optogenetic and thermogenetic activation of specific peptidergic neurons can be performed [ 78 , 79 , 80 ]. Different reporter systems enable functional imaging (see [ 78 , 80 , 81 ]) and establishment of synaptic connections between identified neurons [ 82 , 83 , 84 ]. Thus, today, we can dissect the functional roles of neuropeptides in single cell types, specific neuronal circuits or in cells of other tissues by a large set of techniques.…”