If you want a reminder of the “big picture” of neuroscience, look no further than Liqun Luo’s website. He starts off with a couple fun neuro facts, the first of which is that there are 1011 neurons in the human brain. In other words, we have almost as many neurons as there are stars in the Milky Way. Doesn’t that make you feel kind of special? But even crazier than that is the number of synapses. On average, each neuron makes 103 synaptic connections with other neurons, for a grand total of 1014 synapses (or a hundred trillion if you’d prefer). By the time you go past the power of 1012, the internet has a lot fewer suggestions for how to make that large of a number tangible. The best I could find is that if you stacked a hundred trillion dollar bills, you could reach the moon and back 14 times. The point is we have a LOT of synapses in our brain. So how is it possible for those hundred billion neurons to properly wire those hundred trillion synapses?
Dr. Luo studies this wiring specificity in fruit flies (with a casual 105 synapses) and mice (with a slightly less casual 108 synapses). We know that proper neural circuit assembly requires a spatially and temporally precise chain of developmental events to form precise connections between specific neurons. But what exactly does that entail? In his lab’s recent paper, entitled “Toll Receptors Instruct Axon and Dendrite Targeting and Participate in Synaptic Partner Matching in a Drosophila Olfactory Circuit,” the steps of neural circuit wiring explained so clearly, it could be part of Julia Child’s cookbook…
While the axon guidance part has been well studied, how pre- and post- synaptic binding partners identify each other remains poorly understood. This led Dr. Luo’s lab to conduct a confocal-based RNAi screen of 278 genes through 768 lines to look for wiring specificity molecules in the Drosophila olfactory system. With every knockdown, the lab would use look for resulting developmental targeting defects of Olfactory Receptor Neurons (ORN) and Projection Neurons (PN). Interestingly, they found that after a Toll-6 knockdown, there was a dorsal shift of the VA1d PN dendrites (see Figure 1), which normally arborize at the anterior surface of the antennal lobe, and the VA1d ORN axons, which normally project to the VA1d glomerulus. They also found that the knockdown of Toll-7, another member of the Toll receptor family, led to the medial mistargeting of Va1dPN dendrites and ORN axons (see Figure 1). To confirm the findings of the knockdowns, the targeting of ORN axons was studied in both Toll-6 and Toll-7 null mice and dorsal and medial mistargeting, respectively, were again found (see Figure 1).
Figure 1. Identification of Toll-6 and Toll-7 as Wiring Specificity Molecules in an RNAi Screen. All images are single confocal sections of adult antennal lobes, with magenta showing neuropil staining and other colors showing axons of specific ORN classes and dendrites of specific PN classes as indicated. N is number of antennal lobes tested.
Dr. Luo’s lab conducted an extremely thorough characterization of the role Toll-6 and Toll-7 play in axon targeting, so I’ll just touch on a couple interesting experiments that they did. The Luo lab wanted to know if Toll-7 acts autonomously on VA1d and DA ORNs. So to see if the production of Toll-7 in PNs was necessary, they performed an RNAi knockdown using the PN-specific promoter Mz19-GAL4. The Toll-7 knockdown in PNs had no effect on the axonal targeting of the ORNs or the dendritic targeting of the PNs. However, an ORN-specific knockdown of Toll-7, through the Pebbled-Gal4 promotor, led to VA1d axon mistargeting identical to the pan-neuronal Toll-7 knockdown (see Figure 2). When the antennal lobes of the ORN-specific Toll-7 knockdown flies were stained with an anti-Toll-7 antibody, Toll-7 staining was no longer seen in the anterolateral glomeruli (see Figure 2). These findings suggest that the ORNs are responsible for the production of the Toll-7 wiring specificity molecule. Interestingly, some targeting defects in PN dendrites were seen with the ORN-specific Toll-7 knockdown. As PN dendrites are pre-patterned in the antennal lobe before the arrival of ORN axons, it wasn’t thought their arrival affected dendritic target selection. However, this suggests flexibility in PN dendritic target selection based on ORN axon targeting. Finally, the Luo lab took advantage Mosaic analysis with a repressible cell marker (MARCAM), a technique they had previously developed, to see if Toll-7 acts autonomously on ORNs. With MARCAM, they created and tagged a sub-population of ORNs that were Toll-7 -/- while leaving the remaining ORNs Toll-7+/+. Then, they studied single wild-type ORN axons through a population of Toll-7 null ORNs. Interestingly, there were no targeting defects in the wild-type ORNs growing in the presence of the mutants—indicating that Toll-7 acts autonomously.
Figure 2. Toll-7 Is Expressed in ORN Axons Targeting Anterolateral Glomeruli and Is Required in ORNs
If hearing about any of these crafty experiments or pioneering genetic tools has left you wanting to know more, come to Dr. Linqun Luo’s talk June 9th at 4pm in CNCB. It might be the last seminar of the series, but certainly not the least!
1. Ward A, Hong W, Favaloro V, Luo L (2015). Toll receptors instruct axon and dendrite targeting and participate in synaptic partner matching in a Drosophila olfactory circuit. Neuron 85(5):1013-28.
2. Innumerable Julia Child Photos
Written by Kelsey Ladt, a (currently) sleep deprived 1st year Neuroscience student in Dr. Subhojit Roy’s lab.