Summary: Okay, this one seems really straightforward (and nice and short). Their big question: can you engineer synapses between particular neuron pairs in C. elegans?
As a starting point, they decide to try to create novel electrical synapses (gap junctions), which have been previously created in other systems by overexpressing a single innexin (invertebrates) or connexin (vertebrates). They reason that expressing connexins (again, from vertebrates) in the worm is a better strategy than innexins, as both families of proteins can form gap junctions with a mixture of within-family proteins (e.g. a gap junction may involve several different connexins, or several different innexins, but not a combination of both). Therefore ectopic expression of an innexin would likely lead to all kinds of undesired gap junctions forming due to all the natively expressed innexins already present in neighboring neurons.
In their first experiment, they express a GFP-tagged variant of mouse connexin 36 (Cx36) in the ASER and ASEL neurons. ASER and ASEL do not naturally share any synapses, but come into physical contact with each other at many points. When they expressed Cx36 in both neurons, they observed a handful of discrete fluorescent puncta - some in the cell body, some in the dendrite, and some in the axon. Unfortunately, they only go up to two-color imaging, so the best image they can show is of ASEL (cytoplasmic green label) with red puncta (using mCherry-labeled Cx36), but they can't see ASER in that image. Unfortunately, they don't know how many of these puncta occur at physical interfaces between the two neurons, and how many are elsewhere (and what they might be doing elsewhere!)
ASER is known to detect step-down changes in salt concentration, and ASEL detects steps up. When they express Cx36 in either neuron individually, the GCaMP signals look pretty much identical to wild type. However, when they express Cx36 in both ASER and ASEL, suddenly both neurons respond to a step down in salt concentration, and show almost no response to a step up in salt concentration.
Their next test case was AWC and AIY. AWC normally forms inhibitory chemical synapses onto AIY. AIY suppresses turning and extends runs, meaning AWC activity shortens runs and induces turning. When presented with benzaldehyde, AWC is inhibited, and AIY is activated (inhibiting turning, and enabling chemotaxis towards benzaldehyde). They verify this opposing response with calcium imaging.
When they express Cx36 in AWC and AIY, they find that the calcium signal in both is suppressed, suggesting that they've turned an inhibitory synapse into an excitatory one (I of course use these terms loosely, since they are typically defined with regards to spiking neurons). Here, the Cx36 is again found in puncta, but more of it seems to be localized to the somas. Again, 3-color imaging would have been awesome.
Notably, there are two AWC neurons (on and off), and while they sense most things in phase with each other, they sometimes sense things out of phase (looking back at the Zaslaver paper, which assayed a bunch of stimuli but unfortunately didn't assay benzaldehyde). For most of the paper they don't differentiate which AWC they're measuring (or if they're measuring the average of the two). However, they note that upon benzaldehyde removal, the calcium signal from worms expressing Cx36 in only AWC is smaller than for wild type. They suggest that this is because they have also formed gap junctions between the two AWC neurons. I don't fully understand this explanation, unless they think that only one of the neurons is actually sensitive to benzaldehyde?
At the end of the paper, they speculate that the inverse approach of expressing innexins in vertebrate systems might be a viable approach to engineering gap junctions. However, unlike C. elegans, few vertebrate systems have the genetic tools to drive innexin expression in very specific neurons, so it'll be interesting to see if anyone takes up this challenge.
- What governs where electrical synapses form, or how many of them form?
- How does connexin 36 self-assemble into a gap junction?
- Are heterologously-expressed connexins orthogonal? (e.g. could i express Cx36 in neurons A and B, and Cx37 (?) in B and C, and only get A<--->B<---->C without the connection between A and C?)