Title: Memory in Caenorhabditis elegans Is Mediated by NMDA-Type Ionotropic Glutamate Receptors
Summary: Okay, this is my second paper from Villu Maricq's group, and incidentally, from the same year. Must have been a good year.
In this paper, their question was: are glutamate receptors required for memory in C. elegans? AMPA and NMDA receptors have been implicated in memory in many systems, but not C. elegans. Specifically, in many systems neural activity modulates AMPA and NMDA receptor cycling and thus affects synapse strength.
Their learning paradigm is one of salt avoidance - specifically, worms cultivated with no salt and no food for 4 hours learn to avoid salt, albeit briefly. This is sort of the inverse of the paradigm that was later expanded on by (Kunitomo et al. 2013), and (Luo et al. 2014) in which they show that C elegans generally move towards whatever their previous salt concentration was.
Worms that were 'mock conditioned' (salt- and food-) showed normal chemotaxis up salt gradients, regardless of mutation. However, conditioned worms (salt+ food-) showed significant initial negative chemotaxis, which gradually reversed on a timescale of roughly 50 minutes.*
As with I think every single paper so far I've written about so far, they then ask about how mutants behave. Specifically, they use two AMPA receptor subunit mutants glr-1 and glr-2, and two NMDA receptor subunit mutants nmr-1 and nmr-2 (nmr-2 is novel to this study, and they later electrophysiologically confirm that this mutant eliminates NMDA-mediated currents in AVA). Strikingly, these mutants are viable and don't show any gross motor defects. They don't characterize this much further, but I find this rather striking, given that glutamatergic synapses are so abundant.
However, in the learning paradigm described above, they find that nmr-1 and nmr-2 (but not glr-1 or glr-2) revert more quickly to positive chemotaxis, regaining a positive chemotactic index within 30 minutes instead of the normal 50.
One possibility is that the nmr-1 and nmr-2 mutants are not able to sense starvation, and therefore can't learn to associate starvation with salt. They argue that because mutant and WT worms have similar slowing behavior upon encountering food, and that this behavior is similarly modulated by starvation state, that the worms are likely to be able to sense starvation.
Because either nmr-1 or nmr-2 are sufficient to reduce the apparent memory, they suspect the effect may be driven by neurons that coexpress nmr-1 and nmr-2, of which there aren't too many. Candidates include: RIM, AVG, AVA, AVD, AVE, and PVC. They find that expressing nmr-1 in RIM neurons (under the tdc promoter) in nmr-1 mutants is sufficient to rescue the 'memory' of salt starvation and extend the period of chemotaxis away from salt to the same duration as wild type.
Having determined that input to RIM via glutamate receptors are necessary for this memory, they then ask if RIM's output is also necessary. RIM uses tyramine and octopamine, both of which require tyrosine decarboxylase (tdc) to synthesize. A tdc mutant shows the same memory defects as the nmr-1 and nmr-2 mutants. Thus it looks very likely that RIM is crucially involved in this memory/behavior, as eliminating its inputs or its outputs yields this shortened negative chemotactic behavior.
* This is based on a chemotactic index, which is a pretty crude measure. Basically, divide the plate in half (low NaCl vs high NaCl), and count the number of worms in each half (call these counts A and B). The chemotactic index is (A-B)/(A+B). Unfortunately, this doesn't show where the worms are moving at a given point in time, and probably mostly reflects the worm's past behavior, not its current behavior!