A screen for essential gene function in mature GABA neurons.

<p>(<b>A</b>) Histogram of primary GABA-specific RNAi screen. 79 RNAi clones (green bars) were selected for retesting with aldicarb hypersensitivity above cutoff (dotted line) of two standard deviations above the mean. μ = 14.40%, σ = 12.15%, cutoff = 38.70%. (<b>B</b>) Aldicarb sensitivity of controls from primary screen. Negative control (L4440, blue box) fed worms are significantly less sensitive to aldicarb than positive control (<i>unc-25</i>, green box) fed worms. N = 42 for each condition. Whiskers are min to max values. p<0.0001. (<b>C</b>) Hits from secondary screen. RNAi against 48 genes causes hypersensitivity to aldicarb above primary RNAi screen cutoff in at least 3 independent trials of ∼25 worms each. p<0.0001 for all clones compared to control RNAi.</p

A feeding RNAi-compatible approach to neuron-specific knockdown.

<p>(<b>A</b>) Animals carry a genetic background of <i>lin-15B(n744); eri-1(mg366); rde-1(ne219)</i>, and express wildtype <i>sid-1(+)</i> and <i>rde-1(+)</i> in selected neurons (GABA neurons are depicted). (<b>B</b>) Wildtype <i>sid-1</i> and <i>rde-1</i> are expressed from an artificial operon under various neuron sub-type-specific promoters and inserted as a single copy into the genome by MosSCI.</p

GABA-specific RNAi.

<p>(<b>A</b>) P<i>unc-47</i> (GABA-specific) RNAi strain expressing ubiquitous, nuclear-localized GFP and GABA mCherry after control or <i>GFP</i> RNAi feeding. Arrows mark GABA neurons with nuclear GFP. Stars mark GABA neuron cell bodies. <i>GFP</i> RNAi eliminates GFP in GABA cells, while GFP remains in other cell types. In control RNAi animals, GFP in GABA neurons is reduced relative to other cells due to increased transgene silencing in these neurons (see text). Scale bars = 5 µm. (<b>B</b>) Quantification of the percent of GABA cell bodies with nuclear-localized GFP under control or <i>GFP</i> RNAi conditions. Error bars are SEM, n = 10 worms, *** p<0.0001 (<b>C</b>) Locomotion behaviors in response to RNAi against genes known to function in GABA neurotransmission, in animals sensitive to RNAi in all cells (<i>lin-15B; eri-1</i>) and in the GABA-specific strain (XE1375). In GABA-specific RNAi strain, a GABA-specific shrinker phenotype is observed when pre- but not post-synaptic GABA signaling genes are targeted. (<b>D</b>) Percent of paralyzed animals after 100 min of acute exposure to 750 µM aldicarb. Error bars are SEM, n = 3 trials of ∼25 animals each, ** p = 0.0023, *** p≤0.0003. (<b>E</b>) GABA commissures after control or <i>unc-70</i> RNAi. In <i>unc-70</i> RNAi, the axon has broken and initiated a regenerative growth cone (arrow), and the dorsal nerve cord (DNC) has degenerated. Scale bars = 10 µm.</p

Specific gene knockdown in dopamine, glutamate, and acetylcholine neurons in response to feeding RNAi.

<p>(<b>A</b>) In standard neuron-sensitive strain, RNAi of presynaptic (<i>cat-2</i>) or postsynaptic (<i>dop-3</i>) components of dopamine signaling eliminates basal slowing on a bacterial lawn (gray bars). Error bars are SEM, n = 20 worms, *** p<0.0001. (<b>B</b>) In dopamine-specific strain, RNAi of a presynaptic component of dopamine signaling (<i>cat-2</i>) eliminates basal slowing, while RNAi of a postsynaptic component (<i>dop-3</i>) does not (gray bars). RNAi of presynaptic components of general neurotransmission (<i>unc-13</i> and <i>snb-1</i>) also eliminates basal slowing. Error bars are SEM, n = 20 worms, *** p<0.0001. (<b>C</b>) In standard neuron-sensitive strain, RNAi of presynaptic (<i>eat-4</i>) or postsynaptic (<i>glr-1</i>) components of glutamate signaling diminish the response to nose touch. Error bars are 95% confidence interval, n = 5 trials/animal, 10 animals, *** p<0.0001. (<b>D</b>) In glutamate-specific strain, RNAi of presynaptic components of glutamate neurotransmission (<i>eat-4, unc-13, snb-1</i>) as well as the structural protein <i>unc-70</i> inhibit the nose touch response, while RNAi of a postsynaptic component (<i>glr-1</i>) does not. Error bars are 95% confidence interval, n = 5 trials/animal, 10 animals, *** p≤0.0006. (<b>E</b>) In standard neuron-sensitive strain, RNAi of presynaptic components of general neurotransmission (<i>unc-13</i> and <i>snb-1</i>) slows rate of thrashing. Error bars are SEM, n = 10 worms, *** p<0.0001 (<b>F</b>) In acetylcholine-specific strain, RNAi of presynaptic components of general neurotransmission (<i>unc-13</i> and <i>snb-1</i>) as well as <i>unc-70</i> slows rate of thrashing. Error bars are SEM, n = 10 worms, *** p<0.0001.</p

Essential gene RNAi clones that affect GABA neuron function.

<p>Chr, chromosome.</p>*<p>Conservation based on presence on OrthoList <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003921#pgen.1003921-Shaye1" target="_blank">[42]</a>.</p

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