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. 2020 Jun 5;9:e56942. doi: 10.7554/eLife.56942

Figure 2. Interneurons AIB are crucial for motor state transitions.

(A) Simplified circuit diagram underlying the gentle touch induced escape responses. Neurons were grouped into four modules based on their functional roles and activity patterns. (B) Calcium activity of AIB during spontaneous reversals before type-I (n = 23) and type-II (n = 36) transitions in unrestrained behaving animals (Pinx-1::GCaMP6;Pinx-1::wCherry). Here, data are aligned to the ends of reversals (vertical dashed line, t = 0). Heat map across trials (up) and ΔR(t)/R0 (Mean ± SEM, bottom) are shown. (C) Ramping rate of calcium activity in AIB. Up, raw single trial ΔR(t)/R0 from reversal start to reversal end. The ramping rate is the slope of the red line, fitted by linear regression. Bottom, ramping rates of AIB during type-I and type-II transitions. Each color (Mean ± SEM) represents single animal data across multiple trials. Total nine animals (Pinx-1::GCaMP6;Pinx-1::wCherry) were tested. Very short reversals (less than 1.5 s) are excluded, for some of them have negative ramping rates and the slope estimate is susceptible to noise (but including those trials doesn’t affect our conclusion). **p<0.01, two-way ANOVA. (D) Optogenetic activation of AIB (635 nm, 4.46 mW/mm2, 7 s) or inhibition of AIB (561 nm, 21.71 mW/mm2, 12 s) during ALM/AVM (473 nm, 14.71 mW/mm2, 1.5 s) triggered avoidance behaviors, reversal durations (bar graph) and fractions of animals executing omega turns (pie chart) are shown. Error bars are SEMs. Bar graph, Mann–Whitney U test. Pie chart, χ2 test. *p<0.05, ***p<0.001, ****p<0.0001. Here and below, the actual turning percentages (nturn/ntotal) are noted beside the pie chart and numbers within the bars indicate the number of trials with reversal. (E–F) Reversal length distribution (E) and transition rates (F) during escape responses when AIB were persistently hyperpolarized through an exogenous expression of the potassium channel TWK-18. Control group is from Figure 1C.

Figure 2—source data 1. Source data for Figure 2 and Figure 2—figure supplement 1.
elife-56942-fig2-data1.zip (1,000.3KB, zip)

Figure 2.

Figure 2—figure supplement 1. Local interneurons AIB and RIM in the backward module differentially modulate motor state transitions.

Figure 2—figure supplement 1.

(A) Schematic diagram of the C. elegans nervous system. (B) Multi-neuron calcium imaging in freely behaving animals (Pnmr-1/Plim-4/Plgc-55/Pnpr-9::NLS::wCherry::SL2::GCaMP6). Interneurons in the backward and forward modules were simultaneously imaged using a confocal microscopy (see Materials and methods for details). Data are aligned to the onset of a reversal (up, vertical dashed lines, t = 0) or the end of a turn (bottom, vertical dashed lines, t = 0). Heat map across trials and ΔR(t)/R0 (Mean ± SEM) across trials are shown. (C) Ramping rate of AIB calcium activity during thermally-induced escape responses. Ramping rate is significantly higher in type-II transition (nine animals, each point has more than three trials). *p<0.05, two-way ANOVA. (D–E) Characterization of the probability of type-II transition during optogenetic stimulation of two different interneurons AIB (left) and RIM (right) for varying durations. Longer activation of AIB consistently led to a higher probability of type-II transition (left). The control groups are animals performing spontaneous motor state transitions without optogenetic stimulation. A blue laser (473 nm, 14.71 mW/mm2) was used to stimulate ChR2 expressed neurons. (F–G) Hyperpolarization of RIM (Ptdc-1::Arch) or AIB (Pnpr-9::Arch) both affected reversal duration (bar graph), but only inhibiting AIB affected the probability of type-II transition (pie chart). In F), simultaneous optogenetic activation of AIB (or RIM) (635 nm, 4.46 mW/mm2 for AIB or 473 nm, 14.71 mW/mm2 for RIM) and inhibition (561 nm, 6.80 mW/mm2) of RIM (or AIB) were carried out for 7 (or 5) seconds. In G), RIM were activated for varying durations, while AIB were inactivated through an exogenous expression of the potassium channel TWK-18. (H–I) Reversal length distribution and transition rates after optogenetic ablation of AIB. ALM/AVM were optogenetically activated for 1.5 s to trigger avoidance behavior. AIB were ablated by exogenous expression of a membrane targeted PH-miniSOG. Data from Figure 1C) are used as control. For reversal duration, error bars indicate SEMs. Mann–Whitney U test. For probability of turn, error bars indicate 95% binomial proportion confidence interval. χ2 test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. All multiple comparisons were adjusted using Bonferroni correction.
Figure 2—video 1. AIB neurons are crucial for turn and backward movement, Related to Figure 2 and Figure 2—figure supplement 1.
Download video file (1.6MB, mp4)
Optogenetic activation of AIB neurons in free-moving animals reliably triggered backward movement followed by omega turns. Inhibition of AIB neurons quickly terminated backward movement.