A command-like descending neuron that coordinately activates backward and inhibits forward locomotion

Command-like descending neurons can induce many behaviors, such as backward locomotion, escape, feeding, courtship, egg-laying, or grooming. In most animals it remains unknown how neural circuits switch between these antagonistic behaviors: via top-down activation/inhibition of antagonistic circuits or via reciprocal inhibition between antagonistic circuits. Here we use genetic screens, intersectional genetics, circuit reconstruction by electron microscopy, and functional optogenetics to identify a bilateral pair of larval “mooncrawler descending neurons” (MDNs) with command-like ability to coordinately induce backward locomotion and block forward locomotion; the former by activating a backward-specific premotor neuron, and the latter by disynaptic inhibition of a forward-specific premotor neuron. In contrast, direct reciprocal inhibition between forward and backward circuits was not observed. Thus, MDNs coordinate a transition between antagonistic larval locomotor behaviors. Interestingly, larval MDNs persist into adulthood, where they can trigger backward walking. Thus, MDNs induce backward locomotion in both limbless and limbed animals. Highlights MDN command-like descending neuron induces backward larval locomotion MDN neurons coordinately regulate antagonistic behaviors (forward/backward locomotion) MDN-motor circuit validated at structural (TEM) and functional (optogenetic) levels MDN neurons induce backward locomotion in both limbless larva and limbed adult

4 waves per second (Split1, 0.48; Split2, 0.50; Split3, 0.65 before activation; Split1, 0.48; Split2, 120 0.56; Split3, 0.56 after activation). Conversely, using Split2 or Split3 to express the light-121 inducible neuronal silencer GtACR1 (Mohammad et al., 2017) significantly reduced backward 122 locomotion induced by a noxious head poke (Figure 1G,H). It is likely that these activation and 123 silencing phenotypes arise from the pair of ventral, anterior, medial brain descending neurons 124 common to all three lines, although it is possible that there are different neurons in each Split 125 line that can induce backward locomotion. We distinguish between these alternatives in the 126 next section.

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A single pair of brain neurons can induce a switch from forward to backward locomotion 129 To determine whether Chrimson expression in just one or two of the ventral, anterior, 130 medial brain neurons is sufficient to induce backwards locomotion, we stochastically 131 expressed Chrimson:Venus within the Split2 pattern via the "FLP-out" method ( Figure 2A). We  Figure 2E). We conclude that activation of as few as two of 143 the four MDNs (either both in the same brain lobe or one in each brain lobe) is sufficient to 144 induce a behavioral switch from forward to backward locomotion. 145 If forced activation of MDNs can induce backward locomotion, perhaps the MDNs are 146 normally active specifically during backward locomotion. To test this hypothesis, we used 147 CaMPARI to monitor MDN activity during forward versus backward locomotion within the 148 intact crawling larva. CaMPARI undergoes an irreversible green-to-red conversion upon 149 coincident exposure to elevated Calcium (i.e. neuronal activity) and 405nm illumination (Fosque 150 et al., 2015). We used Split2 to express CaMPARI in MDNs and exposed crawling larvae to 151 405nm illumination for 30 sec during either backward or forward locomotion. We detected little 152 or no activity-induced red fluorescence during forward locomotion, but significant red 153 fluorescence during backward locomotion ( Figure 2F). Note that not all backward crawling 154 larvae activate MDN; we return to this point below. We conclude that MDNs are active during 155 backward but not forward locomotion. 156 5 158 To understand how MDNs induce backward locomotion, we next identified the MDN 159 synaptic partners. To do this, we identified the MDNs in an existing serial section TEM 160 reconstruction of the newly hatched larva (Ohyama et al., 2015). Our first step was to 161 determine the precise morphology of both MDN neurons. We generated individually labeled 162 neurons within the Split2 pattern using MultiColor FlpOut (MCFO) (Nern et al., 2015). These    we have not attempted to reconstruct them; this is beyond the scope of a single paper.

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However, we note that each MDN has similar inputs. We do not detect mono-synaptic sensory 213 input into the MDNs (data not shown), but based on the requirement for MDNs to generate a 214 backward crawl in response to a noxious head touch, we predict that there will be, minimally,   We showed above that MDNs are significantly more active during backward than forward 226 locomotion, raising the question of whether the A18b neurons are also preferentially active 227 during backward locomotion. To answer this question, we performed three experiments. First, 228 we used dual color calcium indicators in a fictive CNS preparation to simultaneously monitor 229 motor neuron activity (GCaMP6m) and A18b activity (jRCaMP1b). We observed robust forward 230 and backward motor waves ( Figure 5D, top), with A18b only active during backward motor 231 waves, not forward motor waves (Figure 5D, bottom). Second, we performed dual color 232 calcium imaging within intact larvae, and again observed that A18b was only active during 233 backward motor waves ( Figure 5E). Third, we used CaMPARI within intact larvae to determine 234 if A18b was preferentially active during backward locomotion. We expressed CaMPARI in A18b 235 and tested for activity-induced green-to-red photoconversion during either forward or 236 backward locomotion. We found that illumination during forward locomotion generated minimal 237 CaMPARI red fluorescence, whereas illumination during backward locomotion resulted in a 238 significant increase in CaMPARI red fluorescence ( Figure 5F). We call the A18b neuron 239 backward-active rather than backward-specific because we do not know its pattern of activity 240 in rolling or other larval behaviors. We conclude that A18b neurons are preferentially active 241 during backward not forward locomotion.

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To determine if MDNs activate A18b, we used Split1 to express Chrimson in MDNs and

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A18b-lexA to express GCaMP6f in A18b in fictive preparations. MDN stimulation led to a 244 significant increase in GCaMP6f fluorescence in A18b, and this was not observed in controls 245 lacking all-trans retinal (ATR), an essential co-factor for Chrimson function ( Figure 5G). 246 Interestingly, MDN activation triggered a backward wave of A18b activity from A2 to A6 ( Figure   247 5G). We propose that MDN activates A18b in segment A1, which is the only segment we 248 detect direct synaptic contacts, and this is transformed into an anterior-to-posterior wave of 249 A18b activity. 250 We showed above that A18b has direct synaptic connectivity to motor neurons and is 251 cholinergic, indicating that is likely to be an excitatory pre-motor neuron. Consistent with this 252 expectation, we observed co-activity of A18b and motor neurons during backward motor 253 waves in fictive preparations ( Figure 5H), and found that A18b stimulation led to a significant 254 increase in GCaMP6f fluorescence in motor neurons, which was not observed in controls 255 lacking ATR ( Figure 5I). 256 We wanted to test whether activation of A18b in segment A1 could induce backward 257 waves of motor neuron activity. Unfortunately, the A18b-Gal4 line is not expressed in A1, 258 precluding this experiment; moreover, it has "off-target" expression in the brain and in the 259 VNC; these off-target neurons don't prevent monitoring A18b activity because they don't 260 overlap with A18b arbors, but they make it impossible to selectively activate or silence A18b. In   (Fushiki et al., 2013). This leads to the hypothesis we test below: MDNs activate Pair1 to inhibit 270 A27h, which terminates forward locomotion.

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Stimulation of MDNs led to a significant increase in Pair1 GCaMP6f fluorescence, and this was 274 not observed in controls lacking ATR ( Figure 6B). We conclude that the MDNs activate Pair1 275 neurons. In addition, we observed that every time MDNs were active, the Pair1 neurons were 276 co-active (n=5; Figure 6C), although Pair1 could be active alone (n=5; Supplement to Figure 6). 277 We conclude that MDNs activate the Pair1 neurons, and that other mechanisms exist for 278 activating Pair1 as well. 279 We next used two methods to determine whether Pair1 neurons are preferentially active  To test whether Pair1 inhibits the A27h neuron, we expressed Chrimson in Pair1 and 301 GCaMP6m in A27h. We used Chrimson to stimulate Pair1 just as A27h activity was rising as 302 part of a forward motor wave, and observed a significant decrease in A27h GCaMP6m 303 fluorescence; this was not observed in controls lacking ATR (Figure 7A,B). Furthermore, we 304 9 found that Pair1 neurons are GABAergic (Figure 7A''), consistent with Pair1 direct repression 305 of A27h activity. In addition, we found that Chrimson stimulation of Pair1 immediately and 306 persistently blocked forward larval locomotion; control larvae lacking ATR briefly paused in 307 response to illumination but rapidly resumed forward locomotion ( Figure 7C; Movie 2).

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Consistent with an inhibitory relationship, we observed that Pair1 and A27h are not co-active 309 (Supplement to Figure 7). We conclude that activation of the GABAergic Pair1 neurons inhibit 310 A27h and prevent forward locomotion.

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Our results suggest that Pair1 suppression of forward locomotion may be an essential Shibire ts blocks vesicle release at 32 o C but not at 25 o C (experiment summarized in Figure 7D). 317 We observed that silencing Pair1 alone had no effect on forward locomotion ( Figure 7E, i-ii),   (Figure 8E,F), whereas the two flies that did not walk 342 backward also did not have ReaChr:citrine expression in moonwalker neurons (data not shown).

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Second, we used 'immortalization' genetics (Harris et al., 2015) to permanently mark larval 344 MDNs and assay their function in the larva and adult (genetics schematized in Figure 8G). We        After eclosion adults were transferred to standard cornmeal fly food supplemented with ATR (0.5mM) for 577 4 days changing to fresh food after two days. Wings were clipped and animals were placed in ring 578 arenas made of 3.0% agar apple juice. The ring arena size was 1.4cm outer diameter, 1.0cm inner 579 diameter and 0.2cm height. After 5 minutes for environmental acclimation, animal behavior was recorded 580 at 5 Hz using an Axiocam 506 mono under low transmitted light for 10 seconds followed by 10 seconds 581 under 0.28 mW/mm 2 red light. This was done three times for each animal. To quantify backward 582 locomotion probability upon light stimulus we divided the amount of times the animal began backward 583 walking within 2 seconds after light stimulus over the total number of times the animals was presented 584 with light. To calculate significance we used Student's t-test unpaired analysis. Adult flies were allowed to lay eggs on standard culture medium that was supplemented with 1µM RU486 588 and 2mM ATR. After 24 hours, light-induced backwards crawling larvae were transferred to culture medium 589 supplemented with 2mM ATR and grown to adulthood. 2-6 day-old adult flies were individually transferred 590 into a 10ml serological pipette for walking assay. Red-orange light from a 617nm high-power LED was fiber-591 coupled to a 200µm core optical cable that was manually triggered via a T-Cube LEDD1B driver (ThorLabs, 592 Newton, NJ, USA). Optogenetic stimulation was measured via a photodiode power sensor (S130VC, 593 ThorLabs) to be ~4.6 µW/mm 2 . We performed same analysis for the intersectional experiment (above) to 594 quantify backward locomotion probability upon light stimulus.