Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding authors Dr. Marta Zlatic ( zlaticm@janelia.hhmi.org ) and Dr. Albert Cardona ( cardonaa@janelia.hhmi.org ).

20xUAS-CsChrimson-mCherry-trafficked in su(Hw)attP1 is gift from V. Jayaraman, unpublished stock (), 13xLexAop2-IVS-GCaMP6s-p10 50.641 in VK000005, is gift from the GENIE project (JANELIA, HHMI), unpublished stock (). LexAop2-myr::TDTomato-p10 (attp40), a gift from D. Mellert () is an myr::TDtomato fragment with AcNPV p10 (). pGP-20XUAS-GCaMP6f-p10.92.693 in VK00005 is a gift from the GENIE project.

These GAL4 combinations (from the Rubin GAL4 collection) were chosen based on stochastic labeling of single cells (using a FLP-based approach) that revealed that above GAL4 combination both contained the cell(s) of interest, namely basin-2, basin-1, drunken-1 and 2, griddle-2, ladder-d. The ‘FLP-out’ approach () for stochastic single-cell is described in detail elsewhere (). In brief, heat-shock induced expression of FLP recombinase was used to excise FRT-flanked interruption cassettes from UAS reporter constructs carrying HA, V5, and Flag epitope tags, and stained with epitope-tag specific antibodies. This labeled a subset of the cells in the expression pattern with a stochastic combination of the three labels.

in attp2 (3L) (JRC-SS00918). To selectively target ladder-d neurons we generated a Split-GAL4 stock R78F07_AD inserted in attp40 (chromosome 2L) and R28E11_DBD in attp2 (3L) (JRC-SS00863). To selectively target drunken-1 and drunken-2 neurons we generated a Split-GAL4 stock R23A05_AD inserted in attp40 (chromosome 2L) and R48D11_DBD in attp2 (3L) (JRC-SS00674). The line for selective targeting of basin-4 was generated as described previously (). AD and DBD combinations were assembled in a wbackground.

In the main text and figures, short names are used to describe genotypes for clarity. The complete genotypes of animals used in this study are shown in Table S3 . We used GAL4-UAS system to direct the expression of effector proteins to specific neuron subtypes. We used UAS-TNT-e () to silence neurons by expressing the tetanus toxin light chain in the GAL4 and Split GAL4 lines we tested, pJFRC12-10XUAS-IVS-myr::GFP (Bloomington stocknumber: 32197 gift from B. D. Pfeiffer, G. Rubin and the GENIE project team (HHMI Janelia Research Campus) to label neurons with green fluorescence and 20xUAS-CsChrimson-mVenus trafficked in attP18 (Bloomington stocknumber: 55134) to activate neurons. Throughout the paper we used as controls the progeny larvae from the UAS-impTNT (II) (gift from J. Simpson, unpublished data) containing the inactive form of TNT (), crossed to appropriate GAL4 or Split GAL4 lines. We used the progeny larvae from the insertion site stock, w;;attp2, w;attP40;attP2 () crossed to the appropriate effector (UAS-TNT-e (II)) for characterizing the behavior (in Figures 1 and S1 ). w;; attP2 and w;attP40;attP2 were selected because they have the same genetic background as the GAL4 and Split Gal4 lines tested respectively. The following strains from the Rubin GAL4/LexA collection were used for the behavioral experiments, immunohistochemistry labeling, flp-out experiments and electrophysiological recordings in the manuscript: R61D08-GAL4, R21B01-GAL4, R72F11-GAL4, R36B06-GAL4, R16B12-GAL4, R21B01-LexA (). To selectively target Basin-2 neurons we generated a Split-GAL4 stock: R72F11_AD inserted in attp40 (chromosome 2L) and R38H09_DBD

Method Details

Behavioral Apparatus Ohyama et al., 2013 Ohyama T.

Jovanic T.

Denisov G.

Dang T.C.

Hoffmann D.

Kerr R.A.

Zlatic M. High-throughput analysis of stimulus-evoked behaviors in Drosophila larva reveals multiple modality-specific escape strategies. Ohyama et al., 2013 Ohyama T.

Jovanic T.

Denisov G.

Dang T.C.

Hoffmann D.

Kerr R.A.

Zlatic M. High-throughput analysis of stimulus-evoked behaviors in Drosophila larva reveals multiple modality-specific escape strategies. Swierczek et al., 2011 Swierczek N.A.

Giles A.C.

Rankin C.H.

Kerr R.A. High-throughput behavioral analysis in C. elegans. Ohyama et al. (2013) Ohyama T.

Jovanic T.

Denisov G.

Dang T.C.

Hoffmann D.

Kerr R.A.

Zlatic M. High-throughput analysis of stimulus-evoked behaviors in Drosophila larva reveals multiple modality-specific escape strategies. Ohyama et al., 2013 Ohyama T.

Jovanic T.

Denisov G.

Dang T.C.

Hoffmann D.

Kerr R.A.

Zlatic M. High-throughput analysis of stimulus-evoked behaviors in Drosophila larva reveals multiple modality-specific escape strategies. The apparatus was described previously (). Briefly, the apparatus comprises a video camera (DALSA Falcon 4M30 camera) for monitoring larvae, a ring light illuminator (Cree C503B-RCS-CW0Z0AA1 at 624 nm in the red), a computer (seefor details); available upon request are the bill of materials, schematic diagrams and PCB CAM files for the assembly of the apparatus) and a hardware modules for controlling air-puff, controlled through multi worm tracker (MWT) software ( http://sourceforge.net/projects/mwt ) (), as described in. Air-puff is delivered as described previously (). Briefly it is applied to a 25625 cm2 arena at a pressure of 1.1 MPa through a 3D-printed flare nozzle placed above the arena (with a 16 cm 6 0.17 cm opening) connected through a tubing system to plant supplied compressed air (0.5 MPa converted to a maximum of 1.4 MPa using a Maxpro Technologies DLA 5-1 air amplifier, standard quality for medical air with dewpoint of 210uC at 90 psig; relative humidity at 25uC and 32uC, ca. 1.2% and 0.9%, respectively). The strength of the airflow is controlled through a regulator downstream from the air amplifier and turned on and off with a solenoid valve (Parker Skinner 71215SN2GN00). Airflow rates at 9 different positions in the arena were measure with a hot-wire anemometer to ensure even coverage of the arena (Extech Model 407119A and Accusense model UAS1000 by DegreeC). The air-puff relay is triggered through TTL pulses delivered by a Measurement Computing PCI-CTR05 5-channel, counter/timer board at the direction of the MWT. The onset and durations of the stimulus is also controlled through the MWT.

Behavioral Experiments Embryos were collected for 8–16 hr at 25°C with 65% humidity. Larvae were raised at 25°C with normal cornmeal food. Foraging 3 instar larvae were used (larvae reared 72-84 hr or for 3 days at 25°C). Larvae with all optogenetic experiments were raised on food supplemented with all-trans retinal. Before experiments, larvae were separated from food using 10% sucrose, scooped with a paint brush into a sieve and washed with water (as described previously). This is because sucrose is denser than water, and larvae quickly float up in sucrose making scooping them out from food a lot faster and easier. This method is especially useful for experiments with large number of animals. We have controlled for the effect and have seen no difference in the behavior between larvae scooped with sucrose and larvae scooped directly from the food plate with a forceps. The larvae were dried and spread on the agar starting from the center of the arena. The substrate for behavioral experiments was a 3% Bacto agar gel in a 25625 cm2 square plastic dishes. Larvae were washed with water at room temperature, the dishes were kept at room temperature and the temperature on the rig inside the enclosure was set to 25°C. The humidity in the room is monitored and held at 58%, with humidifiers (Humidifirst Mist Pac-5 Ultrasonic Humidifier). We tested approximately 50–100 larvae at once in the behavioral assays. The temperature of the entire rig was kept at 25°C. In the assay the larvae were left to crawl freely on an agar plate for 44 s prior the stimulus delivery. The air-puff was delivered at the 45th second and applied for 38 s. After a period of recovery of about 20 s when 10 air-puff pulses, 2 s each, were delivered (with a 8 s separation interval). In the assay with exogenous neuronal activation CsChrimson was activated using a 617-nm wavelength LED, with an irradiance of 296-425 μW/ cm2, as measured from the location of the preparation. The arena was illuminated from below through clear agar. The larvae crawled freely for 30 s prior to light delivery by switching the LED on for 15 s. The MWT software64 ( http://sourceforge.net/projects/mwt ) was used to record all behavioral responses.

Mechanical and Optogenetic Stimulation for Electrophysiology Recordings Mechanical stimulation was generated by arbitrary waveform generator (33220A, Agilent Technologies) and amplified by a stereo power amplifier (PCA3, Pyle Pro). The stimulation signal was delivered to a quick-mount extension actuator (Piezo Systems, Inc.), which was embedded in the sylgard-coated recording chamber. The stimulation was set at 1000Hz, with the intensity of 40 V and duration of 10-50 ms. CsChrimson was activated using a 617-nm wavelength LED, with irradiance of 320 μW/ cm2, as measured from the location of the preparation. The LEDs was on for 10-50 ms. We note there is a drastic difference in context between the optogenetic activation experiments in the dissected preparation and the freely behaving animals. In the dissected preparation, the body wall and the light-sensing organs in the front are damaged, and the animal is not moving. Feedback from proprioceptive neurons and from copies of motor commands is absent, or abnormal, high levels of nociceptive stimulation are present (due to injury of the body wall), and the light stimulus used for optogenetic activation is likely not sensed. The effective light intensity may be much higher, because the light does not need to penetrate through the cuticle before it reaches the CNS (even though the actual light intensities used were very similar, 320 μW/cm2 in electrophysiology, and 296-425 μW/cm2 in behavior). In the freely behaving animals, both proprioceptive feedback and copies of motor commands are present and nociceptive stimulation is absent. Furthermore, larvae do see and react to the red light (617 nm), at intensities used for optogenetic stimulation, by increasing the probability of bending. A large difference in context between the dissected preparation and the freely behaving animal is also present for the mechanosensory stimulation experiments. The absolute magnitudes of mechanical stimulation (g-force 1.12 m/s2 in electrophysiology and behavior) and LED light intensity (ca. 300 - 400 μW/cm2) that evoke reliable behavioral responses and electrophysiological responses are similar. However, it is difficult to compare the effective magnitudes of stimulation in the electrophysiological preparations and freely behaving animals as in the former case the animals is dissected and its body wall is stretched and pinned (which could affect the responses of the mechanosensory neurons) and immersed in a physiological solution, whereas in the second case the animal is intact and the stimulus is delivered through air and from above.

Whole-Cell Patch-Clamp Recordings from Basin Neurons in Ventral Nerve Cord The experiments were performed on third instar larvae at feeding stage. Fillet preparations with ventral nerve cord (VNC) attached were dissected in Baines external solution, which contained (mM): 135 NaCl, 5 KCl, 2 CaCl2.2H2O, 4 MgCl2.6H2O, 5 2-[(2-Hydroxy-1,1-bis(hydroxymethyl)ethyl)amino] ethanesulfonic acid, N-[Tris(hydroxymethyl) methyl] −2-aminoethanesulfonic acid, and 36 sucrose. The pH was adjusted to 7.15 with NaOH, and osmolarity was 310-320. The larvae were cut all the way along the dorsal surface, and the fillet was pinned down at 4 corners onto the sylgard-coated recording chamber using fine wire (0.001 tungsten 99.95% wire; California Fine Wire Company). The guts were removed carefully to avoid nerve damage. To minimize VNC movement during the recordings, a transverse cut was made on the anterior cuticle and body wall retracted toward posterior, so that a tiny piece of parafilm could be placed underneath of VNC. The nerves connecting the cuticle and VNC were “glued” to parafilm using petroleum jelly. The preparation was viewed with a 60 × /1 N.A. water-immersion objective equipped with Olympus microscopy (BX51WI; Olympus). GcAMP6 –labeled basin neurons were visualized with a 470-nm wavelength LED. A small section of the glial sheath above the targeted abdominal basin neurons was ruptured using protease (0.1% Protease XIV; Sigma-Aldrich). Recording electrodes were pulled from thick-wall glass pipet (O.D. 1.5mm, I.D. 0.86mm) using P-97 puller (Sutter Instruments) and fire-polished to resistances of 10–15 MΩ. The Baines intracellular solutions contained (mM): 140 potassium gluconate, 5 KCl, 2 MgCl2.6H2O, 2 EGTA, 20 HEPES. The pH was adjusted to 7.4 with KOH, and the osmolarity was 280. The intracellular solution contained 0.5% Neurobiotin for the further post hoc morphological identification of recorded neurons. The data were acquired and processed using Digidata 1440A, Multiclamp 700B, and Clampex 10.4 software (Molecular Devices). The recording was sampled at 20 kHz and filtered at 6 kHz under current-clamp mode, and 10 KHz and 2 KHz under voltage-clamp mode. The recordings will not be processed for further analysis if the resting membrane potential at cell body became > −45 mV before correcting liquid junction potential (15mV) corrections.

Basin Neuron Identification After the electrophysiology recording, the preparation containing VNC and brain was fixed in 4% paraformaldehyde in 0.1 M phosphate buffer saline (PBS) overnight in refrigerator, and then transferred to PBS until staining. After rinsing in PBS, the CNS preparations were placed in Streptavidin Alexa Fluor 647 (1:200) in PBS-T (overnight, room temperature). After rinsing, the preparations were dehydrated and mounted with DPX. The confocal images were captured with Zeiss 710 confocal laser microscope. Alexa Fluor 647 was excited with a light of 633 nm wavelength, and mcherry-tagged CsChrimson neurons were excited with a light of 567 nm.

Spike Detection in Electrophysiological Recordings of Basin Neurons Burrows and Siegler, 1976 Burrows M.

Siegler M.V. Transmission without spikes between locust interneurones and motoneurones. Hengstenberg, 1977 Hengstenberg R. Spike responses of ‘non-spiking’ visual interneurone. Milde, 1981 Milde J. Graded potentials and action potentials in the large ocellar interneurons of the bee. Pearson, 1976 Pearson K.G. Nerve cells without action potentials. Many insect neurons are non-spiking and influence downstream partners only through graded potentials. Some insect interneurons use, both action potentials and graded potentials, for signal transmission (). It is likely that Basins use both the graded potentials and the APs, for signal transmission and for influencing behavioral output. Gouwens and Wilson, 2009 Gouwens N.W.

Wilson R.I. Signal propagation in Drosophila central neurons. Like most insect neurons, Basin cell bodies are closer to the dendritic tree, than to the axon terminal, but they are separated from both by a long primary neurite. The synaptic potentials generated at the dendritic tree therefore bypass the soma on the way to the main spike initiation zone (SIZ), likely located at the start of the axon, just after bifurcation of the primary neurite into a dendritic and an axonal branch (). The SIZ is much closer to the axon terminals (ca. 24 μm in 3rd instar larva), than to the cell body (ca. 60 μm away in 3rd instar larva). The depolarizations at the axon terminal are likely much larger than the ones we observe at the cell body. Thus, graded potentials observed in Basin neuron cell bodies are likely to propagate all the way to the axon terminal and influence their downstream partners and behavior, and not only APs. Furthermore, because the cell bodies (where the patch-clamp recordings are performed) are very far from the SIZ, it is likely that we do not detect many APs evoked by mechanosensory or optogenetic stimuli, because they are distorted and reduced in amplitude (due to distance). When such APs occur on top of large fluctuating depolarizations it is difficult to detect them. In the current injection experiments APs are much easier to detect, because they are not distorted by riding on large EPSPs.

GABA Histochemistry Labeling To determine the neurotransmitter identification in the interneurons, GABA immune-labeling was performed from the JRC-SS00888 (handle-b), JRC-SS00918 (griddle-2), JRC_SS00674 (drunken-1 and drunken-2), JRC-SS00863 (ladder-d) crossed to pJFRC12-10XUAS-IVS-myr::GFP. The VNC was dissected out from 3rd instar larvae, and fixed with 4% PFA for 30 min. After rinsing in PBS, the CNS preparations were incubated in the rabbit anti-GABA (1:500, Sigma) and chick anti-GFP (1:1000, abcam) in PBS-T, followed by Alexa Fluor 488 goat anti-chick IgG and Alexa Fluor 647 goat anti-rabbit IgG. After rinsing, the preparations were dehydrated and mounted with DPX. The confocal images were captured with Zeiss 710 confocal laser microscope. Alexa Fluor 488 was excited with a light of 488 nm, while Alexa Fluor 647 was excited with a light of 633 nm wavelength.

EM Reconstruction and Wiring Diagrams Schneider-Mizell et al., 2016 Schneider-Mizell C.M.

Gerhard S.

Longair M.

Kazimiers T.

Li F.

Zwart M.F.

Champion A.

Midgley F.M.

Fetter R.D.

Saalfeld S.

Cardona A. Quantitative neuroanatomy for connectomics in Drosophila. Ohyama et al., 2015 Ohyama T.

Schneider-Mizell C.M.

Fetter R.D.

Aleman J.V.

Franconville R.

Rivera-Alba M.

Mensh B.D.

Branson K.M.

Simpson J.H.

Truman J.W.

et al. A multilevel multimodal circuit enhances action selection in Drosophila. Saalfeld et al., 2009 Saalfeld S.

Cardona A.

Hartenstein V.

Tomancak P. CATMAID: collaborative annotation toolkit for massive amounts of image data. Schneider-Mizell et al., 2016 Schneider-Mizell C.M.

Gerhard S.

Longair M.

Kazimiers T.

Li F.

Zwart M.F.

Champion A.

Midgley F.M.

Fetter R.D.

Saalfeld S.

Cardona A. Quantitative neuroanatomy for connectomics in Drosophila. Ohyama et al., 2015 Ohyama T.

Schneider-Mizell C.M.

Fetter R.D.

Aleman J.V.

Franconville R.

Rivera-Alba M.

Mensh B.D.

Branson K.M.

Simpson J.H.

Truman J.W.

et al. A multilevel multimodal circuit enhances action selection in Drosophila. Ohyama et al., 2015 Ohyama T.

Schneider-Mizell C.M.

Fetter R.D.

Aleman J.V.

Franconville R.

Rivera-Alba M.

Mensh B.D.

Branson K.M.

Simpson J.H.

Truman J.W.

et al. A multilevel multimodal circuit enhances action selection in Drosophila. Ohyama et al., 2015 Ohyama T.

Schneider-Mizell C.M.

Fetter R.D.

Aleman J.V.

Franconville R.

Rivera-Alba M.

Mensh B.D.

Branson K.M.

Simpson J.H.

Truman J.W.

et al. A multilevel multimodal circuit enhances action selection in Drosophila. Schneider-Mizell et al., 2016 Schneider-Mizell C.M.

Gerhard S.

Longair M.

Kazimiers T.

Li F.

Zwart M.F.

Champion A.

Midgley F.M.

Fetter R.D.

Saalfeld S.

Cardona A. Quantitative neuroanatomy for connectomics in Drosophila. EM reconstruction followed the procedures described in () and (). Briefly, we performed manual annotation of serial EM sections in a web-based tool CATMAID ( http://www.catmaid.org ) (), which allowed for fast reconstruction of neuronal skeletons, which express the anatomy and topology of neural arbors but lack volumetric information, and connectivity. To ensure accuracy, reconstructions were followed by a later comprehensive review (). To focus on those neurons involved in segmental microcircuits connecting chordotonal sensory terminals and Basin dendrites, we looked at the 1.5 segment first instar volume in which all arbors downstream of chordotonal axons were reconstructed (). This identified the iLNa interneurons described here and a subset of Ladder neurons, but the precise identity of which Ladders could not be determined because key identifying features were located outside of the smaller imaged volume. We continued this work in a second volume spanning the entire first instar CNS () by performing targeted reconstruction of all Ladders, Drunken-1, Griddle-1, and Griddle-2 in segment a1 and any appropriate nearby segments. Manual reconstructions of neuronal anatomy and connectivity were performed and reviewed by author CMSM with significant contributions from Ingrid Andrade, Javier Valdes Aleman, Laura Herren, Waleed Osman, and incidental contributions from fourteen other contributors working in the same dataset. It is possible that additional interneurons between chordotonal and Basin cells may exist if their structure was not uniquely identifiable in the previous volume. Existing reconstructions of chordotonal axon terminals, Basin cells in segments, fbLN-Ha, and fbLN-Hb from segment a1 were taken from prior reconstructions (). Small differences between anatomy and connectivity of previously reconstructed neurons are due to correction of errors that were noticed during subsequent reconstruction, typically in the form of omitted twigs, small branches hosting few synapses that have little impact on the network topology (). For wiring diagram descriptions at the cell type level, we summed the number synapses in a given connection between cell types if that connection was reliably found with 3 or more synapses on both left and right sides of the animal. Individual neuron connectivity can be found in the Supplementary neuronal adjacency matrix. Connectivity was analyzed and visualized with custom Matlab (The Mathworks, Inc) scripts.