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<h2 class="ltx_title_subsection">Dissections</h2><div>The brains of 5 to 9 days old female flies were extracted and laid on a poly-D-lysine coated coverslip (Corning, Corning, NY<b>)</b>. In most experiments, both the brain and the ventral nerve chord (VNC) were dissected out, as we found that having the VNC  attached to the brain increased the mechanical stability of the preparation.  Dissection was performed using the minimum level of illumination possible to avoid spurious activation of CsChrimson. The preparation was bathed throughout in  saline containing (in mM): &nbsp;103 NaCl, 3 KCl, 5 TES, 8 trehalose dihydrate, 10 glucose, 26 NaHCO<sub>3</sub>, 1 NaH<sub>2</sub>PO<sub>4</sub>, 2 CaCl<sub>2</sub>, 4 MgCl<sub>2</sub>, &nbsp;bubbled with carbogen (95% O<sub>2</sub>, 5% CO<sub>2</sub>). Brains were positioned  anterior-side-up, except when the connection tested was thought to be in the PB, in which case they were positioned posterior-side-up to maximize light access close to the assumed synaptic site. Trachea were removed. Only for  experiments involving pharmacology, the glial sheath was gently torn with tweezers to enhance drug access to the neuropiles.&nbsp;</div><h2 class="ltx_title_subsection"></h2><h2 class="ltx_title_subsection">Imaging conditions and trial structure</h2><div>Imaging was performed on an Ultima II 2-photon scanning microscope (Bruker, Billerica, MA) with a Vision II laser (Coherent, Santa Clara, CA). Brains were continuously perfused in the saline used for dissection at 60 mL/hour. Once the sample was placed and centered under the objective, we waited 5 minutes before starting the experiment to avoid any lingering network activation from the dissection or transmission lights. 2-photon excitation wavelength was 920 nm, and power at the sample varied between 3 and 10 mW. CsChrimson was excited with trains of 2 ms long 590 nm light pulses via an LED (M590L3-C1, Thorlabs, Newton, NJ) shone through the objective. The excitation light path contained a 605/55 bandpass filter and was delivered to the objective with a custom dichroic (zt488-568tpc, reflecting between 568nm and 700nm). On the detection arm, a 575nm dichroic beamsplitter and bandpass filters (525/70 nm and 607/45 nm for the green and red respectively) emitted photons to the PMTs (Hamamatsu multi-alkali). Instantaneous power measured out of the objective was roughly 50 μW/mm 2 . Stimulus pulse trains were delivered at 30 Hz and the number of pulses  varied between 1,5,10, 20 and 30 — corresponding to total stimulation durations ranging from 2 ms to 1 s. Imaging fields of view were chosen as to avoid scanning regions containing  CsChrimson-expressing neuropil while being as close as possible to the supposed connection site, &nbsp;as we observed occasional 2-photon stimulated slow activations of CsChrimson-expressing cells  (high-intensity 2-photon stimulation of CsChrimson was used for spatially precise neuronal activation in&nbsp;<cite class="ltx_cite raw v1">\citealt{Kim2017}</cite>). When this was impossible — for example, in self-activation controls or for completely overlapping cell types — we chose a large ROI of which the CsChrimson/GCaMP6m-expressing neuropil represented a small fraction, so as to minimize  duty cycle. ROIs were kept constant throughout the experiment. Each experimental run consisted of 4 repeats each approximately 16 s long. Runs were themselves repeated every 2 minutes. All experiments started with 5 runs corresponding to the five stimulation strengths, in a random order. This was sometimes followed by pharmacological testing. At the end of the experiment, a high intensity 3D stack was acquired to check that the expression patterns were as expected, and the correct region was imaged. At least 6 flies were tested for every pair considered.</div><h2 class="ltx_title_subsection"></h2><h2 class="ltx_title_subsection">Pharmacology</h2><div>For blocking nicotinergic or inhibitory (GABAergic or glutamatergic) transmission, mecamylamine (50 μM) or picrotoxin (10 μM) (Sigma-Aldrich, St Louis, MO) were administered through the perfusion by switching to a different line for 3 minutes, followed by a wash period during which the perfusion was drug-free again. 30 pulses stimulations runs were repeated every 2 minutes, starting 4 minutes before the drug application and throughout the wash. Prior to use, they were kept frozen in 25 mM and 0.3 M aliquots, respectively.</div><h2 class="ltx_title_subsection"></h2><h2 class="ltx_title_subsection">Analysis</h2><div>All analysis was performed in&nbsp;<a href="http://julialang.org/" target="_blank">Julia</a>, using custom-written routines. All data and code is available as an OpenScienceFramework project at&nbsp;<a href="https://osf.io/vsa3z/">https://osf.io/vsa3z/</a>. Code is also centralized in this&nbsp;<a href="https://github.com/romainFr/CX-Functional-Analysis" target="_blank">repository</a> and notebooks recapitulating the analysis can be run straight from the browser&nbsp;<a href="https://mybinder.org/v2/gh/romainFr/CX-Functional-Analysis/master" target="_blank">here</a> (using&nbsp;<a href="https://mybinder.org/" target="_blank">Binder</a>).</div><div></div><h3 data-label="378929" class="ltx_title_subsubsection">Data processing</h3><div>For a given experiment, all movies were aligned to each other to compensate for slow drifts of the sample: for each run, the average image was calculated, and translation drifts between average images were calculated using correlation-based sub-pixel registration (<cite class="ltx_cite raw v1">\citealt{guizar-sicairos_efficient_2008}</cite>,<b>&nbsp;</b>and<b>&nbsp;&nbsp;</b><a href="https://github.com/romainFr/SubpixelRegistration.jl" target="_blank">https://github.com/romainFr/SubpixelRegistration.jl</a><b>&nbsp;</b>for the Julia implementation used here). A region of interest (ROI) was defined for the full experiment: the average image (of all the runs)  between foreground and background was distinguished using k-means clustering. Note that the selection method relies only on average intensity and not activity —a method we chose so as to maintain the same detection method for responsive and non-responsive runs. This also relies on selecting fields of view as unambiguously containing the neuron of interest — and only the neuron of interest — during the experiment.</div><div>ΔF/F<sub>0</sub> =<span class="math ltx_Math v1">\(\frac{\left(F-F_{0\ }\right)}{\left(F_0-B\right)}\)</span>, where F is the raw fluorescence and B the background signal (calculated as the intensity of the&nbsp;10% dimmest pixels of the average image) were then computed for each movie in the ROI. Given that baseline fluorescence could vary widely over the course of an experiment (see Discussion), we defined F<sub>0</sub>  as the median fluorescence in the ROI in the  dimmest 3% of frames of the entire experiment.</div><div></div><h3 data-label="982017" class="ltx_title_subsubsection">Statistics</h3><div>For every experimental repeat, we computed the following statistics  :</div><ul><li>F<sub>peak</sub> the peak fluorescence value, and T<sub>peak</sub> the time after stimulation at which the peak value is reached</li><li>I<sub>toPeak</sub> the integral of the signal up to the peak time</li><li>τ<sub>1/2</sub> the half-decay time</li><li>F<sub>base&nbsp;</sub>the fluorescence baseline before stimulation expressed in ΔF/F0</li></ul><div>Then, for every run, which consists of 4 repeats, we computed:</div><ul><li>R<sub>within-flies</sub> the average correlation between the 4 repeats of the run&nbsp;</li><li>&lt;F<sub>peak</sub>&gt;, &lt;T<sub>peak</sub>&gt;, &lt;I<sub>toPeak</sub>&gt;, &lt;F<sub>base</sub>&gt;&nbsp; and &lt;τ<sub>1/2</sub>&gt; the medians of&nbsp;F<sub>peak</sub>, T<sub>peak</sub>, I<sub>toPeak,&nbsp;</sub>F<sub>base&nbsp;</sub> and τ<sub>1/2</sub>, respectively</li></ul><div></div>