Shared lineage has diverse effects on patterns of neuronal connectivity. In mammalian cortex, excitatory sister neurons assemble into shared microcircuits, whereas throughout the Drosophila nervous system, Notch-differentiated sister neurons diverge into distinct circuits. Notch-differentiated sister neurons have been observed in vertebrate spinal cord and cerebellum, but whether they integrate into shared or distinct circuits remains unknown. Here we evaluate the connectivity between sister V2a/b neurons in the zebrafish spinal cord. Using an in vivo labeling approach, we identified pairs of sister V2a/b neurons born from individual Vsx1+ progenitors and observed that they have similar axonal trajectories and proximal somata. However, paired whole-cell electrophysiology and optogenetics revealed that sister V2a/b neurons receive input from distinct presynaptic sources, do not communicate with each other, and connect to largely distinct targets. These results resemble the divergent connectivity in Drosophila and represent the first evidence of Notch-differentiated circuit integration in a vertebrate system.

This is the raw data for the manuscript.

Cell-attached recordings were targeted to stochastically labeled WT fish with vsx1:Gal4 BAC and UAS:CoChR2-tdTomato plasmid to calibrate firing of vsx1:Gal4;UAS:CoChR2-tdTomato vsx1+ pairs. Whole-cell patch-clamp recordings were performed in Tg(chx10:loxP-dsRed-loxP:GFP) injected with vsx1:GFP and Tg(chx10:GFP;gata3:loxP-dsRed-loxP:GFP) larvae at 4-6 dpf for paired clonal V2a/b and non-clonal V2a/b recordings, respectively. Additionally, whole-cell patch-clamp recordings were performed in stochastically labeled WT fish with vsx1:Gal4 BAC and UAS:CoChR2-tdTomato in downstream targets. Larvae were immobilized with 0.1% α-bungarotoxin and fixed to a Sylgard lined Petri dish with custom-sharpened tungsten pins. Each larva was then transferred to a microscope (Scientifica SliceScope Pro) equipped with infrared differential interface contrast optics, epifluorescence, and immersion objectives (Olympus: 40X, 0.8 NA). One muscle segment overlaying the spinal cord was removed (segments 7-17) using a blunt end glass electrode and suction (Wen & Brehm, 2010). The bath solution consisted of (in mM): 134 NaCl, 2.9 KCl, 1.2 MgCl₂, 10 HEPES, 10 glucose, 2.1 CaCl₂. Osmolarity was adjusted to ~295 mOsm and pH to 7.5.

Patch pipettes (5–15 MΩ) were filled with internal solution for voltage and current clamp and cell-attached composed of (in mM): 125 K gluconate, 2 MgCl₂, 4 KCl, 10 HEPES, 10 EGTA, and 4 Na₂ATP). Whole-cell optogenetic and some paired recordings were performed using internal solution composed of (in mM): 122 cesium methanesulfonate, one tetraehtylammonium-Cl, 3 MgCl₂, 1 QX-314 Cl, 10 HEPES, 10 EGTA, and 4 Na₂ATP. Additionally, Alexa Fluor 647 hydrazide 0.05-0.1 mM or sulforhodamine (0.02%) was included to visualize morphology of recorded cells post hoc. Osmolarity was adjusted to ~285 mOsm and KOH or CsOH, respectively was used to bring the pH to 7.5.  Patch recordings were made in whole-cell configuration using a Multiclamp 700B, filtered at 10 kHz (current clamp) or 2 kHz (voltage clamp). All recordings were digitized at 100 kHz with a Digidata 1440 (Molecular Devices) and acquired with pClamp 10 (Molecular Devices). The following drugs were bath applied where noted: strychnine (10 µM), NBQX (10 µM), APV (100 µM), 18-beta-glycyrrhetinic acid (150 µM), and carbenoxolone disodium salt (500 µM). 

Data collected are .abf files that can be opened using Matlab and ClampFit.