DOI: 10.5061/dryad.ksn02v7jv
title: Phase-specific premotor inhibition modulates leech rhythmic motor output

description: Understanding how motoneuron activity is finely tuned remains an open
 question. Leeches are a highly suitable organism for studying motor
 control due to their well-characterized behaviors and relatively simple
 nervous system. On solid surfaces, leeches display crawling, a rhythmic
 motor pattern that can be elicited in the isolated nerve cord or even in
 single ganglia isolated from it. This study aimed to learn how this motor
 output is shaped by concurrent premotor signals. Specifically, we analyzed
 how electrophysiological manipulation of a premotor nonspiking (NS)
 neuron, which forms a recurrent inhibitory circuit (analogous to that
 formed by vertebrate Renshaw cells), shapes the leech crawling motor
 pattern. The study included a quantitative analysis of putative motor
 units active throughout the fictive crawling cycle, which shows that the
 rhythmic motor output in isolated ganglia mirrors the phase relationships
 observed in vivo. Taken together, the study reveals that the premotor NS
 neurons, under the control of the segmental pattern generator, modulated
 the degree of excitation of motoneurons during crawling in a
 phase-specific manner.
description: # Phase-specific premotor inhibition modulates leech rhythmic motor output
 Dataset DOI:
 [10.5061/dryad.ksn02v7jv](https://doi.org/10.5061/dryad.ksn02v7jv) ##
 Description of the data and file structure ##### Overview This repository
 contains data and code associated with the manuscript “Phase-specific
 premotor inhibition modulates leech rhythmic motor output”. For further
 details, refer to the full manuscript at:
 [https://doi.org/10.7554/eLife.104921.2](https://doi.org/10.7554/eLife.104921.2) ### Files and variables #### File: NS-y-DE3.csv **Description:** As indicated in Materials and Methods, extracellular recordings of DE-3 (dorsal excitor cell 3) motoneurons in DP nerves were combined with NS intracellular recordings during fictive crawling in isolated leech ganglia. Fictive crawling was segmented based on DE-3 bursts, using either the first or the last action potential of each burst to obtain start-to-start versus end-to-end cycles, respectively. For five cycles from each recording (.abf name), the following variables are presented: Variables * recording: .abf file name * cycle: Fictive crawling cycle. Recordings were segmented using the first or the last action potential of each DE-3 burst to obtain start-to-start or -o-end cycles. Both are presented here; the variable name indicates the segmentation type. * dur: DE-3 burst duration.  * ff: DE-3 firing frequency, calculated as the number of spikes relative to burst duration. * pePer-endPeriod from end-to-end. * dc-end: DE-3 duty cycle calculated as burst duration relative to the end-to-end period. * per-start: Period from start to start. * dc-start: DE-3 duty cycle calculated as burst duration relative to the start-to-start period. * elong-pre: Inter-burst duration prior to the DE-3 burst. * elong-post: Inter-burst duration following the DE-3 burst. * hyper-NS: Maximum hyperpolarization of NS membrane potential for the cycle. * amp-NS: NS hyperpolarization amplitude calculated as the minimum membrane potential relative to baseline at the moment of the first spike of the DE-3 burst. #### File: NS-y-motoneurons.csv **Description:** Extracellular recordings from the DP, PP and/or AA nerves were combined with NS intracellular recordings during fictive crawling in isolated leech ganglia. Spike sorting analysis was performed to obtain distinct units, and rhythmic ones were selected using autocorrelation analysis. The rhythmic motor units were tagged according to their activity relative to DE-3 during crawling into three groups: In-Phase, Out-of-Phase, and In-Phase-Early-Onset. Fictive crawling was segmented based on the DE-3 bursts, using the first  action potential of each burst. Depolarizing pulses of +5 nA to +7 nA were injected into the NS soma through the intracellular electrode. Between four and five crawling cycles before and after each depolarizing pulse were left unaffected to obtain control pre and post epochs. ##### Variables * recording: .abf file name. * motoneuron: Motor unit. The number does not indicate identity; only 0 corresponds to DE-3. * nerve: Extracellularly recorded nerve. * tag: Motor unit tag. * cycle: Fictive crawling cycle order for each epoch and recording. * epoch: Pre, depo, post; pre and post are control epochs before or after the test epoch. * treatment: Whether depolarizing current was injected into the NS premotor neuron (depo) or not (ctrl). * per: Period. * 1 - 70: Binned firing frequency along the cycle. #### File: NS-y-DE3-depo.csv **Description:** Extracellular recordings of the DE-3 motoneuron in the DP nerve were combined with NS intracellular recordings during fictive crawling in isolated leech ganglia. Fictive crawling was segmented based on DE-3 bursts, using either the first or the last action potential of bursts to obtain start-to-start versus end-to-end cycles, respectively. During crawling, depolarizing pulses of +5 nA to +7 nA were injected into the NS soma through the intracellular electrode. Between four and five crawling cycles before and after each depolarizing pulse were left unaffected to obtain control pre and post rhythmic epochs. ##### Variables * recording: .abf file name. * pulse: Pulse number of depolarizing current injected into NS for a given recording  (only the first pulse was considered in our project). * cycle: Fictive crawling cycle order for each epoch and recording. Recordings were segmented using the first or the last action potential of each DE-3 burst to obtain start-to-start or -to-end cycles. Both are presented here; the variable name indicates the segmentation type. * treatment: Whether depolarizing current was injected into the NS premotor neuron (NS) or not (CTRL). * ganglia: Ganglion position in the nerve cord. * leech: Individual animals used. * epoch: Pre, depo, post. Pre and post are control epochs before or after the test epoch. The following variables were calculated (the dataframe specifies for each case whether segmentation was start-to-start or end-to-end): * dur: DE-3 burst duration. * ff: DE-3 firing frequency, calculated as number of spikes relative to burst duration. * per: Period. * dc: DE-3 duty cycle, calculated as burst duration relative to the period. #### File: abfs.csv **Description:** Detailed information on the recordings listed in data-abfs.zip, including the identity of the extracellularly recorded nerves, the position of the ganglion in the nerve cord, and the time window considered in the analysis. #### File: data-pklspikes.zip **Description:** .pklspikes files corresponding to all .abf recordings that were analyzed using spike sorting. The spike sorting algorithm is a custom-written algorithm that relies on a template-matching method based on Pouzat et al. (2002), incorporating a t-SNE model (Pedregosa et al., 2011; Van der Maaten and Hinton, 2008). The repository of the algorithm used is available at: [https://github.com/asmerlinsky/PyLeech](https://github.com/asmerlinsky/PyLeech). **Usage notes:** These files are intended to be accessed and interpreted using the activity-cycles-mns.py script included in this dataset. This script provides the necessary loading routines to read the .pklspikes files, extract spike times for each identified motor unit, and compute binned firing frequency. It further detects DE-3 bursts and segments motor unit activity. #### File: data-abfs.zip **Description:** .abf files from the electrophysiological recordings. Details of each recording are provided in the abfs.csv dataframe. #### File: NS_DE3_correlations.R **Description:** Script used for the analysis of NS membrane potential oscillations during crawling. #### File: NS_mns_analysis.R **Description:** Script used for analysing the effect of NS on the activity of the different types of motoneurons during crawling. #### File: activity-cycles-mns.py **Description:** Script used to detect DE-3 bursts and segment the binned firing frequency of all identified motor units into cycles. #### File: analysis-spikesort.py **Description:** Script used for both the automatic and manual steps of the spike-sorting procedure.