DOI: 10.5061/dryad.qv9s4mwr3
title: Interactions between CNS regulation and serotonergic modulation of crayfish hindgut motility

description: Motility is a critical function of the gastrointestinal (GI) system
 governed by neurogenic and myogenic processes. Due to its major role in
 maintaining homeostasis, overlapping mechanisms have evolved for its
 adaptive operation including modulation by the Central Nervous System
 (CNS), Enteric Nervous System (ENS), and intrinsic pacemaker cells. The
 complex interplay of modulatory mechanisms of intestinal motility remains
 poorly understood since mammalian species offer limited accessibility.
 Crayfish provide a tractable ex vivo model to study the interplay between
 CNS and neurochemical regulation of GI motor patterns. Our study
 investigated the effects of CNS denervation and exogenously applied
 serotonin (5-HT) on crayfish hindgut motility. Multiscale spatial
 measurements showed stable motility parameters throughout 90 minutes of
 control conditions. Denervation, i.e., separating the gut from the CNS,
 resulted in a significant decrease in the magnitude and synchrony of
 hindgut contractions, while preserving the underlying frequency and
 directional bias of the waves. Subsequent application of 5-HT to the
 denervated preparation enhanced motility but disrupted spatiotemporal
 coordination. Treatment with TTX (a sodium channel blocker) had minor
 impacts on motility metrics, indicating a prominent role of myogenic
 mechanisms. Our model provides a multiscale analysis framework to dissect
 CNS and interrelated neurochemistry contributions to GI motor dynamics.
description: <strong>Experiments:</strong> Adult male crayfish
 (Procambarus clarkii) were obtained from commercial suppliers and housed
 in communal tanks under controlled conditions (12-hour light/dark cycle,
 22 ± 1.5°C). They were fed twice weekly with shrimp pellets. To
 standardize feeding and ensure empty hindguts before dissection, crayfish
 were isolated in smaller tanks 3-4 days after the last communal feeding,
 given one shrimp pellet upon isolation, and kept unfed for four days
 before experiments. Crayfish were anesthetized on ice for 15–25 minutes
 before dissection. The ventral and dorsal carapaces were cut bilaterally
 to expose the nerve cord and hindgut. The dorsal artery along the hindgut
 was removed, and the nerve cord was carefully separated by cutting the
 lateral nerves, except for nerves 5, 6, and 7 connected to the telson,
 anus, and hindgut musculature. The hindgut, attached to the nerve cord via
 nerve 7, was isolated, cleaned of muscles, arteries, and other nerves, and
 placed in crayfish saline. The preparation was pinned in a Sylgard-lined
 dish, securing the hindgut and nerve cord for further experimentation.
 Video recordings of crayfish preparations were made at 30 frames per
 second for 90 minutes using a monochrome CMOS camera with 10x
 magnification. Control experiments involved continuous superfusion with
 crayfish saline at 5 ml/min to assess viability, with weekly recalibration
 of the superfusion rate. Experiments (excluding TTX) followed three
 sequential conditions (30 minutes each): baseline superfusion with saline,
 hindgut isolation by severing nerve N7, and superfusion of the isolated
 hindgut with either saline (control) or varying 5-HT concentrations (1,
 10, or 100 μM). Electrophysiology experiments with TTX (10^-7 M) were
 conducted to distinguish neurogenic from myogenic contributions to gut
 motility, using both denervated (N=1) and intact hindgut-nerve cord
 preparations (N=5). These experiments included baseline saline superfusion
 (30 minutes), TTX or saline superfusion (30 minutes), and washout with
 saline (1-3 hours), with or without electrical stimulation of the anterior
 nerve cord connectives. <strong>Processing:
 </strong>Time-lapse images were cropped to the hindgut region, and
 transition frames were removed. Frame rate was reduced from 30 to 3 fps,
 and temporal smoothing was applied using a Simoncelli filter. Hindgut
 outlines were extracted using MATLAB, and optical flow analysis with the
 Lucas-Kanade method provided dorsal-ventral motility plots. FFT on
 motility plots revealed frequency components below the Nyquist frequency
 (1.5 Hz). Power was calculated from squared amplitudes, and relative
 rhythmic power near characteristic frequency peaks quantified temporal
 coordination. Lateral waves were identified based on speed, area, and
 length thresholds. Central axes and speeds were calculated, distinguishing
 synchronous "mixed waves" from propagating waves. Directionality
 was labeled as anterior-posterior or posterior-anterior. The last 15
 minutes of phases 1 and 2 and the first 15 minutes of phase 3 were
 analyzed to capture hindgut responses to 5-HT. Statistical tests confirmed
 significant differences across treatments. Shapiro-Wilks tests assessed
 normality of motility parameters for each treatment (N = 6). Friedman
 tests, followed by Wilcoxon signed-rank tests for pairwise comparisons (p
 &lt; 0.05), examined differences across phases (baseline vs. phase 2,
 baseline vs. phase 3, phase 2 vs. phase 3) for each treatment. Saline + N7
 cut movies (N = 24) were pooled to evaluate N7 cut effects relative to
 baseline, while saline + N7 cut + 5-HT movies (N = 18) tested 5-HT effects
 relative to baseline and N7 cut phases. TTX experiments had too few
 replicates for statistical analysis across groups.
description: # Interactions between CNS regulation and serotonergic modulation of
 crayfish hindgut motility Dataset DOI:
 [10.5061/dryad.qv9s4mwr3](10.5061/dryad.qv9s4mwr3) ## Description of the
 data and file structure # README for Gut Motility Dataset This dataset
 contains hindgut motility measurements from crayfish under various
 experimental treatments. Data are stored primarily as MATLAB (`.mat`)
 files, in the form of structured variables such as motility maps, and wave
 metrics for further quantitative analysis. --- ## Data Folder Structure *
 `motility_metrics_by_variable` * `motility_metrics_by_treatment_phase` *
 `dv_motility_kymographs` * `dv_speed_thresholds_all_movies` *
 `motility_statistics_spss` --- ## Key Variables and Units ### Fourier Wave
 Analysis Variables Each `.mat` file stores six metrics derived from
 dorsal-ventral motility maps over two consecutive ~15-minute intervals: *
 `ptot`: Total power in the dorsal-ventral motion spectrum (unit: (mm/s)²)
 * `per_power`: Percent of total power within a narrow band around the
 spectral peak (%) * `fre`: Frequency corresponding to the dominant
 rhythmic peak (Hz) * `norm_peak`: Peak amplitude normalized by total power
 (1/Hz) * `prom`: Peak prominence in the power spectrum ((mm/s)²/Hz) *
 `width`: Width of the dominant spectral peak (Hz) ### Lateral Wave Metrics
 * `n_switch`: Number of directional switching events (AP ↔ PA) * `n_AP`:
 Number of anterior-to-posterior waves traveling &gt;1/5 gut length *
 `n_PA`: Number of posterior-to-anterior waves traveling &gt;1/5 gut length
 * `n_mix`: Number of mixed-direction waves * `nwav`: Total number of
 lateral waves (includes AP, PA, and mixed) * `len`: Duration of the
 interval (min) Waves are identified using top 30% velocity and spatial
 thresholds (≥ 1/5 gut length). --- ## Accessing Each Motility Metric
 **Folder**: `motility_metrics_by_variable` File name starts with metric: *
 `power`, `rhythmic_pow`, `freq`, `n_AP`, `n_PA`, `n_total`, `n_switch`
 File suffix: * `p1`: 15–30 min * `p2`: 45–60 min * `p3`: 60–75 min Each
 column: treatment type Each row: individual movie ### Treatment order: 1.
 Saline + N7 cut 2. Saline 3. Saline + N7 cut + 100 μM 5-HT 4. Saline + N7
 cut + 10 μM 5-HT 5. Saline + N7 cut + 1 μM 5-HT --- ## Accessing Metrics
 by Treatment Phase **Folder**: `motility_metrics_by_treatment_phase`
 Subfolders: * `Fourier analysis parameters` * `Lateral Wave parameters`
 Inside each: folders for `5-HT` and `TTX` experiments ### Lateral Wave
 Parameters * `len`, `n_AP`, `n_PA`, `n_mix`, `nwav`, `n_switch` ###
 Fourier Parameters * `ptot`, `per_power`, `fre`, `prom`, `norm_peak`,
 `width` ### Filename convention: * `exp##_pXX` * `##`: experiment number *
 `pXX`: treatment phase * 5-HT: p11, p21, p31 * N7-cut: p11, p21, p22 *
 Saline: p11, p12, p13 --- ## Accessing DV Motility Kymograph Files
 **Folder**: `dv_motility_kymographs` Each file contains: * `vx2avg`:
 caudal-rostral velocity (pixels/frame) * `vy2avg`: dorsal-ventral velocity
 (pixels/frame) * `wid`: gut width (pixels) Filename: `expt##_p#` For TTX,
 folders are named by experiment date. --- ## Accessing DV Speed Thresholds
 **Folder**: `dv_speed_thresholds_all_movies` * Files: e.g.,
 `spd_thrld_80ptile.mat` (top 20%) * Used: `spd_thrld_70ptile.mat` (top
 30%) median across all movies * Inside `.mat`: column `'thr'` →
 one threshold per treatment window --- ## Accessing Videos of Hindgut
 Motility **Folder**: `videos_by_treatment_phase` Representative 1-minute
 clips from Experiment 26 (Saline + N7 cut + 10 μM 5-HT) stored at 3 fps.
 **Anatomical orientation:** * Right = posterior * Left = anterior * Top =
 ventral * Bottom = dorsal **Videos:** * `Baseline`: pre-denervation
 (29:00–30:00) * `N7 Cut`: post-lesion (58:45–59:45) * `5_HT`: after 5-HT
 application (62:21–63:21) **Supplementary Movie:** *
 `Baseline_Optical_Flow_Movie.avi` (4:15–6:16) * Optical flow arrows: blue
 = upward, red = downward * Playback ~5× speed --- ## Accessing SPSS
 Statistics **Folder**: `motility_statistics_spss` Stats computed for 7
 metrics using: * Normality: Shapiro-Wilk, Kolmogorov-Smirnov * Friedman
 test * Wilcoxon paired signed-rank **Examples:** *
 `descriptive&normality_tests_PAwaves_15int.spv.pdf` *
 `friedman_PAwaves_15int.pdf` * `wilcoxon_PAwaves_15int.pdf` --- ## CSV
 File Descriptions This dataset includes six `.csv` files. 1.
 `5_HT_experiments_overall.csv`: Hindgut motility matrix across 5
 treatments. Rows = movies; Columns = treatments 2.
 `5_HT_and_control_expt.csv`: Annotated list with treatment, anatomy,
 behavioral notes. 3. `TTX_expt_details.csv`: Metadata on date, protocol,
 and preparation. 4. `SpikeCounting_TTX.csv`: Raw spike data (ANC/VNC);
 each row = 1 spike. Columns: Start/End Time (ms), Amplitude (V), Time to
 Peak (ms), Time of Peak(ms) 5. `SpikeCounting_Control.csv`: Same format
 for control experiments (VNC, N7). Includes time windows like “15–30 min”.
 6. `SpikeCounting_Summary.csv`: Aggregated spike statistics by condition.
 Includes prep, protocol, date, and counts. --- ## Notes * All times in
 spike data are in milliseconds (ms) * Voltages in volts (V) * Dates follow
 MM-DD-YYYY * Unnamed columns = empty Excel artefacts (safe to ignore) ####
 File: Crayfish\_Gut\_Data.zip **Description:** This dataset captures
 dorsal-ventral motility dynamics in the crayfish hindgut under various
 experimental treatments, including serotonergic stimulation and
 denervation. Quantitative measurements were derived from optical
 flow-based motility maps and Fourier analysis, yielding metrics such as
 wave frequency, power, and propagation direction. Data are provided as
 MATLAB files and CSV tables, along with supporting kymographs, spike
 recordings, and statistical outputs. This resource enables analysis of gut
 motility patterns in response to neurochemical perturbations. ##
 Code/software The following software was used to view and analyze the
 data: * **MATLAB (R2020a–R2023b)** Used for wave extraction, spectral
 analysis, and statistical post-processing of hindgut motility. Functions
 such as `strel`, `imerode`, `bwlabeln`, `bwareaopen`, and `bwskel` were
 central to image-based segmentation and wave propagation analysis. Access
 was provided via a university license and may require a separate license
 for external use. * **ImageJ (v1.54p)** – *Free and open-source* Used for
 initial video cropping, and frame rate reduction from 30 to 3 fps. *
 **Python (3.8 or later)** – *Free and open-source* Used for removal of
 transitional timepoints (e.g., denervation, 5-HT onset), optical flow
 computation, and visualization of `.csv` outputs. Primary libraries
 included: * `pandas` (data wrangling) * `numpy` (numerical operations) *
 `scipy` (statistical testing) * `matplotlib` (plotting)