Interactions between CNS regulation and serotonergic modulation of crayfish hindgut motility

Pathak, Spandan 1 ; Peña-Flores, Norma1 ; Alvarez, Phillip1 ; Feeley, Jenna1 ; Ghodssi, Reza1 ; Losert, Wolfgang 1 ; Herberholz, Jens1

Published Jun 27, 2025 on Dryad. https://doi.org/10.5061/dryad.qv9s4mwr3

Data files

Jun 27, 2025 version files 5.85 GB

Crayfish_Gut__Motility_Data_Pathak___Peña-Flores_et_al.zip

5.85 GB
README.md

6.86 KB

Abstract

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.

Dataset DOI: 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 >1/5 gut length
n_PA: Number of posterior-to-anterior waves traveling >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:

Saline + N7 cut
Saline
Saline + N7 cut + 100 μM 5-HT
Saline + N7 cut + 10 μM 5-HT
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.

5_HT_experiments_overall.csv:
Hindgut motility matrix across 5 treatments.
Rows = movies; Columns = treatments
5_HT_and_control_expt.csv:
Annotated list with treatment, anatomy, behavioral notes.
TTX_expt_details.csv:
Metadata on date, protocol, and preparation.
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)
SpikeCounting_Control.csv:
Same format for control experiments (VNC, N7).
Includes time windows like “15–30 min”.
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)

Experiments: 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.

Processing: 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 < 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.