Interactions between CNS regulation and serotonergic modulation of crayfish hindgut motility

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.

Methods
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.