MECHANICAL AND OPTOGENETIC STIMULATION OF INTRINSIC PRIMARY AFFERENT NEURONS OF THE MOUSE INTESTINES TRIGGERS SPECIFIC MOTOR PATTERNS

BACKGROUND: Intrinsic primary afferent neurons (IPANs) of the gastrointestinal (GI) tract respond to luminal content including pressure to initiate motor reflexes. Advillin is a marker of mouse IPANs and Avil-CreERT2 mice can drive the expression of optogenetic ion channels. It is unknown whether IPAN activation and luminal pressure initiate the same motor patterns.
AIM: Optogenetically stimulate IPANs to determine their roles in intestinal motility compared to pressure-induced motility.
METHODS: High resolution video recordings were made of jejunum and colon from 10-12 week Avil-CreERT2/FloxSTOPReaChR mice treated with 5mg tamoxifen (PO) at 7 weeks. A Mariotte bottle of glucose-free Krebs applied 0 or 3cmH₂0 intraluminal pressure. Tissue was intermittently and focally stimulated using a 0.5mm fiber optic cable (625-nm, Prizmatix) at 2Hz, 40% duty cycle for 10s. A custom Jupyter Notebook (v6.4.8) was utilized to generate spatiotemporal (ST) maps, manually curate contractions, and extract quantitative features. Chi-Square and Two-way ANOVA tested significance.
RESULTS: Both pressure and light caused more frequent propagating contractions in colon (0.3±0.3, 0cmH₂O; 10±2, 3cmH₂O; 2.2±0.9, 0cmH₂O with light; P<0.01; N=6 mice) and jejunum (0.3±0.3, 0cmH₂O; 2.0±0.9, 3cmH₂O; 1.5±0.6, 0cmH₂O with light; P<0.05; N=8 mice). Both pressure and light increased the frequency (0.011±0.002Hz, 0cmH₂O; 0.020±0.003Hz, 3cmH₂O; 0.022±0.002Hz, 0cmH₂O with light; P<0.05; N=8-19 contractions), and decreased the mean length (2.8±0.5cm, 0cmH₂O; 1.1±0.4cm, 3cmH₂O; 1.5±0.4cm, 0cmH₂O with light; P<0.05; N=6 mice) and duration (303±64s, 0cmH₂O; 92±20s, 3cmH₂O; 117±27s, 0cmH₂O with light; P<0.05; N=8-19 contractions) of ripple contractions in the colon. While pressure had similar effects on ripple contractions in the jejunum compared to colon, optogenetic stimulation had the opposite effect of pressure on ripple contractions in the jejunum, increasing ripple length and duration. Optogenetic stimulation of IPANs at 3cmH₂0 triggered faster and longer propagating contractions with no effect on ripple contractions in the colon where 5/6 tissues contracted orally (0.7±0.2mm) and 4/6 tissues relaxed aborally (0.4±0.3mm) in response to light. Conversely, in the jejunum at 3cmH₂0, light increased mean ripple duration whilst decreasing ripple frequency with no effect on propagating contractions where 6/8 tissues contracted orally (0.4±0.3mm) and 5/8 tissues relaxed aborally (0.3±0.1mm) in response to light.
CONCLUSIONS: Targeted optogenetic stimulation of IPANs triggers peristalsis in most tissues and recapitulates pressure-induced changes in motility in the colon and pressure-induced changes in propagating contractions in the jejunum. This represents a novel approach to dissect IPAN function in intestinal motility and enteric neural reflexes.
FUNDING: NIH grants R01DK129315 and DP2AT010875