CHARACTERIZATION OF MOUSE COLONIC NOCICEPTIVE NEURONS AND THEIR ALTERATION BY INFLAMMATION

MJ Beyak, TMR Stewart, S Vanner

GI Diseases Research Unit, Queen's University, Kingston, Ontario

Abdominal pain is an important cause of morbidity in inflammatory bowel disease. This study examined mechanisms underlying this pain by characterizing properties of sensory nociceptive neurons innervating the mouse colon and the effects of colitis.
METHODS: Dorsal root ganglia (DRG) neurons innervating the mouse colon were identified using the retrograde tracer DiI applied to the serosal surface of the descending colon. Nociceptive neurons were selected by their TTX-insensitive action potentials and small cell size (membrane capacitance < 30 pF). TNBS colitis was induced by the rectal instillation of TNBS in 25% ethanol. Seven days later, control and TNBS animals were euthanized and whole cell recordings were obtained from acutely isolated DRG cells labeled with DiI.
RESULTS: The mean resting membrane potential was 52.2±1.7 mV (n=46). Mean cell capacitance (22.7±1.4 pF), TTX-resistant action potentials (n=5/5) and prominent inflections on the downward phase of the action potential were consistent with nociceptive properties. In voltage clamp, currents underlying neuronal excitability were examined with depolarizing voltage steps. These elicited a fast inward current (Na⁺ and inward rectifier) followed by a transient outward current (I_A) in 80% of cells and a sustained outward current (I_K) in all cells. To examine whether colitis alters neuronal excitability, the rheobase and action potential discharge at one and two times rheobase were compared in control and TNBS animals. The current threshold (rheobase) in the TNBS group was significantly lower (25.3±10.0 vs. 99.4±23.1 p_A, p=0.03). At two times rheobase, the number of cells firing multiple action potentials was significantly greater in the TNBS group (87.5% vs. 33% p=0.01). A subgroup of cells were spontaneously active and were found predominantly in the TNBS group (37.5% vs. 10%, p=0.04).
CONCLUSION: TNBS colitis causes hyperexcitability of mouse DRG nociceptive neurons. Alteration of one or more of the Na⁺ and K⁺ currents likely mediate this effect and this mouse model will permit the use of targeted gene deletion models in elucidating their role.