In humans, detrusor overactivity is present in infants, usually disappears in adults, and frequently reappears with outlet obstruction. Similarly, in rats, spontaneous pacemaker activity, which is responsible for intrinsic smooth muscle contraction, is present in neonatal bladders, absent in adult bladders and re-emerges following bladder outlet obstruction. There have been numerous attempts to characterize bladder pacemaker activity and detrusor overactivity using cystometry, extracellular, intracellular and patch clamp recording techniques. However, whether pacemaker activity originates from restricted specialized sites or occurs randomly, how excitation propagates within the smooth muscle and the mechanism of detrusor over activity remain unclear. We have developed optical imaging techniques to map detrusor action potentials and Ca 2+ fluxes. Our photodiode array system allows us to simultaneously record, at up to 4000 frames/s, action potentials and Ca 2+ transients from 256 sites in the bladder wall. We propose to apply our methodology to investigate the origin(s) and mechanism of propagation of spontaneous activity in the bladders of neonate and adult rats, the involvement of neural modulation, and the mechanism of detrusor over activity following partial outlet obstruction. Rat bladders will be excised, cannulated at the urethra and stained/loaded with the appropriate fluorescent dyes. Intact bladders will be placed in a temperature-regulated bath, connected to a pressure transducer, and imaged from the ventral, dorsal or lateral serosal surface. Alternatively, bladders will be opened and mounted as sheets on a sliding rack that allows us to apply uniform stretch, measure developed tension and acquire optical signals from the entire luminal surface. We will record spontaneous activity, and activity evoked through stimulation of the major pelvic ganglion or direct bipolar stimulation of the serosal or luminal surfaces. In preliminary studies using neonatal bladders, pacemaker activity originated at a single site near the bladder dome and initiated large amplitude (15-30 cm H20) contractions. By contrast, in normal adult bladders, spontaneous activity originated simultaneously at multiple sites and was of low amplitude (2-5 cm H20). In obstructed hypertrophied adult bladders however, focal pacemaker activity re-emerged initiating large amplitude coordinated contractions. Simultaneous blockade of the effects of acetylcholine, ATP, substance P, nitric oxide and alpha- and beta-adrenergic agonists altered but did not completely block spontaneous activity in neonatal, adult or obstructed bladders. However, gap junction blockade (10 mu/M 18beta-glycyrrhetinic acid) reversibly abolished spontaneous activity in neonatal and obstructed bladders, but not normal adult bladders. This suggests that gap junction connectivity is necessary for focally initiated large amplitude spontaneous activity that spreads in a coordinated manner throughout the neonate's bladder, and that this connectivity is lost during development and re-emerges following obstruction. Accordingly, our specific aims are: I.) To investigate the postnatal modulation of intrinsic bladder activity; II.) To study the neural-chemical control of spontaneous detrusor activity; and III.) To determine the origin of detrusor overactivity in partially obstructed bladders.
