Co-Cultures Provide a New Tool to Probe Communication Between Adult Sensory Neurons and Urothelium

Abstract

Purpose Recent evidence suggests the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli that signals to nerve terminals and other cells in the bladder wall. The cellular and molecular basis of neuro-urothelial communication is not easily studied in the intact bladder, which led us to establish a method of co-culturing dorsal root ganglion (DRG) sensory neurons and bladder urothelial cells. Materials and Methods Sensory neurons and urothelial cells obtained from DRG and bladders dissected from adult female Sprague-Dawley rats were isolated by enzyme treatment and mechanical dissociation. These were plated together, or separately, on collagen-coated substrate and cultured in keratinocyte medium for 48–72 h. Retrograde tracer labeling was performed to identify bladder afferents used for functional testing. Results Neurite growth and complexity in neurons co-cultured with urothelial cells was increased relative to neuronal monocultures. The growth-promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor, K252a, but upstream inhibition of NGF signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to ATP (100μM) and activation of TRPV4 (4alpha-PDD, 10μM), but not TRPV1 (capsaicin, 1μM), TRPV3 (farnesyl pyrophosphate, 1μM), or TRPA1 (mustard oil, 100μM). In contrast, co-cultured neurons were activated by all agonists except farnesyl pyrophosphate. Conclusions Co-culturing provides a new methodology for investigating neuro-urothelial interactions in animal models of urological conditions. Our results suggest that neuronal properties are not only maintained in the presence of urothelium but that neurite growth is potentiated by an NGF-independent mechanism.

A Novel Small Molecule GDNF Receptor RET Agonist, BT13, Promotes Neurite Growth from Sensory Neurons in Vitro and Attenuates Experimental Neuropathy in the Rat

“…Cultured lumbar DRG (L3–L5) neurons were prepared using a published method (O'Mullane et al, 2013 ). After plating onto poly-L-ornithine (5 μg/ml)/laminin (5 μg/ml) coated coverslips for 1 h, DRG neurons were cultured for 24–30 h in fresh Neurobasal A/B27.…”

A biophysically detailed computational model of urinary bladder small DRG neuron soma

“…Fig 11 compares the normalized Ca 2+ transient evoked by application of 30 mM [K] o in bladder small DRG neurons generated in experiments [ 111 ] and by our model. Benham et.…”

“… (A) Comparison of normalized Ca 2+ transient obtained from bladder small DRG neurons experimentally (circles, Expt. Calcium Transient, [ 111 ] obtained from fluorescence studies) and our model (Solid line, Sim. Calcium Transient) by application of high [K] o (30 mM) for 50 seconds (indicated by bar).…”

“…For [ Ca ] i validation where normalized simulated [ Ca ] i was compared with that from imaging data [ 111 ] using 30 mM [K] o , Fura-2 was used as an imaging dye with parameters [ B ] tot = 5 μ M [ 111 ], K B = 224 nM [ 112 ] and D dye = 0.1 μ m 2 /s (for free dye diffusion) [ 113 ]. β dye was calculated by Eq 38 .…”

Transient receptor potential channel modulators as pharmacological treatments for lower urinary tract symptoms (LUTS): myth or reality?

“…Initial reports described TRPV1 expression and functional TRPV1 responses in isolated urothelial cells from mice , rats and humans . In contrast, an increasing number of groups have more recently reported the absence of TRPV1 expression and functional TRPV1 responses in guinea‐pig , mouse and human urothelial cells. Moreover, a recent approach using Trpv1 ‐reporter mice, suggested that TRPV1 expression was restricted to primary afferent neurones, with no detectable TRPV1 expression in urothelial cells .…”