Co-cultures provide a new tool to probe communication between adult sensory neurons and urothelium.
OBJECTIVE
Recent evidence suggests that the urothelium functions as a sensory transducer of chemical, mechanical or thermal stimuli and 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. This led us to establish a method of co-culturing dorsal root ganglion sensory neurons and bladder urothelial cells.


METHODS
Sensory neurons and urothelial cells obtained from dorsal root ganglia and bladders dissected from adult female Sprague-Dawley® rats were isolated by enzyme treatment and mechanical dissociation. They were plated together or separately on collagen coated substrate and cultured in keratinocyte medium for 48 to 72 hours. 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 that in neuronal monocultures. The growth promoting effect of urothelial cells was reduced by the tyrosine kinase inhibitor K252a but upstream inhibition of nerve growth factor signaling with TrkA-Fc had no effect. Fura-2 calcium imaging of urothelial cells showed responses to adenosine triphosphate (100 μM) and activation of TRPV4 (4α-PDD, 10 μM) but not TRPV1 (capsaicin, 1 μM), TRPV3 (farnesyl pyrophosphate, 1 μM) or TRPA1 (mustard oil, 100 μM). 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. Results suggest that neuronal properties are maintained in the presence of urothelium and neurite growth is potentiated by a nerve growth factor independent mechanism.
Pain Management Research Institute and Kolling Institute, University of Sydney at Royal North Shore Hospital, St. Leonards, New South Wales, Australia.
Lauren M
LM
O'Mullane
Janet R
JR
Keast
Peregrine B
PB
Osborne
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D002211:Capsaicin / Q000494:pharmacology; D002227:Carbazoles / Q000494:pharmacology; D018920:Coculture Techniques; D013759:Dronabinol / Q000494:pharmacology; D005260:Female; D016257:Fura-2 / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000378:metabolism*; D007150:Immunohistochemistry; D026121:Indole Alkaloids / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D008610:Menthol / Q000494:pharmacology; D023421:Models, Animal; D020932:Nerve Growth Factor / Q000494:pharmacology; D010590:Phalloidine / Q000494:pharmacology; D011106:Polyisoprenyl Phosphates / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011984:Sensory Receptor Cells / Q000378:metabolism*; D012717:Sesquiterpenes / Q000494:pharmacology; D050916:TRPV Cation Channels / Q000096:biosynthesis; D019459:Urothelium / Q000166:cytology*
D002227:Carbazoles; D026121:Indole Alkaloids; D007211:Indoles; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D050916:TRPV Cation Channels; D008610:Menthol; D010590:Phalloidine; C007293:DAPI; C004808:farnesyl pyrophosphate; D013759:Dronabinol; D000255:Adenosine Triphosphate; D020932:Nerve Growth Factor; C049985:staurosporine aglycone; D002211:Capsaicin; D016257:Fura-2
4′,6-diamidino-2-phenylindole dihydrochloride; ATP; CGRP; DAPI; DRG; FPP; Fura-2; KSFM; MEM; NGF; TRP; Tuj1; [Ca(2+)](in); adenosine triphosphate; calcitonin gene-related peptide; coculture techniques; dorsal root ganglion; farnesyl pyrophosphate; fura-2-acetoxymethyl ester; ganglia; intracellular calcium; keratinocyte serum-free medium; minimum essential medium; nerve growth factor; sensory receptor cells; spinal; transient receptor potential; urinary bladder; urothelium; β-tubulin isotype III