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The present study explores the possible involvement of a purinergic mechanism in mechanosensory transduction in the bladder using P2X(3) receptor knock-out (P2X(3)(−)(/−)) and wild-type control (P2X(3)(+/+)) mice. Immunohistochemistry revealed abundant nerve fibers in a suburothelial plexus in the mouse bladder that are immunoreactive to anti-P2X(3). P2X(3)-positive staining was completely absent in the subepithelial plexus of the P2X(3)(−)(/−) mice, whereas staining for calcitonin gene-related peptide and vanilloid receptor 1 receptors remained. Using a novel superfused mouse bladder–pelvic nerve preparation, we detected a release of ATP proportional to the extent of bladder distension in both P2X(3)(+/+) and P2X(3)(−)(/−) mice, although P2X(3)(−)(/−)bladder had an increased capacity compared with that of the P2X(3)(+/+) bladder. The activity of multifiber pelvic nerve afferents increased progressively during gradual bladder distension (at a rate of 0.1 ml/min). However, the bladder afferents from P2X(3)(−)(/−) mice showed an attenuated response to bladder distension. Mouse bladder afferents of P2X(3)(+/+), but not P2X(3)(−)(/−), were rapidly activated by intravesical injections of P2X agonists (ATP or α,β-methylene ATP) and subsequently showed an augmented response to bladder distension. By contrast, P2X antagonists [2′,3′-O-(2,4,6-trinitrophenyl)-ATP and pyridoxal 5-phosphate 6-azophenyl-2′,4′-disulfonic acid] and capsaicin attenuated distension-induced discharges in bladder afferents. These data strongly suggest a major sensory role for urothelially released ATP acting via P2X(3) receptors on a subpopulation of pelvic afferent fibers.

Mutant mice with a hypersensitive serotonin (5-HT)3Areceptor were generated through targeted exon replacement. A valine to serine mutation (V13′S) in the channel-lining M2 domain of the 5-HT3Areceptor subunit rendered the 5-HT3receptor ∼70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT3Bsubunit. Mice homozygous for the mutant allele (5-HT3Avs/vs) had decreased levels of 5-HT3AmRNA. Measurements on sympathetic ganglion cells in these mice showed that whole-cell serotonin responses were reduced, and that the remaining 5-HT3receptors were hypersensitive. Male 5-HT3Avs/vsmice died at 2-3 months of age, and heterozygous (5-HT3Avs/+) males and homozygous mutant females died at 4-6 months of age from an obstructive uropathy. Both male and female 5-HT3Amutant mice had urinary bladder mucosal and smooth muscle hyperplasia and hypertrophy, whereas male mutant mice had additional prostatic smooth muscle and urethral hyperplasia. 5-HT3Amutant mice had marked voiding dysfunction characterized by a loss of micturition contractions with overflow incontinence. Detrusor strips from 5-HT3Avs/vsmice failed to contract to neurogenic stimulation, despite overall normal responses to a cholinergic agonist, suggestive of altered neuronal signaling in mutant mouse bladders. Consistent with this hypothesis, decreased nerve fiber immunoreactivity was observed in the urinary bladders of 5-HT3Avs/vscompared with 5-HT3Awild-type (5-HT3A+/+) mice. These data suggest that persistent activation of the hypersensitive and constitutively active 5-HT3Areceptorin vivomay lead to excitotoxic neuronal cell death and functional changes in the urinary bladder, resulting in bladder hyperdistension, urinary retention, and overflow incontinence.

Inhibitory interneurons play important roles in neuronal circuits, but the synaptic mechanisms that regulate excitatory input onto interneurons remain to be fully understood. We show that ATP-gated presynaptic P2X2 channels facilitate excitatory transmission onto stratum radiatum interneurons but not onto CA1 pyramidal neurons. ATP released endogenously during carbachol-induced oscillations facilitates excitatory synapses onto interneurons. Overall, these data provide evidence for the molecular identity, synaptic function, and interneuron synapse specificity of a presynaptic neurotransmitter-gated cation channel. The findings highlight a novel form of presynaptic facilitation for hippocampal interneurons and suggest a role for extracellular ATP in neuronal networks.