Identification and characterization of rostral ventromedial medulla neurons synaptically connected to the urinary bladder afferents in female rats with or without neonatal cystitis

The neurons in the rostral ventromedial medulla (RVM) play a major role in pain modulation. We have previously shown that early-life noxious bladder stimuli in rats resulted in an overall spinal GABAergic disinhibition and a long-lasting bladder/colon sensitization when tested in adulthood. However, the neuromolecular alterations within RVM neurons in the pathophysiology of early life bladder inflammation have not been elucidated. In this study, we have identified and characterized RVM neurons that are synaptically linked to the bladder and colon and examined the effect of neonatal bladder inflammation on molecular expressions of these neurons. A transient bladder inflammation was induced by intravesicular instillation of protamine sulfate and zymosan during postnatal days 14 through 16 (P14-16) followed by pseudorabies virus PRV-152 and PRV-614 injections into the bladder and colon, respectively, on postnatal day P60. Tissues were examined 96 h postinoculation for serotonergic, GABAergic, and enkephalinergic expressions using in situ hybridization and/or immunohistochemistry techniques. The results revealed that > 50% of RVM neurons that are synaptically connected to the bladder (i.e., PRV-152+) were GABAergic, 40% enkephalinergic, and about 14% expressing serotonergic marker tryptophan hydroxylase 2 (TpH2). Neonatal cystitis resulted in a significant increase in converging neurons in RVM receiving dual synaptic inputs from the bladder and colon. In addition, neonatal cystitis significantly downregulated vesicular GABA transporter (VGAT) with a concomitant increase in TpH2 expression in bladder-linked RVM neurons, suggesting an alteration in supraspinal signaling. These alterations of synaptic connectivity and GABAergic/serotonergic expressions in RVM neurons may contribute to bladder pain modulation and cross-organ visceral sensitivity.