Parasympathetic nerve fibers control the contraction of the bladder musculature. Only a third of the contraction induced by their general activation through EFS is inhibited by antagonists of muscarinic ACh receptors. The remaining contraction is predominantly mediated via purinergic receptors. However, it is unclear whether purinergic ligands are released together with ACh from the same nerve fibers, or from a separate fiber population. We have clarified this question in a transgenic animal model with an optogenetic approach. Here, a light-sensitive channel, ChR2, is expressed in cholinergic neurons so that they can be selectively excited by light at a wavelength of 460 nm.A mouse line expressing a ChR2-dtTomato fusion protein under control of the ChAT promotor was generated by crossing the strains B6N.129S6(B6)-Chattm2(cre)Lowl/J and B6;129-Gt(ROSA)26Sortm1(CAG-COP4*E123T*H134R,-tdTomato)Gfng/J. ChR2-dtTomato expression in the urinary bladder and reference organs was characterized by immunofluorescence microscopy. Detrusor contractions were measured in an organ bath in response to EFS and LED (460 nm) stimulation. As a control group, we used a ChAT-eGFP mouse strain that does not express ChR2. Immunofluorescence microscopy revealed expression of the ChR2-dtTomato fusion protein in cholinergic (ChAT- and VAChT-positive), but not other (noradrenergic, peptidergic sensory) nerve fibers in the urinary bladder and other peripheral organs (e.g. airways and duodenum). EFS led to a frequency-dependent contraction in all strains. LED stimulation evoked a contraction of the urinary bladder only in the ChR2-expressing mouse strain so that thermal effects were excluded. Atropine completely inhibited the muscarine-induced contraction of the bladder but reduced the LED-evoked contraction of the detrusor only by 35%. The application of purinergic receptors antagonists, suramin and PPADS, reduced the LED-induced contraction of the detrusor by 60%. Even after combined cholinergic muscarinic and purinergic inhibition, approximately 20% of the LED-induced contraction remained.For the first time, this optogenetic approach proves cholinergic-purinergic cotransmission in the detrusor by releasing at least two transmitters from the same nerve fiber. The remaining contraction after combined blockade of muscarinic cholinergic and purinergic (P2) receptors may result from a minor ectopic expression of ChR2 in the detrusor muscle or another transmitter system, which can be clarified by further investigations using this model.
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