The technology of optogenetics is useful for understanding the mechanism and developing novel treatments of neurological disorders. There are several reports exhibiting the therapeutic effects of optogenetics for lower urinary tract dysfunction (LUTD). However, their modulating targets were bladder muscle or peripheral nerve, and there is no evidence of optogenetic approach targeting central nervous system.
We already have revealed that the anterior cingulate cortex (ACC) regulates urination; activating excitatory neurons and interneurons induce and suppress urination respectively. In the present study, we evaluated the effect of photostimulation for interneurons in the ACC using urinary frequency model mice.

AAV-DIO-ChR2-EYFP were injected into the ACC of adult PV-Cre mice, resulting in the expression of excitatory opsin in the ACC PV neurons (one of inhibitory interneurons). One month after the virus injection, two operations were performed under urethane anesthesia: cystostomy and bilateral optic fiber implantation in the ACC (Figure 1A).
In cystometry recording, the perfusion was normal saline (control) or 0.1% acetic acid (urinary frequency model). After stabilization, photostimulation was conducted and intercontraction interval (ICI) was measured. In order to minimize a variation of ICIs, we collected 5 ICIs under each condition and took each average of those measurements (Figure1B). We normalized the ICI before manipulation to minimize differences of ICI baseline between individuals. We compared ICIs at pre-stimulation phase, stimulating phase, and post-stimulation phase using one-way ANOVA test (n=6 at each examination). We tested two patterns of stimulation strength; moderate stimulation was 60s burst duration and 90s interval, over stimulation was 120s burst duration and 60s interval.

Photostimulation for ChR2-induced PV-Cre mice prolonged ICI during stimulation phase only and ICI was recovered after photostimulation (stim; 1.23 ± 0.12, post stim; 0.96 ± 0.09) (Figure2A). Otherwise, photostimulation for WT mice demonstrated no significant difference between any phases (stim; 1.04 ± 0.13, post stim; 1.00 ± 0.15) (Figure2B). We identified adequate stimulation, making a comparison to over stimulation. Over stimulation prolonged ICI, but ICI didn’t recover even after stimulation (stim; 2.17 ± 0.91, post stim; 2.19 ± 0.63) (Figure2C). Bladder tension test in organ bath showed that over stimulated bladder had less contractile ability than moderate stimulated. In 0.1% acetic acid cystometry, photostimulation prolonged ICI during stimulation phase (stim; 1.31 ± 0.20, post stim; 1.07 ± 0.22) (Figure2D).

PV neuron selective photoactivation in the ACC succeeded in prolonging ICI. Applying optogenetics to treatment for LUTD have an advantage of temporal specificity, which would be useful for controlling immediate urgency. Too strong stimulation would cause concern of bladder distension. Changeability of stimulation power could avoid an adverse effect of bladder distension. 
In AA model, the ACC would receive signals from activated afferent nerves, and regulate micturition to void frequently. PV neurons activation in the ACC would have suppressed the frequent voiding signals. This is the first study that indicates therapeutic potential of optogenetics targeting central nervous system.

In the present study, we demonstrated that manipulating neural activity in the ACC could regulate micturition even in disease model with cell-type and temporal specificity. We believe that optogenetics could be one of effective therapies for refractory LUTD in the future.