The central nervous system for urination is still unkouwn compared to the detailed elucidation of the peripheral nervous system. Recent human brain imaging studies have shown that ACC (Anterior cingulate cortex) is involved in urination. But compared to well-studied PAG and PMC the ACC mechanisms are not well elucidated. So, we studied how mouse ACC was involved in urination using new technology such as optogenetics.

We performed 4 experiments.
Experiment 1. We injected trans synaptic neuro-tracer WGA (Wheat germ agglutinin) into mouse bilateral ACC. The next day, we perfused the mouse cerebrum and bladder with 4% PFA and excised. 2 days after the operation, we confirmed distribution of WGA. 
Experiment 2. We used Thy1-ChR2 (Channelrhodopsins) transgenic mice whose pyramidal neurons in the 5th layer of cerebral cortex has ChR2. After craniotomy and fixing of light cannula (Fiber Optic Cannula, Ø2.5 mm Ceramic Ferrule, Ø400 µm Core, 0.39 NA, L=2 mm Thorlabs), we performed CMG (cystometrogram) experiments against this mouse under urethane anesthesia. After confirmation of stabilization in the intravesical pressure, we performed blue light stimulation (50Hz) to the bilateral ACC and monitored changes which evoked by light stimulation. We also stimulated ACC by 6 phase stimulation time (0.5s,1s,2s,5s,10s,20s) and compared to WT group data (N=6 mice).
Experiment 3. To confirm the effect of inhibitory neurons, we used AAV (Adeno-associated virus)-DIO (double floxed inverted open-reading-frame)-ChR2 which express ChR2 under existence of Cre protein. We injected this virus into both ACCs of PV(Parvalbumin)-Cre transgenic mice. In this mouse, there are Cre protein in PV neuron, which is one of the inhibitory neurons. This operation allowed ChR2 to be expressed only in ACC PV neurons. Under urethane anesthesia, the light-stimulation was performed for these mice while performing the CMG experiment. (N=6 mice)
Experiment 4. To confirm whether ACC's normal neural activity is involved in urination, we used Thy1-GCaMP transgenic mice. we inserted the light cannula in the vicinity of ACC and performed fiber photometry experiments and CMG experiment at the same time. (N=6 mice)

Experiment1. We confirmed WGA expression in M1 and PAG. WGA was also confirmed in the bladder.
Experiment2. Urination was induced by light-stimulation. Blue light-stimulation every 1 minute for 50 Hz for 20 seconds enabled precise control of voiding pressure. (figure.1)
In Thy1-ChR2 mice, we could see the bladder pressure elevation in all 6 phases stimulation time. At a stimulation time of 20 seconds, the effect of elevated bladder pressure reached a plateau.
Experiment3. The ICI (inter contraction interval) was extended by light stimulation.
Experiment4. We confirmed that the neural activity of ACC was activated at the time of urination.

We elucidated that there is a neural pathway link between ACC and M1, PAG and bladder with using neuro tracer. Thus, experiment 1 showed that stimulation against ACC neuron might affect bladder. Next, we performed selective neural stimulation with using optogenetics. Activation of ACC excitatory neurons induced urination in thy1 ChR2 mouse. In this mouse, light-stimulation controlled ICI accurately. Experiment 2 shows the possibility of being able to manipulate urination. On the other hand, activation of PV neurons in ACC suppressed urination. These results of two experiments may indicate that neural activity of ACC is closely related to urination and that the balance of the neural activity of excitatory neurons and inhibitory neurons in ACC is important for the control of urination. Experiment 2 and 3 were selective neural activation experiments. So, only these two experiments could not clarify the relationship between normal neuronal activity of ACC and urination. Thus, we added fiber photometry experiments. Fiber photometry experiments elucidated that normal neuronal activities of ACC are concerned with urination.

ACC neural activity plays an important role in urination.