The Function And Preliminary Mechanism Of Neural Activity Of PMC Neurons In Urination

Urine release?urination or micturition?serves the physiological purpose of removing metabolic waste and maintaining fluid balance of an organism.Normal bladder function includes a storage phase and a voiding or micturition phase,which are tightly controlled by a highly complex neural network integrated at different levels of the central and peripheral nervous systems.Disruption or diseases in any aspects of this control may cause neurogenic bladder dysfunction?NGB?.Neurogenic bladder dysfunction can be found in many patients with neurological disorders,such as multiple sclerosis,Parkinson's disease,spinal cord injury and spina bifida.NBG can lead to problematic symptoms and complications including urinary incontinence,frequency,and urgency,along with risk for infection of the upper urinary tract.However,our current insights into NGB are very limited and its pathogenesis and pathophysiological process need further study.A better understanding of the neural mechanisms controlling bladder would provide new strategies for the treatment of NGB.Identifying circuits linking the brain and bladder is important for elucidating mechianisms by which the brain regulates bladder function and its role in bladder pathology.A recent advance in neuroanatomical tracing techniques involving the use of pseudorabies virus?PRV?,an alpha herpes virus,has permitted extensive examination of neuronal pathways across synapses and has the potential to reveal pathways that connect various peripheral tissue structures to the brain.Furthermore,the best strategy to uncover the neural mechanism of controlling bladder and urination is to explore the neural coding pattern during the urination and bladder contraction.The development of genetically encoded Ca²⁺indicators?GECIs?and genetically targeted techniques has simplified the use of optical fiber-based Ca²⁺recording?also termed fiber photometry?.Fiber photometry is currently a popular choice for the monitoring of cell type-specific neuronal activities of any brain areas in freely behaving animals.This approach is suitable for the detection of the average spiking activity of a cluster of neurons,and the general consensus is that neuronal Ca²⁺transients reflect neuronal action potential firing.Fiber photometry costs less,and data acquisition and analyses are much simpler than electrophysiology.The purpose of our work is to identify neural circuits linking the brain and bladder,and to reveal the characteristics and mechanism of neural activity of pontine micturition center?PMC?neurons in urination and bladder contraction.We mainly performed the following experiments:?1?To identify the distribution of neurons in the spinal cord and peripheral nerves that participate in bladder control,we injected PRV directly into the bladder wall of adult male mouse.At 4.5-5 d after PRV injection,we observed the distribution of PRV-EGFP labeled cells in the spinal cord sections or bladder sections.We also observed the distribution of PRV-EGFP labeled cells in the spinal cord and bladder using whole-brain sectioning and three-dimensional?3D?reconstruction based on fluorescence micro-optical sectioning tomography?fMOST?.?2?To identify the distribution of neurons in the brain that participate in bladder control,we injected PRV-EGFP directly into the bladder wall of each adult male mouse.At4.5-5d after PRV injection,we observed the distribution of PRV-EGFP labeled cells in the brain sections.We also observed the distribution of PRV-EGFP labeled cells in the brain using whole-brain sectioning and 3D reconstruction based on fMOST.To determine whether the distribution of neurons in the brain involved in bladder control is same or not between male and female mice,we injected PRV-EGFP into the bladder walls of female and male mice respectively.To determine whether the distribution of neurons in the brain involved in bladder control is same or not after bladder was infected by PRV-EGFP at different time points?3d,4d and 4.5-5d?,we obtained brain tissues from different mice respectively.To determine whether the different brain regions could control the different parts of the bladder,we simultaneously injected PRV-EGFP and PRV-RFP separately into the left and right bladder walls of the same mouse.?3?To identify the accurate location of the cortical areas involved in bladder control,we observed the specific location of the cortical regions labeled by PRV-EGFP using whole-brain sectioning and 3D reconstruction based on fMOST.We also observed the specific location of the cortical regions labeled by PRV-EGFP using a scanning confocal microscope.?4?To identify the accurate location of the PMC involved in bladder control,the morphological results obtained by the use of a scanning confocal microscope were compared with the classical mouse brain atlas.To identify the upstream brain regions of the PMC,we injected AAV-Retro-hSyn-EYFP into the PMC.After one month,the mouse brains were sectioned.Then we examined the distribution of EGFP-positive neurons in the brain using of a scanning confocal microscope.?5?To investigate whether the activation of PMC neurons coincides with urination in freely moving mice,we monitored the Ca²⁺activities of PMC neurons by using the optical fiber recording system and urination events simultaneously.?6?To test whether population Ca²⁺activity in the PMC are in fact required for urination,we bilaterally injected muscimol?5?g/?l?locally to suppress neuronal activities in the PMC.Then we examined the changes of urination behavior.?7?To determine if Ca²⁺transients of PMC neurons correlate with bladder detrusor contraction,we simultaneously monitored population Ca²⁺transients of PMC GCaMP6f-positive neurons and urinary bladder pressure in freely moving mice or in anesthetized mice.?8?To test whether population Ca²⁺activity in the PMC are in fact required for bladder detrusor contraction,we simultaneously monitored population Ca²⁺transients of PMC GCaMP6f-positive neurons and urinary bladder pressure in different kinds of anesthetized mice?2.5%isoflurane and urethane?.Neuronal activities in the PMC were blocked by 2.5%isoflurane and then we examined the changes in the bladder detrusor contraction.The main results of our study:?1?The distribution of PRV-EGFP⁺neurons in the spinal cord and peripheral nervesThe nerve fibers in the bladder were labeled by PRV-EGFP.These nerve fibers may originate from neurons in the peripheral ganglia.The majority of PRV-EGFP⁺neurons were found in the L6-S1 within the intermediolateral area?IML?,the dorsal gray commissure?DGC?and the superficial dorsal horn?SDH?.The sacral parasympathetic preganglionic neurons?SPN?located in IML can also be infected by PRV-EGFP.In L1-L2,PRV-EGFP⁺neurons were mainly distributed in the medial lateral column area?IML?and dorsal gray commissure?DGC?,while PRV-EGFP⁺neurons were rarely found in cervical spinal cord?n=5 male mice?.?2?The distribution of PRV-EGFP⁺neurons in the mouse brainPRV-EGFP⁺neurons were mainly distributed in the PMC,gigantocellular reticular nucleus?Gi?,Raphe nuclei,locus coeruleus?LC?,A5 noradrenaline cells,periaqueductal gray?PAG?,Red nucleus,medial preoptic area?MPA?,paraventricular nucleus?PVN?,lateral hypothalamic area?LH?and cortical areas?n=5 male mice?.By contrast,no PRV-EGFP labeled neurons were observed in these regions in control animals with the same PRV injected into the abdominal muscle?n=3 male mice?.Using fMOST and 3D reconstruction method,we have made a whole 3D brain network.Notably,simultaneous injections of PRV encoding EGFP and RFP separately into the left and right bladder walls labeled neurons in the same brain regions.The same brain regions were infected by PRV-EGFP after PRV injection into the female and male mice?n=3 for each sex?.3d after PRV-EGFP injection,only a small number of PRV-EGFP⁺neurons were found in the PMC and Gi regions?n=3 mice?.4d after PRV-EGFP injection,PRV-EGFP⁺neurons were found in all the above brain regions?n=3 mice?.?3?The distribution of PRV-EGFP⁺neurons in the cerebral cortexAt 4.5–5d after PRV injection,we consistently observed a small cluster of PRV-EGFP⁺cortical neurons bilaterally in a specific region that corresponds to primary motor cortex?M1?and to the somatosensory cortex?S1?.Three-dimensional reconstruction and cell counting revealed that the labeled cortical area covered⁹60?m along the anteroposterior axis and contained⁹70 PRV-EGFP⁺neurons?327±9 EGFP⁺neurons in the M1 area and643±20 PRV-EGFP⁺neurons in the S1 area;n=3 male mice?.Moreover,the vast majority of the PRV-EGFP⁺neurons in both S1 and M1?⁹7%?resided in L5 and were projecting pyramidal neurons based on their morphology.At 4d after PRV injection we observed PRV-EGFP⁺neurons in M1 and S1?n=3 mice?.These results suggest that the cortex is the upstream brain region of PMC.?4?The precise localization of PRV-EGFP⁺neurons in the PMC and the distribution of PMC upstream brain regionsThe coordinates for PMC was AP:-5.45 mm;ML:±0.7 mm;DV:-3.15 mm.The upstream brain regions of the PMC included PAG,M1,S1,PVN,LH and prefrontal cortex,in which neurons could be directly projected to the PMC?n=3 mice?.?5?Population Ca²⁺transients of PMC neurons highly correlate with urination events in freely moving miceTo increase the number of urination events,each mouse was treated with an intraperitoneal?i.p.?injection of 2 ml saline or diuretic furosemide?40 mg/kg?prior to recording.A representative example revealed that Ca²⁺transients of PMC neurons were observed in the GCaMP6f-injected group during urination.These urination-related Ca²⁺transients were not observed in the EGFP-injected control group?GCaMP6f-injected group,max?f/f=36.73%±4.90%;EGFP-injected group,max?f/f=2.24%±0.66%;P<0.001?.These experiments indicated that the Ca²⁺transients of the PMC in the GCaMP6f-injected group were not movement artefacts?n=7 mice for each group?.A closer analysis showed that Ca²⁺transients of PMC neurons in the GCaMP6f-injected group started 402±21 ms prior to the onset of urination events.We found that each urination event was 100%associated with Ca²⁺transients in the GCaMP6f-injected group across all the recordings?P<0.001?.Ca²⁺transients of PMC GCaMP6f-positive neurons started before urination onset on average and in single urination events,and this response was absent in shuffled data.Further analyses indicated that the duration of urination events linearly correlated to the full width at the half maximum of Ca²⁺transients.?6?Silencing PMC neurons impairs the urination behaviorTo increase the number of urination events,each mouse was treated with an intraperitoneal?i.p.?injection of 2 ml saline prior to behavior testing.Mice in the experimental group?muscimol-injected group,n=9 mice?were impaired in urination,as shown by reduced urination events?P<0.01?,a delayed onset of urination?P<0.01?,an increased latency to the first urination event?P<0.01?,a decreased volume of single urine deposited?P<0.01?and a decreased total volume of urine deposited?P<0.01?compared to the controls?without muscimol group,n=6 mice;ACSF-injected group,n=6 mice?in the period observed?4h?.Some mice in the experimental group showed acute overflow incontinence,while others showed no micturition.?7?Population Ca²⁺transients of PMC neurons highly correlate with bladder detrusor contraction in freely moving mice or anesthetized miceSimultaneous measurement of neuronal activity and cystometry showed that each spike-like increase in bladder pressure correlated with Ca²⁺transients of PMC neurons in freely moving mice and urethane-anesthetized mice.The onset of Ca²⁺transients preceded the onset of urination and were maintained throughout the entire process of each urination event.We found that each urination cycle was also 100%associated with Ca²⁺transients of PMC GCaMP6f-positive neurons.Cross-correlation of Ca²⁺transients recorded in the PMC and bladder contraction showed that the correlation coefficient of the mean peak value was higher than the shuffled data?n=8 mice;data,0.51±0.08;shuffled,0.09±0.03;P<0.01?.?8?Silencing PMC neurons impairs mouse bladder detrusor contraction.After the neural activity of PMC neurons was blocked by deep gas anesthesia?2.5%isoflurane?,population Ca²⁺transients of PMC neurons were completely absent,and the regular urination cycles were not detected due to dripping overflow incontinence.In the control group?urethane-anesthetized condition?,the neural activity of PMC neurons was not blocked,and each spike-like increase in bladder pressure also could be detected?n=4 mice for each group?.Taken together,the main conclusions of this study are as follows:the brain,spinal cord and peripheral nerves constitute a complex neural network.This neural network is involved in controlling the bladder detrusor.The PMC has multiple upstream brain regions,such as cortex,PAG and LH.Neuronal activities in the PMC highly correlate with urination.Population Ca²⁺transients of PMC neurons correlate with spike-like increases in bladder pressure in freely moving mice or urethane-anesthetized mice.Activation of PMC neurons is the basic condition to initiate urination or bladder detrusor contraction.