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423: Novel model of micturition based on a multi-unit recordings of optogenetically-identified Barrington’s CRH neurons in mice

Speaker
Dr. H. (Hiroki) Ito
Authors
Ito H.1, Sales A.2, Drake M.J.2, Pickering A.E.2
Institutions
1Yokosuka Kyosai Hospital, Dept. of Urology, Kanagawa, Japan, 2University of Bristol, School of Physiology, Pharmacology and Neuroscience, Bristol, United Kingdom
Event
35th Annual EAU Congress - Virtual (EAU20V)
Date – Time - Location
18 July 2020, 16:30 - 17:00, Virtual room 2
Session
Poster Session - Best abstract session: Non-oncology
Topic
Benign Lower Urinary Tract Diseases, Neurogenic bladder - Basic Research & Trials, Urodynamics

Introduction & Objectives

Based on our multiunit recordings of optogenetically-identified BarrCRH neurons we hypothesised that they provided a trigger signal to a primed downstream parasympathetic motor circuit at the spinal level. To address our hypothesis, we optoactivated BarrCRH neurons at a spinal level and developed a novel neuronal model of micturition.

Materials & Methods

To target Barrington’s nucleus in CRH-ires-CRE mice, stereotaxic injections of Cre-inducible vector (AAV-EF1a-DIO-hChR2-mCherry) allowed expression of Channelrhodopsin2 for opto-activation. Under urethane anaesthesia, an optic fibre was placed above Barrington’s and recordings from the Barr-CRH neurons used a 32 channel silicon probe and an open-ephys system. Spike waveforms were clustered in Kilosort and verified in Phy followed by analysis in MATLAB. Bladder pressure and external urethral sphincter activity were recorded while 473nm light was applied above the spinal cord. The model of bladder parasympathetic preganglionic neuronal ‘priming’ was based on an existing preganglionic neuronal model (Briant LJ, et al. J Neurophysiol 2014) constructed within NEURON. The synaptic drive from Barrington’s was modelled by adding a point synapse to the soma which was ‘optogenetically’ driven with trains of action potentials (20Hz x 1 sec). This synapse was subthreshold for action potential generation despite a modest degree of summation (~20%) when driven at 20Hz. A second fast excitatory synaptic drive to model the bladder afferent input was added to a proximal medial dendrite. This was driven with an incrementing frequency of action potentials to model bladder distension derived from recordings of pelvic nerve afferents (Ito H, et al. BJU Int. 2019).

Results

In total 113 neurons were recorded in 3 mice and 12 units were optogenetically-identified as Barr-CRH neurons. A burst of firing in the BarrCRH neurons preceded spontaneous NVC (sNVC) and voiding (20.5 ± 4.1 Hz peak firing frequency) by around 2.5-3 s – suggesting that they were triggered by a signal from the pons. Optogenetic stimuli applied to the BarrCRH axons in the spinal cord reliably induced bladder contractions or full voids. The BarrCRH – parasympathetic - bladder afferent model showed the variation in parasympathetic excitability can result from the incrementing frequency of summating afferent drive as the bladder fills. The resulting output from the parasympathetic neuron when driven by BarrCRH can thus be seen to be strongly dependent upon the phase of the bladder cycle with a ~10-fold increase in gain over the voiding cycle. This mimics the experimental findings that optogenetic stimulus applied at different phases of the cycle can trigger either no response or eNVCs or full voiding contractions.

Conclusions

The BarrCRH neurons can evoke both voiding and eNVC through their spinal projections and novel neural model closely parallels the experimental data.

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