Functional Characterisation of Putative Mechanoreceptive Neurones in the Colorectum

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Conscious control of defecation is an important physiological function for most animals; conveying survival benefits in predation and is essential for territorial marking. Effective sensing of bowel (colorectal) fullness/urge is required to consciously inhibit reflex defecation until it is beneficial to survival, or as in humans, it is socially acceptable. Mechanical distension of the colorectum by passage of faecal matter is transduced by sensory neurones. In ageing, bowel function is impaired with increased rates of constipation and incontinence, concurrent with reduced colorectal sensitivity. Multiple types of sensory neurone innervate the colorectum, but it is unclear which of these populations is responsible for low-threshold (i.e. not painful) mechanosensation linked to sensing fullness and urge to defecate, and how their sensitivity changes in age. Using single-cell RNA-sequencing, we have identified two populations of sensory neurones innervating the colorectum, one expressing the mechanosensitive ion channel Piezo2 at high levels. We hypothesise that the Piezo2-positive (Piezo2+) population are key low-threshold mechanosensors in health and that their function declines with age resulting in impaired defecation.We will use a combination of genetics, physiology and behaviour to determine the contribution of Piezo2 and Piezo2+ neurones to colorectal mechanosensitivity and defecation in age. By using mice lacking Piezo2 specifically in sensory neurones (Piezo2CKO), we will ascertain the role of this ion channel in mechanosensitivity of the colorectum at ages up to 24 months. In addition, mice in which Piezo2+ neurones express GFP/Cre-recombinase (Piezo2GFP) will be used at comparable ages to selectively visualise, modulate and ablate this population of neurones. We hypothesise that wild-type mice will show decreased stool frequency and increased bead expulsion latencies in age, which will both be exacerbated in Piezo2CKO mice due to diminished colorectal mechanosensitivity in this critical neuronal subset. To directly measure colorectal mechanosensitivity, we will use an ex vivo colon-nerve preparation and make electrophysiological recordings in response to colorectal distension and probe/stretch stimuli. To substantiate a role for Piezo2+ neurones, similar experiments will be conducted in Piezo2GFP mice after selective neuronal ablation by colorectal injection of Cre-dependent diphtheria toxin A. To determine Piezo2+ neuronal loss during ageing, we will retrograde label colorectal sensory neurones in Piezo2GFP mice.To fully address how Piezo2+ colorectal sensory neurones contribute to colorectal sensitivity and defecation we will additionally use chemo- and optogenetic methods. Firstly, using Cre-dependent Designer Receptor Exclusively Activated by Designer Drug hM3Dq, we will selectively excite Piezo2+ colorectal neurones in Piezo2GFP mice by injection of the hM3Dq agonist clozapine-N-oxide. By combining behaviours (stool frequency/bead expulsion latency) with measurements of colorectal sensitivity both in vivo (via visceromotor reflex (VMR) to colorectal distension) and ex vivo (colon-nerve electrophysiology), we will investigate how activation of these colorectal sensory pathways impacts defecatory function. Lastly, Piezo2GFP mice will be crossed with mice expressing Cre-dependent channel rhodopsin and a fibre optic cable implanted into the lumbosacral spinal column of the offspring enabling the selective activation of colorectal sensory neurones in freely moving animals. We will determine how this activation modulates stool frequency, bead expulsion latency and VMR.In summary, we will determine how Piezo2 and Piezo2+ neurones subserve low-threshold colorectal mechanosensitivity and contribute to defecation. Importantly, this study will reveal how changes in Piezo2+ neuronal function contribute to ageing-related decline in colorectal mechanosensitivity that may underpin impaired bowel function in geriatric