Functional Characterisation of Putative Mechanoreceptive Neurones in the Colorectum

结直肠中假定的机械感受神经元的功能特征

• 批准号: BB/R006210/1
• 负责人: Ewan Smith
• 金额: $ 53.47万
• 依托单位: University of Cambridge
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2018
• 资助国家: 英国
• 起止时间: 2018 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FR006210%2F1
• 关键词: Functional; Characterisation; Putative; Mechanoreceptive; Neurones

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

有意识地控制排便是大多数动物的重要生理功能;在捕食中传达生存利益，并且对于领土标记至关重要。需要有效感知肠道（结直肠）的充盈/冲动，以有意识地抑制反射性排便，直到它有利于生存，或者像人类一样，它是社会可接受的。感觉神经元传导粪便通过结肠直肠的机械性扩张。随着年龄的增长，肠功能受损，便秘和失禁的发生率增加，同时结肠直肠敏感性降低。多种类型的感觉神经元支配结直肠，但目前尚不清楚这些人群中的哪一个负责与感觉饱腹感和排便冲动有关的低阈值（即不疼痛）机械感觉，以及它们的敏感性如何随年龄变化。使用单细胞RNA测序，我们已经确定了两个群体的感觉神经元支配结肠直肠，一个表达机械敏感离子通道Piezo 2在高水平。我们假设Piezo 2阳性（Piezo 2+）人群是健康的关键低阈值机械传感器，并且他们的功能随着年龄的增长而下降，导致排便障碍。我们将使用遗传学，生理学和行为学的组合来确定Piezo 2和Piezo 2+神经元对结直肠机械敏感性和排便的贡献。通过使用感觉神经元中缺乏Piezo 2特异性的小鼠（Piezo 2CKO），我们将确定该离子通道在24个月大的结肠直肠机械敏感性中的作用。此外，将在相当的年龄使用其中Piezo 2+神经元表达GFP/Cre重组酶（Piezo 2GFP）的小鼠来选择性地可视化、调节和消融该神经元群体。我们假设野生型小鼠随年龄增长会表现出排便频率降低和珠粒排出延迟增加，这两种情况在Piezo 2CKO小鼠中均会加剧，因为这一关键神经元亚群的结肠直肠机械敏感性降低。为了直接测量结直肠机械敏感性，我们将使用离体结肠神经制备物，并对结直肠扩张和探针/拉伸刺激进行电生理记录。为了证实Piezo 2+神经元的作用，在通过结肠直肠注射Cre依赖性白喉毒素A进行选择性神经元消融后，将在Piezo 2GFP小鼠中进行类似的实验。为了确定Piezo 2+神经元在衰老过程中的损失，我们将逆行标记Piezo 2GFP小鼠的结肠直肠感觉神经元。为了充分说明Piezo 2+结肠直肠感觉神经元如何促进结肠直肠敏感性和排便，我们将另外使用化学和光遗传学方法。首先，使用由设计者药物hM 3Dq专门激活的Cre依赖性设计者受体，我们将通过注射hM 3Dq激动剂氯氮平-N-氧化物来选择性地激发Piezo 2GFP小鼠中的Piezo 2+结肠直肠神经元。通过结合行为（排便频率/珠排出潜伏期）与体内（通过内脏反射（VMR）结直肠扩张）和体外（结肠神经电生理学）的结直肠敏感性测量，我们将研究这些结直肠感觉通路的激活如何影响排便功能。最后，将Piezo 2GFP小鼠与表达Cre依赖性通道视紫红质的小鼠杂交，并将光纤电缆植入后代的腰骶脊柱中，使得能够在自由移动的动物中选择性激活结肠直肠感觉神经元。我们将确定这种激活如何调节排便频率，珠驱逐潜伏期和VMR.In总结，我们将确定如何Piezo 2和Piezo 2+神经元subserve低阈值结直肠机械敏感性，并有助于排便。重要的是，这项研究将揭示Piezo 2+神经元功能的变化如何导致年龄相关的结直肠机械敏感性下降，这可能是老年人肠功能受损的基础。

项目成果

Peripheral mechanisms of arthritic pain: A proposal to leverage large animals for in vitro studies.

关节炎疼痛的外周机制：利用大型动物进行体外研究的建议。

• DOI: 10.17863/cam.55596
• 发表时间: 2020
• 作者: Chakrabarti S

Functional characterization of ovine dorsal root ganglion neurons reveals peripheral sensitization after osteochondral defect

绵羊背根神经节神经元的功能特征揭示骨软骨缺损后的外周敏化

• DOI: 10.1101/2021.02.26.432434
• 发表时间: 2021
• 作者: Chakrabarti S

In silico screening of GMQ-like compounds reveals guanabenz and sephin1 as new allosteric modulators of acid-sensing ion channel 3

• DOI: 10.1101/704445
• 发表时间: 2019-07
• 期刊: Biochemical Pharmacology
• 影响因子: 5.8
• 作者: Gerard Callejo;Luke A. Pattison;J. Greenhalgh;Sampurna Chakrabarti;Evangelia Andreopoulou;James R. F. Hockley;E. Smith;Taufiq Rahman