eprintid: 10157063 rev_number: 12 eprint_status: archive userid: 699 dir: disk0/10/15/70/63 datestamp: 2022-12-15 12:11:14 lastmod: 2022-12-15 12:11:14 status_changed: 2022-12-15 12:11:14 type: thesis metadata_visibility: show sword_depositor: 699 creators_name: Pavón Arocas, Oriol title: Biophysical properties and gene expression profile of single periaqueductal gray neurons ispublished: unpub divisions: C08 divisions: D75 divisions: B02 divisions: UCL keywords: Periaqueductal Gray, Midbrain, Biophysics, Transcriptomics, single-cell RNA-sequencing, Patch-clamp recordings, Neuroscience note: Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request. abstract: The midbrain periaqueductal gray (PAG) is a longitudinal columnar structure where instinctive behaviours as diverse as escaping from predators, vocalising, and pup grooming segregate onto distinct anatomical subdivisions. This parallel between behaviour and brain circuit anatomy provides a unique opportunity for investigating how neural mechanisms support the computation of different adaptive actions. In this work, I aimed to characterise the biophysical properties and gene expression profile of single neurons across PAG subdivisions. First, I used loose-seal cell-attached and whole-cell patch-clamp recordings to characterise the biophysical properties of PAG neurons in acute midbrain slices of transgenic mice. I found that, even in the absence of synaptic inputs, GABAergic neurons defined by the expression of the VGAT promoter fire action potentials spontaneously, whereas glutamatergic neurons defined by the expression of the VGluT2 promoter are mostly silent. In addition, VGAT+ neurons had a higher input resistance and a lower action potential threshold than VGluT2+ neurons. Next, to link the expression of ion channels, receptors, and molecular effectors to specific PAG subdivisions, I established a pipeline to perform single-cell RNA-sequencing while preserving the anatomical origin of each neuron. I obtained detailed transcriptomic profiles from VGAT+ and VGluT2+ neurons across PAG subdivisions by individually isolating fluorescently labelled neurons from acute midbrain slices of transgenic mice and processing them with the Smart-seq2 protocol and a target sequencing depth of 4 million reads per sample. Unsupervised clustering of the resulting data revealed putative subpopulations of neurons that mapped onto different PAG subdivisions, whereas differential expression analysis identified candidate genes for setting and regulating key biophysical properties of PAG neurons. By leveraging the unique relationship between PAG circuit anatomy and behavioural output, this work uses anatomical location as an anchor to provide a framework for studying how molecularly defined biophysical properties might underpin behavioural control by the PAG. date: 2022-10-28 date_type: published oa_status: green full_text_type: other thesis_class: doctoral_open thesis_award: Ph.D language: eng primo: open primo_central: open_green verified: verified_manual elements_id: 1981574 lyricists_name: Pavon Arocas, Oriol lyricists_id: OPAVO41 actors_name: Pavon Arocas, Oriol actors_id: OPAVO41 actors_role: owner full_text_status: public pagerange: 37-37 pages: 241 institution: UCL (University College London) department: Sainsbury Wellcome Centre thesis_type: Doctoral citation: Pavón Arocas, Oriol; (2022) Biophysical properties and gene expression profile of single periaqueductal gray neurons. Doctoral thesis (Ph.D), UCL (University College London). Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/10157063/2/Thesis_OriolPavon_221009_final.pdf