Pavón Arocas, Oriol; (2022) Biophysical properties and gene expression profile of single periaqueductal gray neurons. Doctoral thesis (Ph.D), UCL (University College London).

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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.

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