¹Kolling Institute, Pain Management Research, St Leonards, Australia, ²University of Sydney, Faculty of Medicine and Health, Sydney, Australia, ³Brain and Mind Centre, University of Sydney, Faculty of Science, Sydney, Australia

Background: The debilitating condition of chronic pain is a significant health issue that affects 1 in 5 people around the world and is associated with many comorbidities such as depression, anxiety, and sleep disorders. A general understanding of pain pathways in the central nervous system is known, however, knowledge about the contribution of different neuronal subtypes is still emerging. ​A population of glycinergic neurons have been reported in the midbrain ventrolateral periaqueductal grey (vlPAG), a key area involved with descending pain modulation. We have previously demonstrated in mice that vlPAG neurons that express GlyT2, a marker of glycinergic neurons, bidirectionally modulate acute nociception such that their chemogenetic activation increases nociceptive sensitivity whereas inhibition reduces it.

Aims & Objectives: This study aimed to (1) anatomically define the distribution of these neurons and their projections, (2) investigate the brain regions that are activated or inhibited in response to stimulation of these neurons and (3) use a persistent inflammatory pain model to assess the effect of chemogenetic modulation.

Method: Stereotaxic injection of Cre-dependent AAV tracers or DREADDs (hM3D and hM4D) into GlyT2::Cre mice followed by hindpaw injection of an inflammatory agent (Complete Freunds Adjuvant; CFA) and nociceptive and anxiolytic behavioural testing. Tissue was then processed using immunofluorescence and deep-learning algorithms for automatic counting.

Results: First, we demonstrate that GlyT2-PAG neurons are concentrated in the vlPAG, making up ~20% of the region’s inhibitory neuronal population. Chemogenetically modulating the activity of GlyT2-PAG neurons in a GlyT2:Cre mouse model alters nociception, locomotion and anxiety-like behaviours. Furthermore, stimulation of GlyT2-PAG neurons during electrophysiological recordings reveals rather than forming local connections, GlyT2-PAG neurons are projection neurons, and via neuronal activity markers, we determined that they strongly modulate the activity of thalamic and hypothalamic subnuclei, rather than the descending pain pathway. Next, we chemogenetically modulated these neurons in a model of persistent inflammation, and the activity of these neurons was no longer able to modulate nociception but continued to alter anxiety-like behaviours.

Discussion & Conclusions: Together these findings illustrate a novel pain circuit by which GlyT2-PAG neurons potentially contribute to the wider affective pain experience.