Three-dimensional Interrogation of Cell Types and Instinctive Behavior in the Periaqueductal Gray

Abstract

The periaqueductal gray (PAG) is a critical midbrain hub that relays information from the forebrain to motor and autonomic brainstem centers to orchestrate instinctive behaviors. The current organization of the PAG into four main radial columns lacks the resolution needed to account for the vast range of PAG functions. Using spatially resolved single-cell transcriptomic measurements, we uncovered widespread transcriptional heterogeneity in the PAG with >100 excitatory and inhibitory neuronal populations, which further assemble into 19 spatial metaclusters. We explored the transcriptional and spatial logic of PAG function during instinctive behaviors and demonstrated the regional recruitment of cell types for distinct behaviors. Unexpectedly, certain behaviors trigger differential spatial activation patterns within given cell types, illustrating the complexity of PAG molecular and functional 3D organization. The newly uncovered spatial motifs and high precision cellular map of instinctive behavior in the PAG open new avenues for a mechanistic understanding of PAG function.

Pain-related behavioral and electrophysiological actions of dynorphin A (1-17)

“…35 Future research into the role of dynorphin in chronic pain is necessary to unravel its role in nociception and pain-related affective behaviors. Recently published spatial transcriptomics of the periaqueductal gray 36 will be a helpful tool for unraveling the diverse roles of dynorphin in this brain region. While this study provides some aspects of the electrophysiological profiling of vlPAG neurons following intrathecal dynorphin, future work can expand on this to probe deeper into the excitatory and inhibitory neuronal subtypes that exist in the PAG and other levels of the nervous system to interrogate the role of dynorphin in chronic pain.…”

Cellular and circuit diversity determines the impact of endogenous opioids in the descending pain modulatory pathway

“…Deep brain stimulation targeting the vlPAG has been applied therapeutically for treatment-resistant hypertension (Patel et al, 2011 ; O’Callaghan et al, 2014 ), emphasizing the many subcircuits that utilize this region and the importance of understanding whether specific stimuli engage different neuronal subpopulations within the vlPAG. Furthermore, a recent study using single nucleus RNA-sequencing and Multiplexed Error-Robust Fluorescence in situ Hybridization (MERFISH) identified over 100 excitatory and inhibitory neuronal populations (Vaughn et al, 2022 ). In addition to unique transcriptional profiles, these neurons were found to be spatially distributed uniquely along the rostral-caudal axis, and multiple populations were activated in unison by different instinctive behaviors (i.e., mating, aggression, etc.…”

Voltage-Seq: all-optical postsynaptic connectome-guided single-cell transcriptomics

“…The VMH-PAG axons cover a 2-mm-long anterior-posterior (A-P) range of the dorsal, dorsolateral, and lateral (d, dl, l-PAG) portions of the PAG. The cell-type- and circuit-motif-specific routing of VMH information in the local PAG circuitry is poorly understood due to its large anatomical extension and high neuronal diversity 12 . We used the VMH-PAG pathway as a model to optimize the Voltage-Seq methodology, to all-optical voltage image PRTs, and to select specific neurons for somatic harvesting and subsequent scRNA-seq.…”

“…2f,g, Extended Data Fig. 4b,c) and we identified: persistent activity after the Op (cluster 1,4); rhythmic bursting with 3-4 Hz (cluster 8), separate burst upon each Op (cluster 5); time-locked single o-AP upon each Op (cluster 7); strongly depressing (cluster 10,6) and strongly facilitating short-term synaptic plasticity (cluster 9); facilitating paired-pulse (PP) plasticity (cluster 2,4,9,16); depressing PP plasticity (cluster 15); spontaneous firing activity preceding the Op (cluster 3,11,12,13,14); inhibitory responses (cluster 14); inhibitory responses with rebound firing or bursting (cluster 3); subthreshold response (cluster 17); weak or not detectable connection (cluster 18) (Fig. 2h).…”