SCAMPR, a single-cell automated multiplex pipeline for RNA quantification and spatial mapping.

Spatial gene expression, achieved classically through in situ hybridization, is a fundamental tool for topographic phenotyping of cell types in the nervous system. Newly developed techniques allow for visualization of multiple mRNAs at single-cell resolution and greatly expand the ability to link gene expression to tissue topography, yet there are challenges in efficient quantification and analysis of these high-dimensional datasets. We have therefore developed the single-cell automated multiplex pipeline for RNA (SCAMPR), facilitating rapid and accurate segmentation of neuronal cell bodies using a dual immunohistochemistry-RNAscope protocol and quantification of low- and high-abundance mRNA signals using open-source image processing and automated segmentation tools. Proof of principle using SCAMPR focused on spatial mapping of gene expression by peripheral (vagal nodose) and central (visual cortex) neurons. The analytical effectiveness of SCAMPR is demonstrated by identifying the impact of early life stress on gene expression in vagal neuron subtypes.

MET Receptor Tyrosine Kinase Regulates Lifespan Ultrasonic Vocalization and Vagal Motor Neuron Development.

The intrinsic muscles of the larynx are innervated by the vagal motor nucleus ambiguus (nAmb), which provides direct motor control over vocal production in humans and rodents. Here, we demonstrate in mice using the Phox2b Cre line, that conditional embryonic deletion of the gene encoding the MET receptor tyrosine kinase (MET) in the developing brainstem (cKO) results in highly penetrant, severe deficits in ultrasonic vocalization in early postnatal life. Major deficits and abnormal vocalization patterns persist into adulthood in more than 70% of mice, with the remaining recovering the ability to vocalize, reflecting heterogeneity in circuit restitution. We show that underlying the functional deficits, conditional deletion of Met results in a loss of approximately one-third of MET+ nAmb motor neurons, which begins as early as embryonic day 14.5. The loss of motor neurons is specific to the nAmb, as other brainstem motor and sensory nuclei are unaffected. In the recurrent laryngeal nerve, through which nAmb motor neurons project to innervate the larynx, there is a one-third loss of axons in cKO mice. Together, the data reveal a novel, heterogenous MET-dependence, for which MET differentially affects survival of a subset of nAmb motor neurons necessary for lifespan ultrasonic vocal capacity.

Deletion of Stk11 and Fos in mouse BLA projection neurons alters intrinsic excitability and impairs formation of long-term aversive memory.

Conditioned taste aversion (CTA) is a form of one-trial learning dependent on basolateral amygdala projection neurons (BLApn). Its underlying cellular and molecular mechanisms remain poorly understood. RNAseq from BLApn identified changes in multiple candidate learning-related transcripts including the expected immediate early gene Fos and Stk11, a master kinase of the AMP-related kinase pathway with important roles in growth, metabolism and development, but not previously implicated in learning. Deletion of Stk11 in BLApn blocked memory prior to training, but not following it and increased neuronal excitability. Conversely, BLApn had reduced excitability following CTA. BLApn knockout of a second learning-related gene, Fos, also increased excitability and impaired learning. Independently increasing BLApn excitability chemogenetically during CTA also impaired memory. STK11 and C-FOS activation were independent of one another. These data suggest key roles for Stk11 and Fos in CTA long-term memory formation, dependent at least partly through convergent action on BLApn intrinsic excitability.