Molecular signaling during development and maturation of the nervous system

National Institute Of Neurological Disorders And Stroke

Investigators

Linked publications, trials & patents

Abstract

Our first objective for the current year (2020-2021) was to complete the setup of our laboratory at the Bethesda campus of the National Institutes of Health (NIH). Sub-objective 1: Equip and supply the lab. The slice electrophysiology rig installation and training was completed in late 2020. Experiments and training of personnel has been ongoing since then. Our imaging facility is largely complete with the delivery and installation of an Olympus slide scanner and a Nikon confocal / spinning disk microscope in March 2021. Most areas are now operational. Sub-objective 2: Establish our mouse colony. Rederivation of three lines of mice from Stony Brook University to the NIH by the NIMH transgenic core and establishment of productive breeding colonies was completed in late November. Full expansion to provide continuous supply of experimental animals was delayed due to COVID restrictions until early Spring 2021. We are now up and running. Our second objective was to begin experiments aimed at defining the Nrg1 interactome--the galaxy of proteins that physically interact with the Nrg1 protein in neurons. Subobjective 1: Experiments designed to proximity tag interactors followed by affinity purification and proteomics has been put on hold until COVID space restrictions are ended (to be completed in collaboration with Dr. Yan Li, Director of the NINDS proteomics core facility). Subobjective 2: Axonal isoforms of Nrg1 participate in axon-to-nucleus signaling. In order to identify the suite of genes targeted by this signaling mechanism we are analyzing bulk RNAseq data from dentate gyrus, nucleus accumbens, basal lateral amygdala, total hippocampus and frontal cortices of mice carrying either a mutation disrupting expression of axonal Nrg1 (Type III or CRD) isoforms or a mutation in the Nrg1 transmembrane domain that impairs gamma secretase processing and nuclear signaling (and which was initial identified as a psychosis risk gene in a Costa Rican population). Analyses of the dentate gyrus data from the latter animals has been completed (in collaboration with Dr. Kory Johnson, NINDS bioinformatics core). The results predict alterations in cell cycle progression and in dendrite formation. Both of these predictions have been confirmed using other approaches. The second major finding of the RNAseq analyses is highly significant convergence between the mouse mutant data (analyzed via GCNA) and RNAseq differences between schizophrenia patients and controls. Our third objective was to pursue studies aimed to identify the functional heterogeneity of cholinergic neurons. Subobjective 1: Determine the involvement of cholinergic neurons in two distinct BLA dependent behaviors, one in response to an appetitive stimulus, the other in response to learned threat. These two studies (carried out in collaboration with Dr. L Role, NINDS IRP and Dr. M Picciotto, Yale Univ) identify specific populations of cholinergic neurons that participate in distinct types of memory and demonstrate for the first time that cholinergic neurons participate directly in memory engrams (Crouse et al. ELife 2020 and Rajebhosale et al., BioArxiv 2021). These findings provide significantly new insight into the ways that the modulatory, cholinergic system participates in memory formation and retrieval with clear implications to understanding age related cognitive decline that accompanies degeneration of cholinergic brain nuclei. Subobjective 2: We observed that distinct populations of cholinergic neurons were required for threat responsive behaviors depending on whether the threat was learned (cue-dependent conditioning) or was innate (response to predator odor). The former required engagement of cholinergic neurons in the nucleus Basalis, the latter cholinergic neurons in the ventral pallidum two distinct basal forebrain structures. We have initiated a line of investigation focusing on the response of ventral pallidal cholinergic neurons to either innately appetitive or innately aversive olfactory stimuli. Both stimuli broadly activate cholinergic neurons in the ventral pallidum. Using intersectional genetic approaches, we have found that although both stimuli activate cholinergic neurons, the cholinergic neurons activated, represent two distinct, non-overlapping populations. We are now embarking on efforts to understand, at the molecular level, the fundamental properties of these distinct populations.