Circuit development relies on genes and experience for proper assembly and maturation. Defining the mechanisms that drive circuit-specific adaptations during heightened periods of plasticity are key for understanding typical and atypical development. We hypothesize that molecules that regulate timing of maturation modulate experience-dependent development and differential circuit vulnerabilities in neuro- developmental disorders (NDDs). Advanced molecular and connectomics technologies have provided a more complete perspective on the extent of neuronal and circuit diversity in the mature brain, but there is a knowledge gap for the plastic periods of growth and refinement. Insight into this gap has emerged from studies during the current grant period showing that 1) the c-MET receptor tyrosine kinase (MET) regulates timing of excitatory synapse maturation; 2) MET is expressed in discrete subpopulations of intra-telencephalic (IT) and cortico-thalamic (CT) neurons; and 3) dysregulated MET signaling alters the timing of critical period (CP) plasticity for binocularity and disrupts fear learning. Foundational studies of developing molecular and connectivity subtypes and their function in the cortex comprise three specific aims.
In Aim 1, developing medial prefrontal cortex (mPFC) and primary visual cortex (V1) MET+ neurons will be profiled using connectomics and transcriptomics methods. A newly derived transgenic mouse line, MetGFP, will be combined with specific tracing of MET+ connectivity using virally-transduced split-Cre technology that produces Cre-mediated, temporally stable labeling of GFP+ (MET+) neurons and their axonal projections. Injections of fluorescent retrograde tracers in mPFC and V1 targets will label IT or CT neurons. Labeled neurons will be FACS-sorted and profiled by single cell RNA sequencing. Transcriptome data analysis will delineate subtypes of Met+ and Met-neurons, with additional methods used to validate discoveries and determine whether sex and developmental timing are variables for the subtypes.
Aim 2 will test the hypothesis that Met down-regulation is required for structural and functional plasticity during the CP in V1. A new, controllable transgenic mouse (ctg-Met) that sustains MET signaling beyond its endogenous expression period will be used in combination with two-photon dendritic spine imaging to quantify spine genesis and pruning during the V1 CP. Functional circuit connectivity will be assessed by laser scanning photostimulation combined with glutamate uncaging. V1 plasticity will be measured using a classic paradigm of monocular deprivation-induced ocular dominance plasticity.
In Aim 3, the role of MET+ mPFC neurons in mediating the CP for contextual fear memory persistence will be determined using selective expression of DREADDs in GFP+ neurons using the split-Cre approach. The impact of Met deletion or ctg-Met-mediated extended expression will be examined for the developmental emergence of conditioned fear memory persistence. The studies have high impact for determining mechanisms that underlie typical and atypical circuit development and plasticity in the neocortex related to NDDs.
Differences in the vulnerability of specific brain circuits may be responsible for the variation in symptoms for neurodevelopmental disorders. Understanding the development of the most vulnerable circuits and determining the structural and functional differences that control the ability of the circuits to respond to interventions through the process of experience-dependent plasticity is a key goal of the proposed studies. This multi-disciplinary collaboration uses advanced neuroanatomical, molecular and cellular imaging methods, combined with functional assays, to address a knowledge gap that will improve the understanding of the causes of neurodevelopmental disorders, and help develop new intervention strategies.
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