The mammalian brain is a highly diverse structure in which large numbers of cell types, grouped into broad functional areas, serve defined functions according to their developmental origin, shape and connectivity, transcriptional program and intrinsic biophysical properties. A mechanistic understanding of how the brain works, and how dysfunctions lead to neurological disorders, will require a systematic characterization of neural cell types. In turn, such knowledge will transform our ability to analyze and experimentally manipulate specific neuronal populations in normal as well as diseased brains. Our project aims to examine the transcriptome of individual cells still embedded in brain tissue in order to take into account topographical features of individual cells that are lost during cell dissociation, such as information about the positions of cells in brain structures, and their participation in functional circuits. We propose here to develop an unbiased experimental approach and computational toolkit to classify cell types in situ, according to their gene expression profiles, and in the functional context of behaviorally relevant circuits.
In Aim 1 of this grant, we propose to extend MERFISH (Multiplexed Error Robust Fluorescent In Situ Hybridization) to brain tissue, and to develop a computational pipeline to establish a spatially informed cell inventory.
In Aim 2, we will use the methods of in situ single cell transcriptional profiling optimized in Aim 1 to create an inventory of cell types, first in an individual nucleus of the hypothalamus: the paraventricular nucleus (PVN), and next in the entire hypothalamus. We will then validate our approach in a different animal species by exploring the cellular composition of the PVN in the Common Marmoset.
In Aim 3, we will use approaches developed in Aims 1 and 2 to uncover cell types across the hypothalamus that are associated with parenting behavior and infant-evoked aggression. We will further assess changes in transcriptional profiles of this hypothalamic behavior circuit in animals in different physiological states.
In Aim 4, we will build on the approaches optimized in previous aims to characterize transcriptional profiles of neurons of a 3-relay circuit controlling parental behavior across the brain.

Public Health Relevance

To shed light into the basic organizational principles of brain function, a systematic characterization of neuronal cell types is needed both at single cell resolution and in situ. We propose here to develop and employ a new unbiased imaging platform and computational toolkit to classify cell types in situ, according to their gene expression profiles, in the functional context of behaviorally relevant circuits, and according to the animal's internal physiological state.

National Institute of Health (NIH)
National Institute of Mental Health (NIMH)
Research Project (R01)
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Special Emphasis Panel (ZMH1-ERB-L (04))
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Freund, Michelle
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Harvard University
Schools of Arts and Sciences
United States
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Moffitt, Jeffrey R; Bambah-Mukku, Dhananjay; Eichhorn, Stephen W et al. (2018) Molecular, spatial, and functional single-cell profiling of the hypothalamic preoptic region. Science 362:
Moffitt, Jeffrey R; Hao, Junjie; Bambah-Mukku, Dhananjay et al. (2016) High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proc Natl Acad Sci U S A 113:14456-14461