So far, it has proven a significant challenge to isolate specific sub-populations of adult or aged neurons of the central nervous system (CNS) under conditions that allow transcriptome analysis from single or limited numbers of neurons, ideal to perform unbiased studies of the aging brain circuits. Recently, we have optimized methods that allow flow cytometric purification of neurons from the CNS of adult (3 months) and aged (8 months old) mice followed by accurate quantification of gene expression changes from a limited number (< 200) of rare and specific neuronal populations by RNA sequencing or high-throughput microfluidic qRT-PCR analysis in individual mice.
In general, these methods can be applied to study different neuronal populations from normal and/or diseased adult and aged mouse brain, which is central to understanding how mature/aged neurons and brain circuits adapt to numerous stimuli.
In particular, these methods allow us to initiate molecular dissection of signalling pathways activated by brain derived neurotrophic factor (BDNF) through its high affinity receptor TrkB contributing to maintain basal ganglia circuit homeostasis during aging in a normal and/or diseased mouse brain.
Previous methods were limited to the isolation of neurons from immature, juvenile/young adult mouse brain, and/or required a large number of cells for gene expression analysis making multiple comparison analysis unfeasible and masking phenotypic heterogeneity of both cells and individuals.
At the same time, we have also optimized microfluidic analysis from limited number of adult and aged sorted neurons (50 cells) to validate the RNA sequencing data.
This analysis is robust enough to be performed as a stand-alone method to assay gene expression changes across cells, tissues, and disease states.
Studying BDNF/TrkB Signaling: Transcriptome Analysis from a Limited Number of Purified Adult or Aged Murine Brain Neurons. Vatanashevanopakorn C., Grover A., Nath A.R., Clark K., Sopp P., Nerlov C., Minichiello L. (2017). Neuromethods. Humana Press (DOI: 10.1007/7657_2017_3).
Studying BDNF/TrkB Signaling: High-Throughput Microfluidic Gene Expression Analysis from Rare or Limited Samples of Adult and Aged Central Neurons. Nath A.R., Drissen R., Guo F., Nerlov C., Minichiello L. (2017). Neuromethods. Humana Press (DOI: /10.1007/7657_2017_4).