1Omics Science Center, RIKEN Yokohama Institute, 1-7-22, Suehiro Cho, Tsurumi, Yokohama, Japan
2Lab for Neuronal Circuit Development, RIKEN Brain Science Institute
3RIKEN Genome Science Laboratory, 2-1 Hirosawa, Wako-shi, Saitama, Japan
*A.S. and A.W. contributed equally to this work
***This affiliation is not merged with the Omics Science Center at the RIKEN Yokohama institute.
**M.F. and T.K.H. present address: FM Kirby Neurobiology Center, Children's Hospital Boston, Harvard Medical School, 300 Longwood Ave, Boston MA 02115 USA
BioTechniques, Vol. 52, No. 6, June 2012, pp. 381–385
Efficient isolation of specific, intact, living neurons from the adult brain is problematic due to the complex nature of the extracellular matrix consolidating the neuronal network. Here, we present significant improvements to the protocol for isolation of pure populations of neurons from mature postnatal mouse brain using fluorescence activated cell sorting (FACS). The 10-fold increase in cell yield enables cell-specific transcriptome analysis by protocols such as nanoCAGE and RNA seq.
The mammalian brain consists of a variety of projection neurons, local interneurons, glial cells and extracellular matrix factors entangled in a complex network. Mature neurons are severely damaged by enzymatic dissociation and mechanical trituration, causing extensive cell death thus hampering downstream analyses. Collection of pure neuronal subtypes for RNA extraction and expression profiling has, therefore, been challenging, especially in the case of adult brain tissue. Gene expression profiling of isolated neuronal sub-types has the power to provide a catalog of genes expressed in neuronal sub-populations. Such catalogs would not only provide the means to understand normal neuronal development and function, but also permit distinctions between healthy and diseased states, establish biomarkers for diagnostic and prognostic applications, and facilitate the design of targeted molecular therapies (1, 2).
The proliferation of mouse strains with fluorescently labeled neuronal subpopulations has provided a means of identifying individual neuron sub-types in the intact brain tissue (3-6). Current technologies that enable the collection of pure populations of individual fluorescent neurons, such as laser capture micro-dissection (LCM) (7, 8), manual sorting (9, 10) or automated fluorescence activated cell sorting (FACS) have considerable limitations. LCM, conducted on fixed tissues, collects few cells of partial purity, while manual sorting also yields low cell numbers, and both provide inadequate amounts of high quality RNA required for whole transcriptome analysis. Automated FACS yields adequate numbers of pure cells and has been used to purify post-mitotic neurons from embryonic brains (5, 6, 11), as well as adult neural stem cells (12, 13) and more recently astrocytes (14). FACS has also been used in the microarray profiling of purified post-mitotic embryonic 5HT neurons (15). Although FACS was considered inapplicable for the isolation of pure neuronal populations from adult brain (1) due to neuronal fragility and shearing forces during sorting (9), at least four reports demonstrate the applicability of FACS to isolate neuronal subtypes (16-19). Arlotta et al. analyzed the array profile of FACS sorted, retrograde labeled corticospinal motor neurons during development (from E18 to P14) and compared them with callosal projection neurons and corticotectal projection neurons (16). Lobo et al. purified genetically labeled medium spiny neuron subtypes of the basal ganglia from striatal slices of up to 2 month old mice and reported a recovery of 5000–10,000 neurons yielding 3–10 ng of RNA (19). Guez-Barber et al. successfully isolated, from fixed immunolabeled cells, 200,000 NeuN positive cell bodies without neuronal processes, from striata or midbrains of adult rats (17, 18). To obtain high numbers of intact adult neurons, current protocols require gradient separation and FACS to isolate neuronal cell bodies, followed by culturing with appropriate factors over a few weeks to regenerate neuronal processes (20). Thus sequence based expression profiling of specific neurons isolated from the adult mouse brain has not been conducted to date.
Here, we aimed to overcome the poor isolation rate of single intact neurons dissociated from adult mouse brain by supplementation with trehalose and essential modifications of existing methodology. Trehalose is a disaccharide and its role in maintaining cell viability during heat stress and cryopreservation is well documented (21). Trehalose helps maintain catalytic activity of proteins at high temperatures and is known to act as a chaperonin-like small molecule (22). Upon induction of stress in bacteria, yeast, fungi and invertebrate cells, trehalose synthesis is induced intracellularly (23, 24), but endogenous trehalose is absent in mammalian cells. Although trehalose has been shown to be essential on both sides of the plasma membrane to provide cryoprotection (25), extracellular trehalose alone can improve cell viability and membrane integrity of mammalian cells (26, 27). In Dauer larvae of C. elegans, intracellular trehalose is essential to confer protection against dessication (24). A role for trehalose has been proposed in barotolerance (28), however recent simulations on membrane integrity during mechanical stress did not reveal a protective role for 2 Molal Trehalose solution under the experimental conditions (29).
We reasoned that trehalose might stabilize stressed cells and cell membranes during tissue digestion and dissociation. Accordingly, we supplemented trehalose at concentration of 0.132M in all solutions during tissue digestion and neuron disaggregation to maintain cellviability. Here, we present substantial improvements to the protocol for the isolation of fluorescent neurons from adult mouse cortex by FACS sorting and total RNA extraction for whole transcriptome sequencing applications. Our protocol consistently yields high numbers of viable cells in a single-cell suspension and good quality and quantity of RNA.