Fig. 13: Three-plex detection of glutaminergic neurons, Vglut1 (Red), Vglut2 (Green), and GABAergic neurons Vgat (white) expression in the FFPE mouse brain with RNAscope® Multiplex Fluorescent Assay v2. Nuclei were labeled with DAPI (Blue).
Fig 1: Detection of different cell types in mouse brain hippocampus using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen samples: Rbfox3 (neurons, Green), Aif1 (microglia, Red) and Gfap (astrocytes, White). Cells are counterstained with DAPI.
Fig 2: Visualization of two distinct striatal neuronal populations expressing either Drd1 (Red) or Drd2 (Green) in mouse brain striatum using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen tissue samples. Cells are counterstained with DAPI.
Fig 3: Detection of lncRNAs in mouse brain hippocampus using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen tissue samples: Neat1 (Red) and Malat1 (Green). Cells are counterstained with DAPI.
Fig 4: Detection of G protein-coupled receptors (GPCRs) in mouse brain striatum using RNAscope®2.5 HD Duplex Chromogenic assay on FFPE tissue samples: Cnr1 (Red) and Drd1 (Green). Cells are counterstained with hematoxylin.
Fig 5: Detection of G protein-coupled receptors (GPCRs) in mouse brain hippocampus using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen tissue samples: Chrm3 (Red) and Drd2 (Green). Cells are counterstained with DAPI.
Fig 6: Detection of G protein-coupled receptors (GPCRs) in mouse brain hippocampus using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen tissue samples: Cnr1 (Green) and Drd1 (Red). Cells are counterstained with DAPI.
Fig 7: Detection of G protein-coupled receptors (GPCRs) in mouse brain hippocampus using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen tissue samples: Cnr1 (Green) and Drd1 (Red). Cells are counterstained with DAPI.
Fig 8: Fluorescent detection of ion channels. Detection of Asic1 (Green) and Kcnj3 (Red) in mouse brain hippocampus using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen tissue samples.
Fig 9: Detection of transcription factor immediate early gene and activity marker Cfos (Green) and GPCR Chrm3 (Red) in mouse hippocampus using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen samples. Cells are counterstained with DAPI.
Fig 10: Detection of the effector immediate early gene and plasticity marker Arc (Green) and GPCR Drd1 (Red) in mouse brain striatum using the RNAscope® Multiplex Fluorescent assay on Fresh Frozen tissue samples.
Fig 11: Detection of G protein-coupled receptors (GPCRs) in normal mouse brain hippocampus using RNAscope®2.5 HD Duplex Chromogenic assay on FFPE tissue samples: Cnr1 (Red) and Drd1 (Green). Cells are counterstained with hematoxylin.
Fig 12: Detection of G protein-coupled receptors (GPCRs) in normal mouse brain hippocampus using RNAscope®2.5 HD Duplex Chromogenic assay on FFPE tissue samples: Cnr1 (Red) and Drd1 (Green). Cells are counterstained with hematoxylin.
The nervous system, esp. the brain, is the most complex tissue composed of numerous cell types and subtypes organized with delicate topological characteristics, presenting unique challenges to traditional gene expression analysis techniques. The RNAscope® in situ hybridization assay uniquely addresses these challenges by enabling highly sensitive and specific gene expression analysis at single-transcript detection level and single-cell resolution while preserving spatial and morphological context. In particular, the multiplexing capability for the simultaneous detection of multipleneuronal cell markers enables robust gene expression analysis and visualization of distinct cell populations within the nervous system (see Image gallery). Furthermore, our new BaseScope™ assay goes beyond gene expression detection by allowing for the detection of specific exon-exon junctions in alternatively spliced transcripts in the tissue environment. Alternative splicing is especially prominent in the brain, generating much of the complexity of the brain transcriptome.
"RNAscope® has streamlined in situ RNA hybridization to the point were it can become a standard technique easily implemented in most labs, even by individuals with no experience with this often difficult technique. Moreover the ability to multiplex ISH to analyze simultaneously mRNA levels of up to three different genes allowed the study of changes in gene expression in specific cell populations in the CNS in response to different experimental conditions. Also this technique has been useful to analyze mRNA sub-cellular localization, which may prove important to understand disease mechanisms in neurodegenerative diseases”.
Miguel Sena-Esteves, PhD, Associate Professor, University of Massachusetts Medical School
Neuroscience Applications of the RNAscope® assay:
- Detection, characterization, and (co-) localization of mRNAs in the central and peripheral nervous systems including cell type-specific and subcellular target localization
The RNAscope® assay can be used for the visualization of multiple target co-expression patterns or the co-expression of the target(s) of interest with desired cell type markers that characterize particular types of neurons or glia. Commonly used cell type markers to distinguish neuronal and glial target expression patterns include Rbfox3, Aif1, and Gfap for the detection of neurons, microglia and astrocytes, respectively (figure 1 in image gallery). The striatum harbors two distinct neuronal populations that either express Dopamine Receptor D1 (Drd1) – the striatonigral pathway – or Dopamine Receptor D2 (Drd2) - the striatopallidal pathway (figure 2 in image gallery). These different neuronal cell types can be accurately detected and visualized using the RNAscope® technology. Images for the detection of these different cell type markers can be found in our image gallery.
In their research for perineuronal nets in hippocampal CA2 neurons, Carstens et al. J Neurosci 2016 (1) used the RNAscope® assay to demonstrate the co-localization of Aggrecan mRNA – a perineuronal net component - and Pcp4 mRNA - a marker of hippocampal CA2 neurons. Moreover, their ISH results were consistent at the protein level for co-localization of Aggrecan and Pcp4 protein.
The single-molecule resolution of the RNAscope® technology coupled with morphological context allows for the sub-cellular localization of mRNA expression in the cell body, axons or dendrites. Using the RNAscope® assay to confirm RT-PCR findings, Gervasi et al. RNA 2016 (2) provided the first direct evidence that TH mRNA is trafficked to the axon and that the mRNA is locally translated in the distal axons of SCG neurons.
- Detection of long non-coding RNAs (lncRNAs) in the central and peripheral nervous systems
Recent studies have suggested the involvement of lncRNAs in neurodevelopment and brain function. Despite their functional importance, the mechanisms by which lncRNAs control cellular processes are still elusive. The RNAscope® technology can shed light on the brain-enriched lncRNA functionality by visualizing tissue-restricted expression patterns, localization of lncRNAs to distinct subcellular structures, and regulated expression. Examples for the detection of lncRNAs NEAT1 and MALAT1 can be found in our image gallery (figure 3).
Validation of target mRNA expression after high-throughput gene expression analysis
The RNAscope® technology can be used to validate gene expression profiling studies or RT-PCR results. Lake et al. Science 2016 (3) used both RNAscope® chromogenic and multiplex fluorescent ISH for a set of selected markers and confirmed subtype-and layer-specific expression patterns in the cortex revealed by single-cell transcriptomics for the human brain. Quantitative analysis showed that gene expression levels determined by the RNAscope® assay RNAs were consistent with RNA-seq data. Greer et al. Cell 2016 (4) employed the RNAscope® technology to validate RNA profiling expression data and interrogate the expression pattern of 12 members of the Ms4a family in necklace sensory neurons.
Validation of (cell type-specific) genetic modifications
RNAscope® ISH can be used to validate knock-out models or transgene expression patterns. In their study investigating the influence of the orphan GPCR Gpr88 in A2AR-expressing Drd2 neurons on anxiety-like behaviors, Meirsman et al. eNeuro 2016 (5) used multiplex fluorescent RNAscope® ISH for Gpr88, Drd1 and Drd2 to verify the specific excision of Gpr88 in striatal Drd2-medium spiny neurons of the conditional A2AR-driven Gpr88 knock-out. Seidemann et al. eLife (6) reports results of a new method using the genetically encoded calcium indicator GCaMP6f for chronic imaging of neural population responses in the primary visual cortex V1 of behaving monkeys. The RNAscope® Multiplex Fluorescent assay was used for the spatial analysis and validation of virus-mediated GCaMP expression in tissue samples from a GCaMP6f expressing site in a macaque 10 weeks after viral injection. They showed that 80% of cells were CaMKIIa-positive and all neurons expressing GCaMP were CaMKIIa-positive (attesting to promotor fidelity).
Detection of mRNA in the nervous system when no (reliable) antibodies are available
IHC is a well-established method for a broad range of applications from discovery to diagnostic and prognostic tests. However, raising antibodies to G-protein coupled receptors (GPCRs) has been challenging due to problems in obtaining suitable antigen accessibility since GPCRs are often expressed at low levels in cells and are very unstable when purified (Hutchings et al. 2010 (7)). Ion channels, another class of membrane proteins, also constitute a challenging class of targets for antibody discovery since they must remain membrane-associated to maintain their native conformation. The RNAscope® technology is an ideal method to visualize these targets within their morphological context in the central and peripheral nervous systems. Examples for the detection of GPCRs and ion channels can be found in our image gallery (figure 4 to 8).
Visualization of neuronal network activity and plasticity
RNAscope® ISH can be applied for the visualization of activity and plasticity markers in the brain by means of detecting immediate early genes. Immediate early genes (IEGs) are genes which are transiently and rapidly activated at the transcriptional level in response to a wide variety of stimuli without the need for de novo protein synthesis (Guzowski et al.(8) 2002). The indirect detection of neuronal activity in the mouse brain hippocampus is shown by using a probe for Cfos which is a proto-oncogene and transcription factor. To detect neuronal plasticity, the expression of the effector immediate early gene Activity-Regulated Cytoskeleton-associated protein Arc (also known as Arg3.1) can be mapped. Images for these two markers can be found in the image gallery (figure 9 and 10).
- Carstens KE, Phillips ML, Pozzo-Miller L, Weinberg RJ, Dudek SM. Perineuronal Nets Suppress Plasticity of Excitatory Synapses on CA2 Pyramidal Neurons. The Journal of Neuroscience 2016; 36(23):6312– 6320.
- Gervasi NM, Scott SS, Aschrafi A, Gale J, Vohra SN, Macgibeny MA, et al. The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons. RNA 2016; 22:1–13.
- Blue LB, Ai R, Kaeser G., Salathia N, Yung YC, et al. Neuronal subtypes and diversity revealed by single-nucleus RNA sequencing of the human brain. Science 2016; 352(6293), 1586-1590.
- Greer PL, Bear DM, Lassance JM, Bloom ML, Tsukahara T, Pashkovski S, et al. A Family of non-GPCR Chemosensors Defines an Alternative Logic for Mammalian Olfaction. Cell 2016; 165(7): 1734-1748.
- Guzowski JF. Insights into immediate-early gene function in hippocampal memory consolidation using antisense oligonucleotide and fluorescent imaging approaches. Hippocampus 2002; 12:86-104.
- Seidemann E, Chen Y, Bai Y, Chen SC, Mehta P, et al. Calcium imaging with genetically encoded indicators in behaving primates. Elife 2016; 5: e16178.
- Hutchings C, Koglin M, Marshall F. Therapeutic antibodies directed at G protein-coupled receptors. mAbs 2010; 2(6), 594-606.
- Meirsman AC, Robé A, de Kerchove d’Exaerde A, Kieffer BL. GPR88 in A2AR-neurons enhances anxiety-like behaviors. Eneuro 2016; 3(4), ENEURO-0202.