Molecular Methods and Techniques of Neuroscience
From the second half of the 20th century, the amazing advances in molecular biology changed biology and medicine. The construction of recombinant DNA, the emergence of rapid DNA sequencing technology, and the maturity of gene editing have pushed molecular biology to a higher level, allowing researchers to have a deeper understanding of medical conditions. And one of the main problems in biology today is still discovering how our brains work. Therefore, the analysis of the mechanism of neuroscience from the molecular biology level is full of unknowns and expectations.
Molecular Methods of Neuroscience
The neural circuit is composed of a network of multiple neuron types, each of which has its unique morphology and intrinsic physiological characteristics, connectivity, and biochemical properties. How do unique neuronal sub-units composed of different types of neurons promote brain function? By using molecular and genetic techniques to target gene expression to specific neuron types in complex neuronal circuits, the expressed transgenes can be used to clarify and manipulate these circuits with unprecedented precision and control, linking perception, cognition, and behavior with the brain’s specific components.
- DNA Recombination Technology
- Bacterial Artificial Chromosome (BAC)
- Track specific cell type connections
- Selective neuron inactivation
- Viral vector gene delivery
The one-step modification BAC allows the reporter gene to be detected in the central nervous system; the two-step modification BAC is used for the insertion, deletion, and mutation of the target gene.Fig.1 Viral vectors target specific neuron types for reversible inactivation experiments. (Callaway, 2005)
Use wheat germ agglutinin (WGA) / tetanus toxin C fragment (TTC) / specific antibody / green fluorescent protein (GFP) to locate specific neuronal cells.
Selective neuron inactivation can be performed by overexpressing K+ channels and expressing tethered toxins that can block the function of ion channels.
Use adenovirus (AAV) vector / lentivirus vector / herpes simplex virus (HSV) vector to express the gene encoding specific neurons.
- Gene Sequencing Technology
The nervous system is one of the most complex systems in nature. Next-generation sequencing (NGS) and high-throughput brain network research together to analyze the whole gene transcriptome will be the main driving force to promote the understanding of brain function and neurological dysfunction. NGS technology including:
Fluorescence in situ hybridization (FISH)
Microarray pull method
Fluorescence in situ RNA sequencing (FISSEQ) Cross-linked immunoprecipitation sequencing
(CLIP-seq) / Genome run-on sequencing (GRO-seq) Natural extension transcript sequencing (NET-seq) Chromatin immuno-precipitation sequencing (ChIP-seq)
RNA single-molecule fluorescence in situ hybridization (smFISH)
Fig.2 Use smFISH to map the space of transcriptional cells in tissues. (Lein, 2017)
Molecular biology provides new tools for more accurately characterizing the structure and function of neurobiological systems. The systematic mapping of brain development through single-cell RNA-seq and spatial transcription will help to discover the key principles of brain development and help understand the origin of the developing nervous system. Spatial transcription methods will help to gain insights into cell types and neuronal circuits in the developing, adult, and diseased brains.
For detailed molecular applications in neuroscience, please click the following links:
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- Callaway, E.M.; A molecular and genetic arsenal for systems neuroscience. Trends in neurosciences. 2005, 28(4): 196-201.
- Lein, E.; et al. The promise of spatial transcriptomics for neuroscience in the era of molecular cell typing. Science. 2017, 358(6359): 64-69.