Biochemical Methods and Techniques of Neuroscience
How do we understand neuronal signals in the brain, how neurons control behavior, or how signals fail during disease? Understanding chemical changes are necessary to analyze neuronal communication. Analysis and measurement go beyond traditional neurochemical substances, such as oxygen and dopamine, into new molecular research, such as small molecule neuromodulators, peptides, proteins, and lipids. Although no single technique can fully analyze neurological issues, by using information from many technologies in tandem, a better understanding of the nervous system can be achieved.
The analysis of neuron signals often involves sample collection, and due to the complexity of neurons, the components of interest need to be separated. Therefore, we mainly summarize the biochemical methods involved in three aspects: neuron collection, separation, and neurotransmitter detection.
Neuron Sampling Methods
- Microdialysis
- Low flow perfusion sampling (LFPS)
Microdialysis is currently the most common method for harvesting neuronal cells from the brain, such as depositing astrocytes directly on the microdialysis probe for sampling.
LFPS has been used to study gender differences in the concentration of amino acids in the brains of mice.
Separation Technology
- Capillary liquid chromatography (cLC) and electrochemical detection (EC)
- High performance liquid chromatography-mass spectrometry (HPLC-MS)
- Liquid Chromatography Mass Spectrometry (LC-MS)
- Capillary electrophoresis (CE)
- Microchip Electrophoresis (MCE)
It was used to quickly separating dopamine in the brain.
Seventeen total neurotransmitters (mainly amino acids and monoamines) were isolated from zebrafish homogenated by HPLC-MS.
It was routinely used for the separation of neuropeptides.
It was a technique for separating neutral molecules based on their electrophoretic mobility.
It was one of the most popular neurotransmitter separation technologies, which can simultaneously measure the release of glutamate, dopamine, and serotonin.
Electrochemical Detection of Exocytosis
In the process of exocytosis, biochemical messengers such as neurotransmitters, hormones, or neuropeptides stored in the vesicles are secreted after the vesicles fused with the cell membrane. The traditional techniques for detecting extracellular secretion were amperometric and cyclic voltammetry. The development of microarray manufacturing technology made it possible to monitor multiple exocytosis processes at the same time.
- Carbon fiber microelectrodes (CFMEs) method
- Electrochemical cell detection method
- Microelectrode array (MEA)
It was an effective method to selectively measure the release of chemicals at the individual cell level, suitable for traditional cell models such as PC12 cells, chromaffin cells, or neurons.
It was a hybrid microfluidic-capillary electrophoresis-electrochemical detection platform, single vesicles were separated by capillary electrophoresis. When the vesicles were in contact with the vesicles, the electrodes at the ends detected the neurotransmitters in the vesicles, measure the content of the transmitter by recording multiple instantaneous values.
Compared with CFMEs, MEA has high spatial resolution and used a single microelectrode in an array to simultaneously record extracellular events of single cells or cell clusters.
Fig.1 Schematic diagram of electrochemical detection of neurotransmitter exocytosis. (Ganesana, 2017)
Detection of Neurotransmitters and Neuromodulators in the Brain
- Fast-scanning cyclic voltammetry (FSCV)
- Fast-scanning controls the adsorption voltammetry (FSCAV)
- High performance liquid chromatogram-electrochemical detection (HPLC-EC)
Due to its high spatial resolution and ability to monitor chemical changes in real time, it was one of the most widely used technologies for monitoring neurotransmitters and neuromodulators in the brain.
It can directly measure the absolute concentration of electroactive neurotransmitters in vitro.
It is used to measure the levels of dopamine, serotonin, epinephrine, norepinephrine and their precursors and metabolites in rodent brain homogenates.
Due to the complexity and diversity of neural samples, separation is often required in neuroscience analysis. Advances in sampling technology have improved the time resolution of the separation process and the viability of the sampled tissue, making it possible to measure the chemical complexity of brain samples. Although biochemical methods are not usually presented quickly on time and space scales like imaging techniques, they can obtain abundant chemical information at the same time, which is an indispensable technical means in neuroscience research.
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Reference
- Ganesana, M.; et al. Analytical techniques in neuroscience: recent advances in imaging, separation, and electrochemical methods. Analytical chemistry. 2017, 89(1): 314-341.
