Applications of Calcium Imaging Technology in Nervous System Research
The key to brain science research is to realize the real-time observation of the activity of neuron clusters and to analyze the function and structure of neural circuits on the whole brain scale through the structure tracking of specific neural circuits and their activity manipulation.
Calcium imaging technology refers to the use of calcium indicators to monitor the concentration of calcium in tissues. It is often used in the study of the nervous system to indicate changes in calcium in neurons and reflect neuronal activity.
In organisms, calcium is the basis for cell signal generation and plays an important role in many biological functions. In the mammalian nervous system, calcium is an important type of neuron intracellular signal molecule. In the resting state of the neuron, the intracellular calcium concentration is about 50-100 nM. When the neuron is active, the intracellular calcium concentration can rise 10-100 times; calcium is essential for the release of synaptic vesicles. Neurons will burst out a short calcium concentration peak when they are firing, which means that the calcium concentration of neurons can represent synaptic transmission and neuronal activity .
Fig.1 Neuron signal transmission.
Therefore, the principle of neuronal calcium imaging technology is to rely on the strict correspondence between calcium concentration and neuronal activity. By using special fluorescent dyes or protein fluorescent probes (calcium indicator), the concentration of calcium in neurons is displayed by fluorescence intensity and captured by a microscope to achieve the purpose of monitoring neuronal activity.
The commonly used gene-encoded calcium indicator is to combine the fluorescent protein derived from green fluorescent protein (GFP) and its variants (such as cyclic array GFP, YFP, CFP) with calmodulin (CaM) and myosin light chain Kinase M13 domain fusion. When the calcium concentration increases, it will cause M13 to bind to CaM, thereby changing the conformation of cpEGFP, changing it from a non-fluorescent state to green fluorescence.
Fig.2 The basic structure and principle of GCaMP protein (Ali Gheisari, 2017)
- GCaMP6 Series
- jGCaMP7 Series
- XCaMP Series
- jRGECO1a & RCaMP
The sixth-generation GCaMP protein (GCaMP6) has three different subtypes: GCaMP6s, GCaMP6m, and GCaMP6f, which have different characteristics and need to be selected according to experimental requirements .
|GCaMP6s||Ultrasensitive GCaMP variant; slow kinetics||Suitable for low-frequency signals|
|GCaMP6m||Ultrasensitive GCaMP variant; medium kinetics||Suitable for high-frequency signals|
|GCaMP6f||Ultrasensitive GCaMP variant; fast kinetics||Wide range applications|
jGCaMP7 (Janelia GCaMP7, different from G-CaMP7), including four types of proteins with different characteristics: jGCaMP7s, jGCaMP7f, jGCaMP7b, jGCaMP7c . The jGCaMP7 sensors were tested in vitro and in vivo, and show substantially better performance than the GCaMP6 sensors.
|jGCaMP7s||Sensitive and slow||The sensitivity is more than five times that of GCaMP6s, which is suitable for the detection of single action potentials.|
|jGCaMP7f||Fast kinetics||The sensitivity is more than five times that of GCaMP6f, which is suitable for the detection of single action potentials.|
|jGCaMP7b||Brighter baseline fluorescence||The sensitivity is 3 times that of GCaMP6s, and the fluorescence brightness is increased by 50%. It is suitable for detecting neuronal processes or nerve fibers.|
|jGCaMP7c||High contrast with low baseline fluorescence||Clear signal, enhanced contrast, suitable for wide-zone imaging|
XCaMP is obtained through a series of mutation screening using CaMKK as the backbone. XCaMP includes four different colors: blue XCaMP-B, green XCaMP-G, orange XCaMP-O, and red XCaMP-R. Among them, the fluorescence intensity of XCaMP-G is stronger than that of GCaMP6, and the performance of calcium response induced by action potential stimulation is also significantly improved than that of GCaMP6. Combined with specific neuron-specific expression methods, the activities of three different neuron types in specific behaviors can be simultaneously monitored in the state of free activity; combined with two-photon microscope, microstructure functional imaging can be realized to achieve the Two-color imaging of the rear structure at the same time .
A new type of calcium imaging technology that can take into account both the global and the micro, including CaMPARI and CaMPARI2 (second generation)
Principle: CaMPARI protein emits green fluorescence under normal conditions, and if this protein is treated with high-concentration calcium and ultraviolet light at the same time, it will irreversibly and permanently transform into another confirmation that emits red fluorescence. The researchers introduced this new type of protein into the nervous system of experimental animals through genetic modification and then irradiated the animal’s brain with high-intensity ultraviolet light. By checking the fluorescence, red fluorescent neurons can be found, which are the neurons that are active during ultraviolet light irradiation. Since ultraviolet light can irradiate the whole brain, in theory, CaMPARI can be used to image the whole brain .
The red calcium-sensitive protein can be used with GCaMP to label neurons with different activities in two types of cells in the same brain area of the same mouse .
The non-damaging calcium probe can protect the excitation-transcription coupling that depends on the L-type calcium channel from interference, while still showing some of the excellent Ca2+ sensing properties of GCaMP. It is suitable for the monitoring of ultra-long-term calcium signals .
Calcium Imaging Virus Vector List at Creative Biolabs
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