Glial Ion Homeostasis: A Fluorescence Microscopy Approach

In an active synapse, the ion gradients at the pre-synapses and post-synapses were constantly changing and used for signal transmission. The glial cells around the synapse are mainly represented by the fine protrusions of astrocytes, which enter the vicinity of the neuron synapse to maintain the normal ion environment outside the cell. Synaptic transmission activated several ion flux pathways that existed in the process of glial-neuron reaction. The first was the Na⁺ gradient across the membrane, but also related to the transmembrane communication with other ions such as K⁺, H⁺/OH^-, and Cl^-. In addition, astrocytes express ionic and metabolic receptors, and the activation of these receptors is mainly related to the intracellular Ca²⁺ signal from the endoplasmic retina (ER).

Fluorescence Imaging to Monitor the Homeostasis of Glial Ions

Glial cells regulate the balance of ions and neurotransmitters, support neuronal metabolism, and monitor synaptic activity, which is the basis for the progression of neurological diseases. Fluorescence imaging technology tracks the dynamic positioning of glial ions and has been widely used in the study of neuronal processes. Following are several applications based on fluorescence imaging to maintain the homeostasis of glial ions:

Fig.1 Brain tissue is largely divided between domains of astrocytes. (Parpura, 2012)

• Genetically encoded Ca²⁺ indicators (GECIs)

GECIs track neuronal activity and synaptic transmission based on changes in intracellular Ca²⁺ concentration. GECIs do not directly detect membrane potentials or action potentials but track Ca²⁺ signals to monitor nerve transmission. Fig.1 showed the combination of R-CaMP2 (a new red fluorescent protein-derived biosensor) and a green Ca²⁺ indicator to obtain two-color Ca²⁺ imaging of the brain activity of behavioral animals.

• High-resolution immunofluorescence technology

Physiological evidence indicated that cultured astrocytes could release glutamate through a Ca²⁺-dependent mechanism. In addition, glutamate released by astrocytes in the hippocampus interferes with synaptic neurotransmission, and glutamate is the main excitatory neurotransmitter of the central nervous system. Therefore, the application of immunofluorescence technology combined with a high-performance deconvolution microscope (with a resolution of <200 nm), allows the evaluation of the double labeling of a single vesicle, which proved that astrocytes contained glutamate-containing exocytotic vesicles, and was closely related to the central nervous system diseases.

• Quantum dots (QDs)

The accumulation and deposition of β-amyloid (A+) peptides and the formation of neurofibrillary tangles were classic features of Alzheimer's disease (AD). Increased levels of A+ peptides in the brain and their subsequent deposition in A+ plaques lead to the activation of surrounding microglia and astrocytes. As shown in Fig.3, this activation was detected in AD patients using quantum dot ligands targeting receptors that activate glial cells. After activation, glial cells and astrocytes released various pro-inflammatory and anti-inflammatory mediators, thereby producing chronic inflammation in the brain. The emergence of this technology has played an important role in determining the correlation between encephalitis and AD.

Fig.2 Astroglia assembling around a plaque in an amyloid precursor protein/presenilin 1 transgenic mouse. (Parpura, 2012)

• Two-photon laser scanning microscope (2PLSM)

2PLSM provided the necessary spatial and temporal resolution to study glial cells in intact living brains. It clarified the important role of glial ion homeostasis in normal brain function and debilitating neurological diseases. It was found that ammonia can quickly damage the potassium buffer of glial cells, increasing the concentration of extracellular potassium, and over-activate the Na⁺-K⁺-2Cl^- cotransporter 1 (NKCC1) in neurons, causing neurological dysfunction and epilepsy.

Fig.3 Representative images of astrocytes containing calcium indicator rhod-2 under 2PLSM. (Thrane, 2013)

Ion gradients and changes in neurons, astrocytes, and extracellular spaces are the basic elements of the initiation, accompanying, and subsequent processes of synaptic transmission. Like neurons, some ion changes in astrocytes were homeostasis. Through the newly developed fluorescence microscopy technology to observe the imaging of glial ions in vivo (cells) and in vitro (tissues or body fluids), to explore the relationship between neuronal activity and glial metabolism, it provided an effective tool for the diagnosis and targeted therapy of neurological diseases.

Creative Biolabs has decades of experience and advanced technology in neuroscience research. We can provide you with different optical imaging methods for the study of glial and other nerve cells. If you are interested or have any questions, please feel free to contact us.

References

1. Parpura, V.; et al. Glial cells in (patho) physiology. Journal of neurochemistry. 2012, 121(1): 4-27.
2. Thrane, V.R.; et al. Ammonia triggers neuronal disinhibition and seizures by impairing astrocyte potassium buffering. Nature medicine. 2013, 19(12): 1643-1648.