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Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators
Published on: November 19, 2014
[Optogenetic activation of dorsal hippocampal astrocytic Rac1 blocks the learning of associative memory]
Xiao-Mu Guo1, Zhao-Hui Liao1, Ye-Zheng Tao1
1The State Key Laboratory of Medical Neurobiology and Pharmacology Research Center, School of Basic Medical Sciences, Fudan University, Shanghai 200032, China.
Abstract:
Rac1 belongs to the family of Rho GTPases, and plays important roles in the brain function. It affects the cell migration and axon guidance via regulating the cytoskeleton and cellular morphology. However, the effect of its dynamic activation in regulating physiological function remains unclear. Recently, a photoactivatable analogue of Rac1 (PA-Rac1) has been developed, allowing the activation of Rac1 by the specific wavelength of light in living cells. Thus, we constructed recombinant adeno-associated virus (AAV) of PA-Rac1 and its light-insensitive mutant PA-Rac1-C450A under the control of the mouse glial fibrillary acidic protein (mGFAP) promoter to manipulate Rac1 activity in astrocytes by optical stimulation. Primary culture of hippocampal astrocytes was infected with the recombinant AAV-PA-Rac1 or AAV-PA-Rac1-C450A. Real-time fluorescence imaging showed that the cell membrane of the astrocyte expressing PA-Rac1 protruded near the light spot, while the astrocyte expressing PA-Rac1-C450A did not. We injected AAV-PA-Rac1 and AAV-PA-Rac1-C450A into dorsal hippocampus to investigate the role of the activation of Rac1 in regulating the associative learning. With optical stimulation, the PA-Rac1 group, rather than the PA-Rac1-C450A group, showed slower learning curve during the fear conditioning compared with the control group, indicating that activating astrocytic Rac1 blocks the formation of contextual memory. Our data suggest that the activation of Rac1 in dorsal hippocampal astrocyte plays an important role in the associative learning.
Insights
Activating Rac1 in astrocytes using light-sensitive proteins impairs associative learning and memory formation in the hippocampus. This suggests Rac1 plays a key role in regulating cognitive functions.
Area of Science:
- Neuroscience
- Cell Biology
- Molecular Biology
Background:
- Rac1, a Rho GTPase, is crucial for brain function, influencing cell migration and axon guidance by regulating cytoskeleton dynamics.
- The precise role of dynamic Rac1 activation in physiological functions, particularly in learning and memory, remains incompletely understood.
Purpose of the Study:
- To investigate the role of astrocytic Rac1 activation in regulating associative learning and memory formation.
- To develop a method for temporally controlling Rac1 activity in astrocytes using optogenetics.
Main Methods:
- Construction of recombinant adeno-associated viruses (AAVs) encoding a photoactivatable Rac1 (PA-Rac1) and a light-insensitive mutant (PA-Rac1-C450A) under the mGFAP promoter.
- Infection of primary hippocampal astrocytes and in vivo injection into the dorsal hippocampus.
- Optical stimulation to activate PA-Rac1 and assessment of astrocyte morphology and fear conditioning performance.
Main Results:
- Real-time imaging confirmed light-induced membrane protrusion in astrocytes expressing PA-Rac1, but not in those with PA-Rac1-C450A.
- Mice receiving AAV-PA-Rac1 exhibited a slower learning curve in fear conditioning upon optical stimulation compared to controls and the PA-Rac1-C450A group.
- Activation of astrocytic Rac1 via optical stimulation blocked contextual memory formation.
Conclusions:
- The dynamic activation of Rac1 in dorsal hippocampal astrocytes is essential for the formation of associative memories.
- Optogenetic manipulation of astrocytic Rac1 activity provides a powerful tool to study its role in cognitive processes.
- Targeting astrocytic Rac1 signaling may offer novel therapeutic strategies for memory-related disorders.
