AAV DJ-mCherry

Although acid-sensing ion channels (ASICs) are widely expressed in the central nervous system, their distribution and role in axonal growth cones remain unclear. Here, researchers examined ASIC localization and function in axonal growth cones of cultured immature hippocampal neurons. Immunocytochemical data showed that both native and overexpressed ASIC1a and ASIC2a were localized in the growth cones of cultured young hippocampal neurons. Calcium imaging test results showed that ASICs (mainly ASIC1a) present in growth cones mediated calcium influx despite the addition of voltage-gated Ca2+ channel antagonists and depletion of intracellular calcium stores. Electrophysiological test results showed that a rapid drop in extracellular pH at the growth cones of voltage-clamped neurons induced an inward current, which was blocked by bath application of the ASIC antagonist amiloride, indicating that the ASICs expressed in growth cones are functional. Subsequent immunocolocalization detection results showed that in mature hippocampal neurons, ASIC1a and ASIC2a were colocalized with Neurofilament-H and Bassoon, indicating that ASIC1a and ASIC2a were distributed in axons and presynaptic membranes after maturation.

Cultures were transduced at DIV0 and imaged and immunoblotted at DIV6. Overexpression of ASIC1a and ASIC2a was quantified by Western blot analysis. The researchers observed that, at the protein level, ASIC1a and ASIC2a expression was significantly increased in the AAV-dj-ASIC1a-mCherry and AAV-dj-ASIC2a-eGFP cultures compared with that observed in the AAV-dj-mCherry and AAV-dj-eGFP control cultures (Figure 1). The molecular weights of ASIC1a and ASIC2a are approximately 60 and 80 kD, respectively. In addition to protein band enhancement, the expression of proteins fused to mCherry (28.8 kD) and eGFP (27 kD) was higher and clearer than that in the respective controls. ASIC1a and ASIC2a fluorescence signals were detected in growth cones at DIV6. In addition to the soma, dendrites and axons, ASIC1a and ASIC2a are also distributed in growth cones.

Figure 1. Transduced ASIC1a and ASIC2a is expressed at the axonal growth cones. (Liu X, et al., 2020)