Figure 1.

ClopHensorN: a genetically-encoded Cl⁻ and pH sensor optimized for use in the nervous system. (A) Biolistic transfection of hippocampal slices was used to test different fusion proteins for use as a simultaneous Cl⁻ and pH sensor in the nervous system. These were composed of E²GFP (green) fused to one of three different red fluorescent proteins (RFPs; red) via a flexible linker (gray). The RFP was either DsRed (“ClopHensor”; top), mCherry (middle), or TdTomato (“ClopHensorN”; bottom). (B) A hippocampal CA3 pyramidal neuron expressing ClopHensor. The E²GFP (left) shows uniform expression but the DsRed (right) revealed marked intracellular aggregation and poor co-localization with the E²GFP. (C) A CA3 pyramidal neuron expressing the E²GFP-mCherry fusion protein. Pronounced aggregation issues also affect this construct. (D) A CA3 pyramidal neuron expressing the E²GFP-TdTomato fusion protein “ClopHensorN.” ClopHensorN displayed homogenous expression of both E²GFP and TdTomato, and complete co-localization of the two fluorophores. (E) Population data from hippocampal slices 2 days after biolistic transfection with four different ClopHensor variants; E²GFP-DsRed (original ClopHensor), PalmPalm-E²GFP-DsRed (ClopHensor with a membrane targeting sequence—PalmPalm-ClopHensor), E²GFP-mCherry and E²GFP-TdTomato (ClopHensorN). The majority of cells expressing E²GFP-DsRed (19 of 26 cells imaged), PalmPalm E²GFP-DsRed (39 of 62 cells), and E²GFP-mCherry (19 of 26 cells) demonstrated dense aggregation of the red fluorophore. In contrast, no aggregates were detected in any of the cells expressing ClopHensorN (0 of 58 cells) and the two fluorophores exhibited uniform and corresponding expression. (F) 5 days post transfection, almost all cells expressing E²GFP-DsRed (20 of 24 cells), PalmPalm E²GFP-DsRed (24 of 24 cells) and E²GFP-mCherry (50 of 50 cells) contained dense aggregates. In contrast, all cells expressing ClopHensorN (0 of 36 cells with dense aggregates) continued to demonstrate uniform expression. (G) In utero electroporation of ClopHensorN in mouse embryos resulted in long-term and uniform expression in cortical neurons. After 4 weeks in vivo, expression was assessed using acute brain slices. A schematic of a coronal mouse brain slice at P21 and low magnification fluorescent image (left) indicate the position of the ClopHensorN-expressing cortical cells. High magnification confocal images collected following excitation at 458, 488, and 594 nm, respectively (right), show that ClopHensorN displays homogenous expression and co-localization of the two fluorophores in mature cortical neurons.