To explore the dynamic range of PIRK’s effect on neuronal Dinaciclib clinical trial firing, we measured the firing frequency from each neuron over a range of current injections (0–70 pA). Cells that did not fire throughout the current range were excluded. With small current injections (<40 pA), the firing frequency decreased significantly upon UV light activation of PIRK channels (Figure 5A). With larger current injections (40–70 pA) and higher firing

frequencies, however, there was no significant change in firing frequency following UV light illumination of PIRK-expressing neurons. This ceiling effect can be explained by the native properties of strong inwardly rectifying Kir2.1 channels, which conduct little outward current at positive membrane potentials (Ishii et al., 1994 and Kubo et al., 1993). Kir channels are well known for their selleck inhibitor ability to hyperpolarize membranes and increase the threshold for firing an action potential. To examine this, we measured the minimum amount of current required to

evoke an action potential (referred to as rheobase). The rheobase increased in PIRK-expressing neurons following UV light exposure (Figure 5C). UV light activation of PIRK also hyperpolarized the resting membrane potential of PIRK-expressing neurons by −17 mV, whereas UV light had no effect on the resting potential of control neurons (Figure 5D; Figure S4G). Having successfully expressed PIRK channels in dissociated hippocampal neurons, we next attempted to express PIRK channels in vivo. Genetically encoding Uaas using orthogonal tRNA/synthetase has great potential

to address challenging biological questions in vivo, but this technology has yet to be applied in mammals. There were two main challenges for in vivo incorporation of Uaas in mammals: (1) efficient delivery and expression of the genes for the orthogonal tRNA/synthetase and the target protein into specific found tissue or cells; and (2) sufficient bioavailability of the Uaa at the target tissue and cells. We chose mouse embryos for genetically incorporating Uaas into the brain because of the ability to introduce cDNA and chemicals in utero and then to prepare brain slices pre- and postnatally (Mulder et al., 2008, Saito, 2006 and Tabata and Nakajima, 2001). We started addressing the first challenge by attempting to incorporate Leu, an endogenously available amino acid, into GFP through UAG suppression in mouse embryonic brain. The GFP_Y182  TAG reporter gene was encoded on the same plasmid with the orthogonal tRNACUALeu ( Figure 6A). Three copies of this tRNA expression cassette driven by the H1 promoter were included to increase the UAG suppression efficiency, as we previously demonstrated in mammalian cells ( Coin et al., 2011). A red fluorescent protein, mCherry, was coexpressed with the orthogonal LeuRS through the internal ribosome entry site (IRES) on the other plasmid to indicate successful gene delivery in vivo.