Unlike the excitatory Selleckchem KRX-0401 channelrhodopsins, NpHR is a true pump and requires

constant light in order to move through its photocycle. Moreover, although optogenetic inhibition with NpHR was shown to operate well in freely moving worms and in mammalian brain slices ( Zhang et al., 2007) as well as cultured neurons ( Zhang et al., 2007 and Han and Boyden, 2007), several years passed before mammalian validation of any inhibitory optogenetic tool was obtained by successful application to behavioral studies in intact mammals ( Witten et al., 2010 and Tye et al., 2011), due to membrane trafficking problems that required additional engineering ( Gradinaru et al., 2008, Gradinaru et al., 2010 and Zhao et al., 2008). At high expression levels, NpHR-EYFP-expressing cells were found to show accumulations of intracellular fluorescence that colocalized with endoplasmic reticulum (Gradinaru et al., 2008). Addition of an ER export motif from the Kir2.1 potassium channel (ER2—identified PD0332991 cost after

a screen of many possible corrective motifs; Gradinaru et al., 2008) improved the surface membrane localization of NpHR and yielded eNpHR2.0 (Gradinaru et al., 2008 and Zhao et al., 2008), with higher currents suitable for use in intact rodent tissue (Sohal et al., 2009 and Tønnesen et al., 2009) as well as in human and nonhuman primate tissue (Busskamp et al., 2010 and Diester et al., 2011). Next, eNpHR3.0, which additionally contains a neurite trafficking sequence from the Kir2.1 potassium channel, showed further enhanced photocurrents (nanoampere scale at moderate light intensities, < 5 mW/mm2) that can be used to drive inhibition by yellow- or far-red-shifted wavelengths (up to 680 nm at the infrared Endonuclease border; Gradinaru et al., 2010). eNpHR3.0 ultimately enabled the loss-of-function

side of optogenetics for behavior in freely moving mammals (Witten et al., 2010 and Tye et al., 2011), complementing the engineered channelrhodopsins that had enabled gain-of-function in freely moving mammals (Adamantidis et al., 2007). eNpHR3.0 was first used along with bilateral optical fiber devices to inhibit the cholinergic neurons of the nucleus accumbens and elucidate a causal role for these rare cells in implementing cocaine conditioning in freely moving mice, which appears to operate via enhancing inhibition of inhibitory striatal medium spiny neurons (Witten et al., 2010). eNpHR3.0 was also used in a two-fiber approach to inhibit a specific intra-amygdala projection in freely moving mice, implicating a defined neural pathway in aspects of anxiety and anxiolysis (Tye et al., 2011).