Targeting and readout strategies for fast optical neural control in vitro and in vivo

Interesting integrated applications of these targeting and readout methods could go beyond simply driving specific populations of cells to driving specific cells in different brain regions (e.g., among thalamus, cortex, hippocampus, and striatum) with precise relative timing (synchrony or asynchrony with well defined phase shifts). For example, the motor behavioral studies (Fig. 2D) suggest that specific cortical layers in a region could be turned on or off with high temporal precision in behaving subjects, a possibility unapproachable with electrode-based, pharmacological, or genetic methods. These experiments are within reach now, but there are also many refinements of microbial opsin technology still pending that could enhance the power of the approach. For example, the independence (in mammals) from exogenous cofactors is a positive aspect, but it remains possible that some mammalian cells will not have sufficient retinoids to fully support opsin function; in these cases, cointroduction of enzymes governing rate-limiting steps in retinoid uptake and metabolism (T. Wang et al., 2007; Yang and O'Tousa, 2007) (Tracey, personal communication) might be helpful in driving maximally efficacious opsin function. The subcellular-targeting approach (Fig. 1D) suggests additional strategies to target axons, somata, and synaptic terminals. Other molecular modifications may result in usefully shifted spectral properties [e.g. redshifted opsins for better tissue penetration and multicolor optical activation experiments (Luecke et al., 2001; Shimono et al., 2003)], greater or lesser Ca2+ permeance, or improved conductance properties (Nagel et al., 2005). We find in hippocampal neurons that H134R mutation in ChR2 expressed under the synapsin I promoter gives rise to a 2.37 ± 0.07-(p < 0.005) fold enhancement in steady state current and 1.73 ± 0.10 (p < 0.005) fold enhancement in peak current (n = 5; comparison with wild-type ChR2). It is important to remember that in principle, ChR2 and NpHR will contribute to small, brief, and reversible elevations in intracellular Ca2+ and Cl-, respectively; likewise, native excitatory transmission also elevates intracellular Ca2+ via Ca2+-permeable glutamate receptors and voltage-activated Ca2+ channels, and native inhibitory transmission typically also elevates intracellular Cl-. Because elevations in Ca2+ can drive intracellular biochemical changes, and elevations in Cl- can modulate the effects of endogenous GABAergic neurotransmission, these factors should be considered in experimental design. Findings obtained from microbial opsin work in animal models may ultimately inform novel clinical approaches, even without introduction of opsins into patients. Clinically compatible, noninvasive interventions that are likely to modulate neural activity, including radiation, ultrasound, and magnetic methods, all can be depth targeted in various ways (for example by using stereotactically guided accumulation of energy at subcentimeter resolution, delivered from multiple calculated trajectories as with the radiation-based CyberKnife/Gamma Knife, or depth focused as with high-intensity focused ultrasound). Energy can be depth targeted in other ways as well, for example with fiber optics or implanted transducers (Adamantidis et al., 2007). In principle, many focusing methods could be used to directly transduce energy, or even to activate custom stereotactically or endovascularly delivered micro-"antennas" that locally modulate radiation sensitivity, ultrasound response, and magnetic susceptibility, at specific circuit nodes in the clinical setting that had previously been implicated with optogenetic animal models (Zhang et al., 2007b). However, work in animal models will be a primary focus of the field, and in itself can promote understanding of the circuit basis of neuropsychiatric diseases like narcolepsy (Adamantidis et al., 2007). Looking toward the future, plants and many fungi and microbes are highly dependent on light and therefore have developed highly specialized light-sensing proteins that will likely continue to provide novel and powerful tools for perturbing and interrogating the intact nervous system. Optical activation of intracellular signaling via modified opsins (Kim et al., 2005) and blue-light-activated adenylate cyclases (PACs) from the flagellate Euglena gracilis (Iseki et al., 2002; Ntefidou et al., 2003) can trigger rapid and reversible increases in cAMP levels in vitro and modulate behavior in Drosophila in vivo (Schroder-Lang et al., 2007). Phototropins, photo-regulated protein kinases (Huala et al., 1997; Briggs et al., 2001), might also provide useful tools for biochemistry studies of phosphorylation events within a cell that could complement ChR2/NpHR modulation of biochemical signaling, along with other light-activated compounds yet to be identified. Perhaps only a small fraction of the potential opsins and other tools identified in macrogenetic approaches (e.g., Venter et al., 2004) will be immediately useful, as the experience with the comparatively unstable Halobacterium salinarum halorhodopsin (HsHR) revealed (Zhang et al., 2007a), and the broad action spectra of these naturally occurring proteins may limit the number of independently addressable optical control channels achievable, but identification of new players may refine the microbial opsin approach, and molecular optimizations have the potential to incrementally further unlock latent utility. Together, these novel tools, targeting techniques, and readout technologies will extend the utility of the microbial opsins in bidirectional, reversible, and spatiotemporally precise control of activity in targeted neurons. Copyright © 2007 Society for Neuroscience.