Temporally precise in vivo control of intracellular signalling

Abstract

In the study of complex mammalian behaviours, technological limitations have prevented spatiotemporally precise control over intracellular signalling processes. Here we report the development of a versatile family of genetically encoded optical tools ('optoXRs') that leverage common structure-function relationships among G-protein-coupled receptors (GPCRs) to recruit and control, with high spatiotemporal precision, receptor-initiated biochemical signalling pathways. In particular, we have developed and characterized two optoXRs that selectively recruit distinct, targeted signalling pathways in response to light. The two optoXRs exerted opposing effects on spike firing in nucleus accumbens in vivo, and precisely timed optoXR photostimulation in nucleus accumbens by itself sufficed to drive conditioned place preference in freely moving mice. The optoXR approach allows testing of hypotheses regarding the causal impact of biochemical signalling in behaving mammals, in a targetable and temporally precise manner.

Light induced stimulation and delay of cardiac activity

“…The transfection was analyzed using a standard 25 eYFP filter set in combination with the fluorescence microscope. As Ch2 is fused to eYFP, the expression level should be directly linked to the fluorescence and only cells with a high fluorescence level were used for experiments (Fig.1a), even though fluorescence does not give information 30 about the amount of ChR2 formed from Ch2 or the amount of ChR2 inserted into the membrane. After seeding, the cells formed a confluent layer after 2-4 days on our custom made MEAs.…”

“…The time constant for a channel 200 µm away from the stimulation point, was 58.5 ± .4 and 111 ± .7 ms, for on-and off-kinetics respectively. With increasing distance from the origin, the 30 time constant increases and the signal visibly fades. In addition to the passive processes, the active AP propagation speed was analyzed in the form of a distance versus time shift plot and fit linearly (Fig.5e).…”

“…Applications for treatment of several kinds of diseases or disorders such as loss of sensory perception [1,2,3] central nervous system 25 disorders or loss of limbs [4,5,6] would all benefit from bidirectional communication with electrogenic networks of cells. Micro electrode arrays (MEAs) have been established as an electrical recording system that is non-invasive for 30 electrogenic cells [7]. MEA recording does not disturb cell signaling and generates scar tissue only upon initial placement.…”

“…For these reasons we seek reliable alternatives to electrode stimulation for artifact-free, controllable, and specific cell stimulation [27,28]. Promising results were achieved using 20 light triggerable ion channels that depolarize a cell and subsequently cause action potentials [29,30,31]. Nagel et al found and established channelrhodopsin-2 (Ch2), a directly blue-light-gated cation channel, which binds retinal to form the ChR2 complex [32,33].…”

“…Stimulation can be applied 25 rapidly and non-invasively to a limited subset of genetically defined cells reproducibly [34,35]. Though genetic engineering can be tricky and requires sophisticated techniques for high efficiency or influence free transfection, two main approaches exist in vivo; viral transfection [36], 30 with high efficiencies but increased biological safety risk, or electrofection, with high efficiencies but limited application regions [37,38,39]. Several tissues have been successfully transfected with Ch2 allowing optical stimulation including retina [40], somatosensory neurons [41], and cortex [42] and 35 devices for light delivery have been designed [43].…”