The release of chemicals at the synaptic connections between two neurons is basic for the formation of neuronal networks and for intercellular communication. Many neuronal disorders such as schizophrenia, Alzheimer’s and epilepsy, are, at least in part, due to failure in the transmission of information between cells. The classical approaches to mitigate the symptoms of these diseases rely on pharmacologically active chemicals that can substitute the endogenous missing molecule. Optogenetics poses an alternative strategy, in which the use of light is explored as a means to control neuronal activity. Many applications have been described so far in this growing field, both in vitro and in vivo, with animal models like mice, nematodes or flies. One handicap for in vivo applications, though, is that the light source needs to be surgically implanted close enough to the defective neurons to facilitate their activity, an invasive procedure that translates into considerable challenges for practical implementations, especially in deep brain regions. In PhAST, photons are emitted directly from the pre-synaptic cell and received by the post-synaptic neuron, via engineered light-activated ion channels that allow the transmission of the signal.
But how do we use light to signal between two cells? We took advantage of genetically encoded NanoLanterns (NL), enzymes originally derived from the deep-sea shrimp Oplophorus gracilirostris, but genetically engineered in vitro1. These enzymes catalyze the oxidation of a metabolite to generate photons. However, this only happens in presence of the high calcium concentrations characteristic of neuronal signaling, specifically triggering light emission with the endogenous activity of the pre-synaptic neuron. Furthermore, NL are the fusion of the light generating enzyme and a fluorescent protein, which allows for the emission in different colors by simply using a different fluorescent protein. This way, the light can be tuned to spectrally match the ion channel of interest in the post-synaptic neuron.
On the postsynaptic neuron, we used a very sensitive channelrhodopsin that can evoke behavioral responses with a low amount of photons (as little as 10.000 photons). As cool as it may sound, though, working with photons was quite challenging. Caenorhabditis elegans (C. elegans), the soil nematode we use as animal model, needed to be kept in the dark and to visualize photon production in single, pre-synaptic neurons, we required new optical technology. At ICFO, we conceived and built a new low light microscope2 and adjusted it to capture the ‘low’ output of photons coming out of the neurons, which was more than enough to open the ion channels in the post-synaptic cell, but not for the detectors we usually find in the standard cameras on conventional microscopes.
However, the imaging of the light coming out of the worm was not the only challenge we needed to address. C. elegans is a fantastic model to work with. Not only is it transparent, allowing us to easily look inside and follow the flow of information by bioluminescence and fluorescence microscopy, but also permits the generation of transgenic animals by many methods. Yet, it has a thick and dense cuticle that is hard to cross and complicates any assay with drugs on this nematode. We encountered this problem when providing the substrate for the luciferase. Luckily, we could solve this matter with newly generated molecules that are both more efficient to oxidize and more soluble in different media.
With these improvements and some more, in a first approach we generated a defect (with the CRISPR/Cas9 genome editing technique) specifically on the cell that is the main responsible for avoidance upon touch on the nose, the ASH nociceptor. The expression of a highly efficient blue luciferase and an ion channel with a large operational light sensitivity effectively overcomes such defect and even increases the naturally low response to nose touch of males in this sexually dimorphic circuit to almost the levels of the hermaphrodites. To provide further evidence, we visualized neuronal activity downstream of the photon-source using a novel microfluidic device, which successfully mimics the nose touch classically performed by the navigation of a freely moving worm onto an eyebrow hair conveniently placed in front of it. In this device, single animal is trapped inside a channel and presented in front of a flexible rubber membrane, while a pneumatic pressure pushes the membrane into the animal’s nose. Thus, we called it Trap’N’Slap. This stimulus was enough to reproduce the touch response and facilitated simultaneously recording changes in the fluorescence of the genetically encoded neuronal activity indicator.
Having proven the efficiency of PhAST in closely located neurons, we the plan to expand it to reach targets at medium and long distances. In light of the achievements in the recent work, this method has not only proved very powerful, but also very versatile and may pave the way to techniques alternative to the ones currently in use, increasing specificity and decreasing invasiveness. With this, we envision a future in which PhAST helps to alleviate neurological disorders, but also defects in other types of communication that arise during physical trauma, e.g. regeneration or brain-machine interfaces.
1 Suzuki K, Kimura T, Shinoda H, Bai G, Daniels MJ, Arai Y, Nakano M, Nagai T. Five colour variants of bright luminescent protein for real-time multicolour bioimaging. Nat Commun. 2016 Dec 14;7:13718. doi: 10.1038/ncomms13718. PMID: 27966527
2 Morales-Curiel LF, Gonzalez AC, Castro-Olvera G, Lin LL, El-Quessny M, Porta-de-la-Riva M, Severino J, Morera LB, Venturini V, Ruprecht V, Ramallo D, Loza-Alvarez P, Krieg M. Volumetric imaging of fast cellular dynamics with deep learning enhanced bioluminescence microscopy. Commun Biol. 2022 Dec 3;5(1):1330. doi: 10.1038/s42003-022-04292-x. PMID: 36463346; PMCID: PMC9719505