Arthur W. English,1,2,* Ken Berglund,3 Dario Carrasco,1 Katharina Goebel,1 Robert E. Gross,3 Robin Isaacson,1 Olivia C. Mistretta,1 and Carly Wynans1
"1Department of Cell Biology, Emory University School of Medicine, Atlanta, GA 30322, USA; [email protected] (D.C.); [email protected] (K.G.); [email protected] (R.I.); [email protected] (O.C.M.); [email protected] (C.W.)
2Department of Rehabilitation Medicine, Emory University School of Medicine, Atlanta, GA 30322, USA
3Department of Neurosurgery, Emory University School of Medicine, Atlanta, GA 30322, USA; [email protected] (K.B.); [email protected] (R.E.G.)
*Correspondence: [email protected]"
Functional recovery after peripheral nerve injury (PNI) is poor, mainly due to the slow and incomplete regeneration of injured axons. Experimental therapies that increase the excitability of the injured axons have proven remarkably successful in promoting regeneration, but their clinical applicability has been limited. Bioluminescent optogenetics (BL-OG) uses luminopsins, fusion proteins of light-generating luciferase and light-sensing ion channels that could be used to increase neuronal excitability if exposed to a suitable substrate. Excitatory luminopsins were expressed in motoneurons of transgenic mice and in wildtype mice transduced with adeno-associated viral vectors. Intraperitoneal administration of coelenterazine (CTZ), a known luciferase substrate, generated intense bioluminescence in peripheral axons. This bioluminescence increased motoneuron excitability. A single administration of CTZ immediately after sciatic nerve transection and repair markedly enhanced motor axon regeneration. Compound muscle action potentials were 3–4 times larger than controls by 4 weeks after injury. The results observed with transgenic mice were comparable to those of mice in which the luminopsin was expressed using viral vectors. Significantly more motoneurons had successfully reinnervated muscle targets four weeks after nerve injury in BL-OG treated mice than in controls. Bioluminescent optogenetics is a promising therapeutic approach to enhancing axon regeneration after PNI.
Patch-clamp recordings were conducted under an upright wide-field fluorescence microscope (Scientifica HyperScope) equipped with an LED (CoolLED pE-300ultra, CoolLED, Andover, MA, USA), a multi-bandpass filter cube (Chroma 89402, Chroma Technology, Bellows Falls, VT, USA), water immersion objective (Nikon CFI75 LWD 16X W 0.8NA), a scientific CMOS camera (OptiMOS, QImaging, Surrey, BC, Canada), a micromanipulator (Scientifica PatchStar, Scientifica, Clarksburg, NJ, USA), a patch-clamp amplifier (Axon MultiClamp 700B, Molecular Devices, San Jose, CA, USA), and a digitizer (Axon Digidata 1550B, Molecular Devices) at room temperature.
Product Associated Features
The broad spectrum pE-300 ultra offers TTL triggering, individual channel control and software integration. This makes it ideal for fast pulsing at differing durations and irradiance, such as in these optogenetic protocols.
International Journal of Molecular Sciences
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