Visualizing the lipid dynamics role in infrared neural stimulation using stimulated Raman scattering.

Infrared neural stimulation (INS) uses pulsed infrared light to yield label-free neural stimulation with broad experimental and translational utility. Despite its robust demonstration, INS's mechanistic and biophysical underpinnings have been the subject of debate for more than a decade. The role of lipid membrane thermodynamics appears to play an important role in how fast IR-mediated heating nonspecifically drives action potential generation. Direct observation of lipid membrane dynamics during INS remains to be shown in a live neural model system. We used hyperspectral stimulated Raman scattering microscopy to study biochemical signatures of high-speed vibrational dynamics underlying INS in a live neural cell culture model. The findings suggest that lipid bilayer structural changes occur during INS in vitro in NG108-15 neuroglioma cells. Lipid-specific signatures of cell stimulated Raman scattering spectra varied with stimulation energy and radiation exposure. The spectroscopic observations agree with high-speed ratiometric fluorescence imaging of a conventional lipophilic membrane structure reporter, 4-(2-(6-(dibutylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)pyridinium hydroxide. The findings support the hypothesis that INS causes changes in the lipid membrane of neural cells by changing the lipid membrane packing order. This work highlights the potential of hyperspectral stimulated Raman scattering as a method to safely study biophysical and biochemical dynamics in live cells.

Stimulation of water and calcium dynamics in astrocytes with pulsed infrared light.

Astrocytes are non-neuronal cells that govern the homeostatic regulation of the brain through ions and water transport, and Ca2+ -mediated signaling. As they are tightly integrated into neural networks, label-free tools that can modulate cell function are needed to evaluate the role of astrocytes in brain physiology and dysfunction. Using live-cell fluorescence imaging, pharmacology, electrophysiology, and genetic manipulation, we show that pulsed infrared light can modulate astrocyte function through changes in intracellular Ca2+ and water dynamics, providing unique mechanistic insight into the effect of pulsed infrared laser light on astroglial cells. Water transport is activated and, IP3 R, TRPA1, TRPV4, and Aquaporin-4 are all involved in shaping the dynamics of infrared pulse-evoked intracellular calcium signal. These results demonstrate that astrocyte function can be modulated with infrared light. We expect that targeted control over calcium dynamics and water transport will help to study the crucial role of astrocytes in edema, ischemia, glioma progression, stroke, and epilepsy.