Journal: iScience

Article Title: Optical Interrogation of Sympathetic Neuronal Effects on Macroscopic Cardiomyocyte Network Dynamics

doi: 10.1016/j.isci.2020.101334

Figure Lengend Snippet: High Throughput All Optical Interrogation of Neurocardiac Cultures (A) Schematic representation of the in vitro co-culture system, neonatal rat cardiac stellate sympathetic neurons, and neonatal rat ventricular cardiac confluent monolayers cultured to test sympathetic-cardiac interactions. The stellate sympathetic neurons were infected with hChR2-eYFP. Schematic representation of the co-cultures with different neuron dosing regimes (neuron-myocyte ratios in 1:5, 1:20, 1:100, 1:100,000). (B and C) “Optoelectric” versus “optochemical” stimulation of neurons. (B) Optogenetic neural stimulation of cardiac tissue via Channelrhodopsin-2 (ChR2), selectively expressed only in the neurons. Co-cultures of neurons and myocytes (loaded with dye Di-4-ANBDQBS spectrally compatible with ChR2). Optical stimulation (470 nm) was provided at pulse lengths of 3 s, at 0.5 Hz, using irradiance of 0.5–1 mW/mm 2 . (Bi) Post-processed traces using custom-written MATLAB software. Traces showing baseline no activity and followed by long light pulse stimulation, action potentials are evoked indirectly in the myocytes via the ChR2-light-sensitized neurons. Blue is the trace after baseline subtraction after median filtering, red indicates detected spike times, black is an indicator of when light is present (black down = light off). (Bii) The number of neurons innervating the myocytes affects the firing frequency of myocytes in cultures with different neuron to myocyte ratios (0 = myocytes/controls, 1 × 10 −5 = 1:100,000, 0.01 = 1:100, 0.05 = 1:20 and 0.2 = 1:5); the number of experiments (n) for each group were 14, 4, 23, 28, and 12 and confidence values (p) (against null hypothesis of zero effect; Wilcoxon signed rank test) were 0.7131, 0.6250, 0.1091, 0.0000, and 0.0352. We observe a dose-dependent effect (i.e. the greater the number of neurons innervating the myocytes, the greater the effect, with values greater than 0 indicating an increase in beat rate). (C) Photo-uncaging of nicotine using a flash of blue light may lead to the release of noradrenaline by the sympathetic neurons resulting in increase in myocyte beat rate. Panel A was created with Servier Medical Art according to a Creative Commons Attribution 3.0 Unported License guidelines 3.0. See also Figure S4 for immunohistochemistry; see also . SN: sympathetic neuron; N: neuron, CM: cardiomyocyte, HT: high throughput.

Article Snippet: Optical stimulation (470 nm) was provided at pulse lengths of 3 s, at 0.5 Hz, using irradiance of 0.5–1 mW/mm 2 . (Bi) Post-processed traces using custom-written MATLAB software.

Techniques: High Throughput Screening Assay, In Vitro, Co-Culture Assay, Cell Culture, Infection, Software, Activity Assay, Immunohistochemistry