Electrode- and Label-Free Assessment of Electrophysiological Firing Rates through Cytochrome C Monitoring via Raman Spectroscopy

Abstract

In vitro neurotoxicology aims to assess and predict the side effects of exogenous chemicals toward the human brain. Among the exploited approaches, electrophysiological techniques stand out for the high spatiotemporal resolution and sensitivity, with the patch clamp considered the gold standard technique for such purposes. However, structural toxicity and metabolic effects may elude detection when only the electrical activity is measured, highlighting the need for integrating electrophysiological recordings with complementary approaches such as optical methods. In this study, we describe an integrated platform for recording neuronal electrical activity and performing chemical analysis with a noninvasive label-free optical imaging, Raman spectroscopy. Specifically, we developed a protocol that maximizes the signal-to-noise ratio while avoiding the crosstalk of the electrical and spectroscopical readouts and any phototoxicity associated with the laser exposure. Synchronous and sequential electrical-optical measurements were carried out and compared, with the sequential approach being more suitable for the longitudinal investigation and correlation of the neuronal electrical activity to the intracellular content of reduced cytochrome C, lipids, proteins, and nucleic acids. Data analysis shows a strong correlation between the metabolic status of the single cells and the overall neuronal firing rate, suggesting the electrode- and label-free assessment of the neuronal firing rates through the monitoring of cytochrome C via Raman spectroscopy when multielectrode array devices with high electrical noise and impedance are used. Conversely, the neuronal firing rate and the reduced cytochrome C content were not correlated to lipids, proteins, and nucleic acids. Thus, this study demonstrates the crosstalk of the neuronal firing rate and reduced cytochrome C as downstream and upstream features of the neuronal metabolic activity and that through the monitoring of the de novo synthesis of lipids, proteins, and nucleic acids, Raman spectroscopy provides additional information for a more accurate assessment of the acute and chronic neurotoxicity.

Keywords: Raman spectroscopy; cytochrome C; electrophysiology; firing rate; multielectrode array; neurotoxicity.

Figure 1

Development of a protocol for repeated (A) sequential or (B) synchronous acquisition of electrophysiological and spectroscopic data.

Figure 2

Effect of the laser illumination toward the neuronal firing rate. The neuronal firing rate before (Wo₁), during (W₂), and following (Wo₂) the laser exposure (532 nm). The neuronal firing rate was measured at the beginning of the time lapse and then prior to any laser exposure (NBA) and the neuronal firing rate following 10 laser exposures (NFA).

Figure 3

Raman fingerprints of neuronal rat cells in relation to the subcellular environments. (A) Raman map indicative of mitochondria and associated with the Raman ratio 750/1004 cm^–1, (B) Raman map indicative of the cytoplasm and associated with the Raman ratio 1450/1004 cm^–1, and (C) Raman map indicative of the nucleus and associated with the Raman ratio 782/1004 cm^–1.

Figure 4

Molecular changes associated with the neuronal firing rate. (A) Representative longitudinal average Raman spectra associated with a high and a low neuronal firing rate plotted against (B) their respective neuronal firing rate; we illustrate the representative sample for the high and low neuronal firing rate in red and blue, respectively. Data processing and analysis were carried out according to Figures S2 and S3. (C) Correlation matrix between the neuronal firing rate, the reduced cytochrome C content (750 cm^–1), proteins (1660 cm^–1), lipids (1450 cm^–1) and nucleic acid content (782 cm^–1). In hot colors and blue colors, we expressed the high- and low-correlation Pearson values, respectively. The correlation is calculated over seven different measurements and more than three different neuronal cell cultures.