Evaluating safe infrared neural stimulation parameters: Calcium dynamics and excitotoxicity thresholds in dorsal root ganglia neurons

Background: As a promising neural stimulation technique, infrared neural stimulation (INS) has recently gained significant attention due to its ability to stimulate neuronal activities without needing exogenous agents. NIR light is absorbed by water of the tissue producing local thermal effects. Therefore, INS is a suitable candidate for localized and targeted neural stimulation. However, despite the wide variety of research studies on INS applications, limited studies have focused on identifying and optimizing the stimulation parameters to avoid potential excitotoxicity. This study evaluates the dorsal root ganglia (DRG) neurons' response under INS with varying intensities and illumination time. New method: Here, DRG neurons are cultured and labeled by the CamkII-GCaMP6s virus. The neurons were exposed to infrared laser pulses (2.01 µm wavelength, different powers of 2.5 mW, 5 mW, 7.5 mW, and 10 mW) for durations of 300 s and 400 s. The light was delivered through a silica optical fiber aligned and stabilized within a free-space optical setup. Simultaneous with INS, neuronal activity was evaluated by calcium imaging through a fluorescence microscope. This method allowed real-time monitoring of neuronal calcium dynamics under different stimulation conditions, preparing an overview of the safe thresholds for INS. Results: It was found that calcium saturation has happened for the neurons in exposure to light intensities (7.5 mW and 10 mW) for 300 s, representing potential excitotoxicity. In contrast, with the same exposure time, lower light intensities (2.5 mW and 5 mW) did not show significant signs of calcium saturation or neuronal damage. Moreover, in some neuronal networks, the peripheral neurons of the illuminated area revealed indirect activation, indicating inter-neuronal communication effects. Comparison with existing methods: Compared to previous studies that have explored the use of INS on DRG neurons, our work introduces a systematic approach to evaluate the light intensity-dependent INS, while addressing the critical issue of potential thermal injury. While earlier research has demonstrated the ability of INS to modulate neuronal activity and reduce electrical artifacts in electrophysiological recordings, concerns regarding excitotoxicity and neuronal damage remain insufficiently investigated. We examined a range of laser intensities (2.5 mW to 10 mW) to determine the safe exposure thresholds and optimize the photothermal impact. Furthermore, by utilizing CamKII-GCaMP6s virus-modified neurons, we enhance sensitivity in detecting calcium influx, providing a more precise evaluation of neuronal responses to INS. Therefore, here, we provide the knowledge for safe INS. Conclusions: This work identifies the required laser stimulation parameters, particularly intensity and illumination time of the tissue for efficient and safe INS. We concluded that higher intensities (7.5 mW and 10 mW) can cause calcium saturation and potential neuronal injury, while lower intensities (2.5 mW and 5 mW) are safe for prolonged exposure. Moreover, the observed peripheral neuronal activation suggests indirect stimulation through inter-neuronal connections, offering further insights into the effects of INS on neural networks. These findings contribute valuable information towards safe neuromodulation methods with potential use in clinical settings.