Optogenetics allows light activation of genetically defined cell populations and the study of their link to specific brain functions. While it is a powerful method that has revolutionized neuroscience in the last decade, the shortcomings of directly stimulating electrodes and living tissue with light have been poorly characterized. Here, we assessed the photovoltaic effects in local field potential (LFP) recordings of the mouse hippocampus. We found that light leads to several artifacts that resemble genuine LFP features in animals with no opsin expression, such as stereotyped peaks at the power spectrum, phase shifts across different recording channels, coupling between low and high oscillation frequencies, and sharp signal deflections that are detected as spikes. Further, we tested how light stimulation affected hippocampal LFP recordings in mice expressing channelrhodopsin 2 in parvalbumin neurons (PV/ChR2 mice). Genuine oscillatory activity at the frequency of light stimulation could not be separated from light-induced artifacts. In addition, light stimulation in PV/ChR2 mice led to an overall decrease in LFP power. Thus, genuine LFP changes caused by the stimulation of specific cell populations may be intermingled with spurious changes caused by photovoltaic effects. Our data suggest that care should be taken in the interpretation of electrophysiology experiments involving light stimulation.

Optogenetics is revolutionizing Neuroscience, but an often neglected effect of light stimulation of the brain is the generation of heat. In extreme cases, light-generated heat kills neurons, but mild temperature changes alter neuronal function. To date, most in vivo experiments rely on light stimulation of neural tissue using fiber-coupled lasers of various wavelengths. Brain tissue is irradiated with high light power that can be deleterious to neuronal function. Furthermore, absorbed light generates heat that can lead to permanent tissue damage and affect neuronal excitability. Thus, light alone can generate effects in neuronal function that are unrelated to the genuine "optogenetic effect." In this work, we perform a theoretical analysis to investigate the effects of heat transfer in rodent brain tissue for standard optogenetic protocols. More precisely, we first use the Kubelka-Munk model for light propagation in brain tissue to observe the absorption phenomenon. Then, we model the optothermal effect considering the common laser wavelengths (473 and 593 nm) used in optogenetic experiments approaching the time/space numerical solution of Pennes' bio-heat equation with the Finite Element Method. Finally, we then modeled channelrhodopsin-2 in a single and spontaneous-firing neuron to explore the effect of heat in light stimulated neurons. We found that, at commonly used light intensities, laser radiation considerably increases the temperature in the surrounding tissue. This effect alters action potential size and shape and causes an increase in spontaneous firing frequency in a neuron model. However, the shortening of activation time constants generated by heat in the single firing neuron model produces action potential failures in response to light stimulation. We also found changes in the power spectrum density and a reduction in the time required for synchronization in an interneuron network model of gamma oscillations. Our findings indicate that light stimulation with intensities used in optogenetic experiments may affect neuronal function not only by direct excitation of light sensitive ion channels and/or pumps but also by generating heat. This approach serves as a guide to design optogenetic experiments that minimize the role of tissue heating in the experimental outcome.

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The development of computational tools is essential for the development of new technologies, including experimental designs needed for behavioral neuroscience research. The computational tool developed in this study is based on the convolutional neural networks and the You Only Look Once (YOLO) algorithm for detecting and tracking mice in videos recorded during behavioral neuroscience experiments. The task of mice detection consists of determining the location in the image where the animals are present, for each frame acquired. In this work, we propose mice tracking using the YOLO algorithm, running on an NVIDIA GeForce GTX 1060 GPU. We analyzed a set of data composed of 13622 images, made up of behavioral videos of three important researches in this area. The training set used 50% of the images, 25% for validation and 25% for the tests. The results show that the mean Average Precision (mAP) reached by the developed system was 90.79% and 90.75% for the Full and Tiny versions of YOLO, respectively. It has also been found that the use of the Tiny version is a good alternative for experimental designs that require real-time response. Considering the high accuracy of the results, the developed work allows the experimentalists to perform mice tracking in a reliable and non-evasive way, avoiding common system errors that require delimitations of regions of interest (ROI) or even evasive luminous identifiers such as LED for tracking the animals.

Optogenetics is revolutionizing neuroscience but an often neglected effect of light stimulation of the brain is the generation of heat. In extreme cases, light-generated heat kills neurons but mild temperature changes alter neuronal function. In this work, we investigated heat transfer in brain tissue for common optogenetic protocols using the finite element method. We then modeled channelrhodopsin-2 in a single-and a spontaneousfiring neuron to explore the effect of heat in light stimulated neurons. We found that, at commonly used intensities, laser radiation considerably increases the temperature in the surrounding tissue. This effect alters action potential size and shape and cause increase in spontaneous firing frequency in a neuron model. However, the shortening of activation time constants generated by heat in the single firing neuron model produce AP failures in response to light stimulation. We also found changes in the power spectrum density and a reduction in the time required for synchronization in an interneuron network model of gamma oscillations. Our findings indicate that light stimulation with intensities used in optogenetic experiments may affect neuronal function not only by direct excitation of light sensitive ion channels and/or pumps but also by generating heat. This approach serves as a guide to design optogenetic experiments that minimize the role of tissue heating in the experimental outcome.PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27248v1 | CC BY 4.0 Open Access | rec: