Measurement, modeling, and prediction of temperature rise due to optogenetic brain stimulation.

Optogenetics is one of the most important techniques in neurophysiology, with potential clinical applications. However, the strong light needed may cause harmful temperature rises. So far, there are no methods to reliably estimate brain heating and safe limits in actual optogenetic experiments. We used thermal imaging to directly measure such temperature rises at the surface of live mouse brains during laser illumination with wavelengths and intensities typical for optogenetics. We then modeled the temperature rise with a simple logarithmic model. Our results indicate that previous finite-element models can underestimate temperature increases by an order of magnitude. We validate our empirical model by predicting the temperature rise caused by pulsed stimulation paradigms. These predictions fit closely to the empirical data and constitute a better estimate of real temperature increases. Additionally, we provide a web-based app for easy calculation that can be used as a tool for safe design of optogenetic experiments.

Crossmodal propagation of sensory-evoked and spontaneous activity in the rat neocortex.

In the cortex, neural responses to crossmodal stimulation are seen both in higher association areas and in primary sensory areas, and are thought to play a role in integration of crossmodal sensations. We used voltage-sensitive dye imaging (VSDI) to study the spatiotemporal characteristics of such crossmodal neural activity. We imaged three cortical regions in rat: primary visual cortex (V1), barrel field of primary somatosensory cortex (S1bf) and parietal association area (PA, flanked by V1 and S1bf). We find that sensory-evoked population activity can propagate in the form of a distinct propagating wave, robustly in either crossmodal direction. In single trials, the waveforms changed continuously during propagation, with dynamic variability from trial to trial, which we interpret as evidence for cortical involvement in the spreading process. To further characterize the functional anatomy of PA, we also studied the propagation of spontaneous sleep-like waves in this area. Using a novel flow-detection algorithm, we detected a propagation bias within PA of spontaneous waves--these tend to propagate parallel to the crossmodal axis, rather than orthogonal to it. Taken together, these findings demonstrate that intracortical networks show pre-attentive crossmodal propagation of activity, and suggest a potential mechanism for the establishment of crossmodal integration.

Normalization of voltage-sensitive dye signal with functional activity measures.

In general, signal amplitude in optical imaging is normalized using the well-established DeltaF/F method, where functional activity is divided by the total fluorescent light flux. This measure is used both directly, as a measure of population activity, and indirectly, to quantify spatial and spatiotemporal activity patterns. Despite its ubiquitous use, the stability and accuracy of this measure has not been validated for voltage-sensitive dye imaging of mammalian neocortex in vivo. In this report, we find that this normalization can introduce dynamic biases. In particular, the DeltaF/F is influenced by dye staining quality, and the ratio is also unstable over the course of experiments. As methods to record and analyze optical imaging signals become more precise, such biases can have an increasingly pernicious impact on the accuracy of findings, especially in the comparison of cytoarchitechtonic areas, in area-of-activation measurements, and in plasticity or developmental experiments. These dynamic biases of the DeltaF/F method may, to an extent, be mitigated by a novel method of normalization, DeltaF/DeltaF(epileptiform). This normalization uses as a reference the measured activity of epileptiform spikes elicited by global disinhibition with bicuculline methiodide. Since this normalization is based on a functional measure, i.e. the signal amplitude of "hypersynchronized" bursts of activity in the cortical network, it is less influenced by staining of non-functional elements. We demonstrate that such a functional measure can better represent the amplitude of population mass action, and discuss alternative functional normalizations based on the amplitude of synchronized spontaneous sleep-like activity. These findings demonstrate that the traditional DeltaF/F normalization of voltage-sensitive dye signals can introduce pernicious inaccuracies in the quantification of neural population activity. They further suggest that normalization-independent metrics such as waveform propagation patterns, oscillations in single detectors, and phase relationships between detector pairs may better capture the biological information which is obtained by high-sensitivity imaging.