Advantages, Pitfalls, and Developments of All Optical Interrogation Strategies of Microcircuits in vivo.

The holy grail for every neurophysiologist is to conclude a causal relationship between an elementary behaviour and the function of a specific brain area or circuit. Our effort to map elementary behaviours to specific brain loci and to further manipulate neural activity while observing the alterations in behaviour is in essence the goal for neuroscientists. Recent advancements in the area of experimental brain imaging in the form of longer wavelength near infrared (NIR) pulsed lasers with the development of highly efficient optogenetic actuators and reporters of neural activity, has endowed us with unprecedented resolution in spatiotemporal precision both in imaging neural activity as well as manipulating it with multiphoton microscopy. This readily available toolbox has introduced a so called all-optical physiology and interrogation of circuits and has opened new horizons when it comes to precisely, fast and non-invasively map and manipulate anatomically, molecularly or functionally identified mesoscopic brain circuits. The purpose of this review is to describe the advantages and possible pitfalls of all-optical approaches in system neuroscience, where by all-optical we mean use of multiphoton microscopy to image the functional response of neuron(s) in the network so to attain flexible choice of the cells to be also optogenetically photostimulated by holography, in absence of electrophysiology. Spatio-temporal constraints will be compared toward the classical reference of electrophysiology methods. When appropriate, in relation to current limitations of current optical approaches, we will make reference to latest works aimed to overcome these limitations, in order to highlight the most recent developments. We will also provide examples of types of experiments uniquely approachable all-optically. Finally, although mechanically non-invasive, all-optical electrophysiology exhibits potential off-target effects which can ambiguate and complicate the interpretation of the results. In summary, this review is an effort to exemplify how an all-optical experiment can be designed, conducted and interpreted from the point of view of the integrative neurophysiologist.

Visual stimulation with blue wavelength light drives V1 effectively eliminating stray light contamination during two-photon calcium imaging.

BACKGROUND: Brain visual circuits are often studied in vivo by imaging Ca2+ indicators with green-shifted emission spectra. Polychromatic white visual stimuli have a spectrum that partially overlaps indicators´ emission spectra, resulting in significant contamination of calcium signals. NEW METHOD: To overcome light contamination problems we choose blue visual stimuli, having a spectral composition not overlapping with Ca2+ indicator´s emission spectrum. To compare visual responsiveness to blue and white stimuli we used electrophysiology (visual evoked potentials -VEPs) and 3D acousto-optic two-photon (2P) population Ca2+ imaging in mouse primary visual cortex (V1). RESULTS: VEPs in response to blue and white stimuli had comparable peak amplitudes and latencies. Ca2+ imaging in a Thy1 GP4.3 line revealed that the populations of neurons responding to blue and white stimuli were largely overlapping, that their responses had similar amplitudes, and that functional response properties such as orientation and direction selectivities were also comparable. COMPARISON WITH EXISTING METHODS: Masking or shielding the microscope are often used to minimize the contamination of Ca2+ signal by white light, but they are time consuming, bulky and thus can limit experimental design, particularly in the more and more frequently used awake set-up. Blue stimuli not interfering with imaging allow to omit shielding. CONCLUSIONS: Together, our results show that the selected blue light stimuli evoke responses comparable to those evoked by white stimuli in mouse V1. This will make complex designs of imaging experiments in behavioral set-ups easier, and facilitate the combination of Ca2+ imaging with electrophysiology and optogenetics.

Goal-dependent tuning of muscle spindle receptors during movement preparation.

Voluntary movements are believed to undergo preparation before they are executed. Preparatory activity can benefit reaction time and the quality of planned movements, but the neural mechanisms at work during preparation are unclear. For example, there are no overt changes in muscle force during preparation. Here, using an instructed-delay manual task, we demonstrate a decrease in human muscle afferent activity (primary spindles) when preparing to reach targets in directions associated with stretch of the spindle-bearing muscle. This goal-dependent modulation of proprioceptors began early after target onset but was markedly stronger at the latter parts of the preparatory period. Moreover, whole-arm perturbations during reach preparation revealed a modulation of stretch reflex gains (shoulder and upper arm muscles) that reflected the observed changes in spindle activity. We suggest that one function of central preparatory activity is to tune muscle stiffness according to task goals via the independent control of muscle spindle sensors.

Sensory deprivation during early development causes an increased exploratory behavior in a whisker-dependent decision task.

Stimulation of sensory pathways is important for the normal development of cortical sensory areas, and impairments in the normal development can have long-lasting effect on animal's behavior. In particular, disturbances that occur early in development can cause permanent changes in brain structure and function. The behavioral effect of early sensory deprivation was studied in the mouse whisker system using a protocol to induce a 1-week sensory deprivation immediately after birth. Only two rows of whiskers were spared (C and D rows), and the rest were deprived, to create a situation where an unbalanced sensory input, rather than a complete loss of input, causes a reorganization of the sensory map. Sensory deprivation increased the barrel size ratio of the spared CD rows compared with the deprived AB rows; thus, the map reorganization is likely due, at least in part, to a rewiring of thalamocortical projections. The behavioral effect of such a map reorganization was investigated in the gap-crossing task, where the animals used a whisker that was spared during the sensory deprivation. Animals that had been sensory deprived performed equally well with the control animals in the gap-crossing task, but were more active in exploring the gap area and consequently made more approaches to the gap - approaches that on average were of shorter duration. A restricted sensory deprivation of only some whiskers, although it does not seem to affect the overall performance of the animals, does have an effect on their behavioral strategy on executing the gap-crossing task.

Relating information, encoding and adaptation: decoding the population firing rate in visual areas 17/18 in response to a stimulus transition.

Neurons in the primary visual cortex typically reach their highest firing rate after an abrupt image transition. Since the mutual information between the firing rate and the currently presented image is largest during this early firing period it is tempting to conclude this early firing encodes the current image. This view is, however, made more complicated by the fact that the response to the current image is dependent on the preceding image. Therefore we hypothesize that neurons encode a combination of current and previous images, and that the strength of the current image relative to the previous image changes over time. The temporal encoding is interesting, first, because neurons are, at different time points, sensitive to different features such as luminance, edges and textures; second, because the temporal evolution provides temporal constraints for deciphering the instantaneous population activity. To study the temporal evolution of the encoding we presented a sequence of 250 ms stimulus patterns during multiunit recordings in areas 17 and 18 of the anaesthetized ferret. Using a novel method we decoded the pattern given the instantaneous population-firing rate. Following a stimulus transition from stimulus A to B the decoded stimulus during the first 90ms was more correlated with the difference between A and B (B-A) than with B alone. After 90ms the decoded stimulus was more correlated with stimulus B than with B-A. Finally we related our results to information measures of previous (B) and current stimulus (A). Despite that the initial transient conveys the majority of the stimulus-related information; we show that it actually encodes a difference image which can be independent of the stimulus. Only later on, spikes gradually encode the stimulus more exclusively.