The ability to simultaneously detect and control neuronal activity grants the capacity to unravel the causal relationships behind brain processing. Nevertheless, scaling-up this technology to the whole encephalon of a vertebrate is a challenging task, and this impedes reaching a global comprehension of how the brain works. To this aim, we developed an optical system that enables fast noninvasive functional imaging of the whole brain of the zebrafish larva and, at the same time, allows us to optogenetically stimulate the activity of arbitrary sets of neurons in the volume. Our preliminary results show that with this optical system we can both consistently evoke activation of neurons in the stimulation site and identify distantly-located functionally-connected neurons placed downstream in the activated circuits. The expansibility of this concept paves the way for the brain-wide mapping of functional connectivity in the zebrafish larva.
We present the development of a custom-made two-photon light-sheet microscope optimized for high-speed (5 Hz) volumetric imaging of zebrafish larval brain for the analysis of neuronal physiological and pathological activity. High-speed volumetric two-photon light-sheet microscopy is challenging to achieve, due to constrains on the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarization, and we implemented remote scanning of the focal plane to record without disturbing the sample. Two-photon illumination is advantageous for zebrafish larva studies since infra-red excitation does not induce a visual response, that otherwise would affect the neuronal activity. In particular, we were able to record whole-brain neuronal activity of the larva with high temporal- and spatial-resolution during the nocturnal period without affecting the circadian rhythm. Analyzing the spatially resolved power spectra of GCaMP signal, we found significant differences for several frequency bands between the day/night phases in various brain regions. Moreover, we studied the fast dynamics that characterize the acutely induced pathological epileptic activity of the larvae, identifying the brain structures that are more susceptible to the action of the epileptogenic drug. In conclusion, the high speed two-photon light-sheet microscope that we developed is proving to be an important tool to study both the physiological and the pathological activity of the zebrafish larval brain without undesired visual stimulation.
KEYWORDS: In vivo imaging, Optogenetics, Cortical activation, Calcium, Brain mapping, Organisms, Photostimulation, Radiofrequency ablation, Actuators, Control systems
Neuronal networks in living organisms are highly interconnected. Usually, to study their functional roles in healthy conditions, task-evoked neuronal responses are correlated with the behavioral readout in freely moving or head-fixed animals. Recently, optogenetics proved to be a useful tool to manipulate targeted neuronal circuits using light. Optogenetic photostimulation of different cortical motor areas revealed distinct and reproducible motor movements: Rostral Forelimb Area (RFA) is critically involved in controlling grasping-like movements, while Caudal Forelimb Area (CFA) has a role in tap- or locomotion-like movements. In parallel, the development of red-shifted genetically encoded calcium indicators (red-GECIs) like jRCaMP1a allowed to reduce the spectral overlap with the most common optogenetic actuator, channelrhodopsin-2 (ChR2). Therefore, by combining these optical tools it is possible to develop all-optical systems, which are smart approaches for long-term low-invasive studies of neuronal patterns.
Here, in order to understand the functional role that cortical ensembles play in motor generation and control, we developed a cross-talk free large-scale all-optical system for unraveling cortical neuronal patterns associated with optogenetically-evoked movements. We demonstrated that the motor cortex exhibits precise inter-regional patterns during movement initiation of grasp- or locomotion-evoked movements. Moreover, the cortical activation covers most of the related light-based optogenetic maps, revealing that a strong local neuronal connectivity is associated with optogenetically-evoked complex movements. To confirm the relevance of local connectivity for the generation of complex movements we used both optogenetic interference and pharmacological inhibition, showing that movement disruption is linked to reduced cortico-cortical coactivation.
We developed an all-optical system that couples large-scale cortical imaging with chronic light-based motor mapping in awake mice. By AAV-mediated cortical transfection, we induced the co-expression of the red-shifted genetically encoded calcium indicator and a light-sensitive optogenetic actuator ChR2 over both the rostral and caudal forelimb areas, which was stable over several months. No evidence of cross-talk was detected during illumination of ChR2+ neurons with the light source used for RCaMP1a excitation. Light-based motor mapping coupled with wide-field imaging of neuronal activation in awake mice revealed spatiotemporal patterns of cortical activation specific for movement category.
Although it is well known that zebrafish display the behavioural signature of sleep, the neuronal correlates of this state are not yet completely understood, due to the complexity of the measurements required. For example, when performed with visible excitation light, functional imaging can disrupt the day/night cycle due to the induced visual stimulation. To address this issue, we developed a custom-made two-photon light-sheet microscope optimized for high-speed volumetric imaging. By employing infra-red light (not visible to the larva) for excitation, we are able to record wholebrain neuronal activity with high temporal- and spatial-resolution without affecting the sleep state. In two-photon light-sheet microscopy the maximum achievable frame rate is limited by the signal-to-noise ratio. To maximize this parameter, we optimized our setup for high peak power of excitation light, while finely controlling its polarisation, and we implemented remote scanning of the focal plane to record without disturbing the sample. Using this setup, as a preliminary result, we characterized the intensity spectra of neuronal calcium traces of 4 days post fertilisation larvae during the day/night phases. We aim to extend these results to multiple brain regions and frequency bands.
Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal–oxide–semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast.
In the last few years, optogenetic tools and optical functional indicators are increasingly used together to perform simultaneous manipulation and recording of neuronal activity. Nevertheless, this method has still some limitations mainly due to the spectral cross-talk between optogenetic actuators and functional sensors [1;2]. To address this issue, red variants of genetically encoded calcium indicators (red-GECIs) have been recently developed [3;4]. The main goal of this project is to develop a full-optical system that allows effective interrogation of brain circuits. To this aim, we combined a red-shifted calcium indicator (jRCaMP1a), with the most common blue-light activated opsin, Channelrhodopsin II (ChR2). The results presented here show: (I) extended expression of the full-optical system that covers all the motor areas, (II) functional correlation between the laser power and the evoked neuronal activity, (III) segregation of the cortical functional areas of two different forelimb evoked movements. The future perspective of this project concerns the study of the functional areas correlation during optogenetically-evoked forelimb complex movements.
In this study, we developed a wide-field all-optical system based on a red-shifted GECI (RCaMP1a) combined with channelrhodopsin II (ChR2) for simultaneous stimulation and readout of neuronal activity. Our results show that RCaMP1a transfection in primary motor cortex extends all over the cortical motor areas. The RCaMP1a and ChR2 reporter expression largely overlap, thus allowing the stimulation and readout from the same functional areas. Furthermore, we characterized the functional response by performing single pulse optogenetic stimulation and we observed that evoked calcium signals increase at increasing laser power. In order to study the cortical activation underlying a specific motor behavior, we performed optogenetic-stimulation of the Rostral Forelimb Area (RFA) with a train of lasers pulses. We observed that during 1s of 16 Hz train stimulus the animals suddenly start grasping with the contralateral forelimb. Cortical dynamics recorded during the optogenetically-triggered motor task show correlated activity between the RFA and the nearby motor areas. The all-optical system optimization and the possibility to link the neuronal population activity with the animal behavior would be a key point in understanding the network activity underlying a specific behavior.
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