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.
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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