Research On Monitoring And Controlling Neural Circuits Basing On Femtosecond Laser And Random Access Scan

In the nervous system, all types of brain function are encoded in dynamic patterns of activity in neural circuits. Although breakthroughs have been achieved in some respects, but the operating principles in information processing of various kinds of cells consisting the neural networks remain enigmatic. Although multi-electrode extracellular recordings enable simultaneous recordings of the activities of hundreds of neurons, such recordings sample from widely distributed cell populations with poorly defined cell types and spatial relationships. The temporal resolution of commercial two-photon laser scanning microscopy cannot satisfy the requirement for the detection of fast functional signal, yet the prospect of direct brain control has been limited by lacking of suitable techniques. A random access two-photon fluorescence microscope constructed based on acousto-optic deflectors(AODs) and a femtosecond laser is an alternative and complimentary technique which enables high-resolution functional imaging of neural circuits. However, the selection and loading of calcium dyes as well as the analysis of calcium signals lack of perfect methods. In this paper, we introduce a systematical method consisting of calcium dyes selection and loading, calcium signals analysis and neural activities stimulation for the real-time monitoring and controlling activities of the neural circuits.We demonstrated the high spatial-temporal resolution of our constructed system by calcium imaging of the brain slice. By comparing the random access scanning mode to line scanning mode via simulation data, static fluorescence sample and calcium intensities of neurons, it is identified that random access scanning mode is more flexible in the signal detection and in the adjustment of signal to noise ratio.According to the optoelectronic detection results of membrane voltages variance and calcium transients to electronic stimuli, Fluo-5F and Fura-2 were chosen as the calcium dyes for indicating neural activities. Through injection calcium dyes to each individual neuron via somatic patch pipettes in sequence, the neuron population calcium loading was achieved. High frequency response parameter (HFRP) of different locations revealed that apical dendrite 50～100μm from the soma was favorable for detecting high frequency neural activities. The optical scaled template method was applied to detect calcium transients of neurons indicated with Fura-2 and the electronic activities of neuron population were reconstructed. A new method based on difference calculation was developed to analysis calcium signals of neurons marked with Fluo-5F and reconstruct the electronical activities. The efficiencies of the methods could be estimated by the corresponding evaluation methods.In addition, it is necessary to manipulate, stimulate or silence specific sets of neurons when determine their role in brain function. The femtosecond laser was used to stimulate neuron circuits in this study due to its ultrahigh peak power. Our results showed that the optical stimulation of a single neuron triggered significant calcium responses in the neighboring neurons. According to the responses, neural connections were estimated and the functional topology of the neural circuit was mapped. The optical identification of neural connections proved to be non-contact, non-destructive and highly reproducible, which would enable the characterizing of the dynamics of neural circuits.