To Establish A Cranial Window Method For Two-photon Imaging In Awake Mice

The complex neural network within the brain is formed by a large number of neurons connected by synapses.The transmission and processing of information between neurons is the functional basis of brain cognition and memory.In vivo imaging technology,combined with functional markers to record neuronal dynamic activity,has become one of the main methods of brain function research.The cranial window method solves the problem of skull opacity and provides technical support for direct observation of cortical nerve activity.However,the cranial window method also has problems such as fixed instability and cranial window obscuration,which hinders the study of imaging of conscious animals.Specifically,first,the mouse head is fixed.In the experiment,the mouse head is usually fixed by a fixed rod or a fixed piece,but the soft tissue remaining on the surface of the skull may cause the contact surface of the fixation device and the soft tissue to be unstable.This instability can result in reduced mechanical stability of the fixture when the awake mouse is active.At the same time,the lack of stability of the mechanical structure of the head can also cause the focal plane sway of the imaging link,and even the mechanical structure to fall off.Second,the sealing problem of the cranial window.In cranial window surgery,glue bonding is often used to seal the gap between the glass piece and the skull.This method is not strong because of the tight sealing of the cranial window,which causes the glue to enter the inside of the cranial cavity and cause inflammation of the brain.This not only destroys the phenomenon.The physiological environment inside the cranial window,and will cover or even cover the target area.These problems affect the functional imaging of awake mice,resulting in poor stability and low success rates for long-term observations.In response to the above problems,this paper established a cranial window method for awake mouse two-photon imaging.The mechanical stability of the fixation device was improved by the improved fixation method.The optimized surgical procedure enhanced the airtightness of the cranial window and reduced the occurrence of inflammation,thus successfully achieving long-term stable observation of the functional signals of cortical neurons in awake mice.Firstly,a new type of head fixing device is used to process a large amount of soft tissue on the surface of the skull based on the mechanical structure of the fixing device,and the fixing device and the rigid skull having the soft tissue removed are firmly connected to each other,thereby ensuring long-term high mechanical stability..Secondly,the direct contact between the glue and the glass is avoided by using the combination of the cranial window glass and the titanium ring;the cranial window mold is made according to the size of the cranial window glass to accurately grasp the size of the cranial window;and the processing is performed during the installation process of the cranial window glass.The fixed bracket fixes and compacts the cranial window glass,achieving high airtightness of the cranial window glass and the skull,thereby preventing the glue from entering the cranial cavity and inflammation.Finally,long-term functional imaging of the brain of awake mice was achieved.Using the above optimization methods of head fixation and cranial window surgery,we performed cranial window surgery on different strains of transgenic mice and performed in vivo imaging of cortical neurons of mice 14 days after operation.For awake mice,the neuron bodies and dendritic structures at the depth of 350 um in the cortex were clearly observed,which confirmed that the method had good mechanical stability and transparency of the cranial window.At the same time,we also applied this scheme to the study of neuron function.Mouse motor vision was used as a model to observe the neuronal soma at 10 and 57 days after operation.When visual stimulation was given,the calcium response signal could be recorded steadily.This paper improves the cranial window operation method,establishes a complete cranial window operation scheme,and based on this scheme can record the long-term functional signals of neurons.The establishment of the method provides a perfect cranial window surgery solution,which solves the jitter of the awake mouse during imaging and the occlusion of the inflammatory tissue inside the cranial window,and improves the success rate of neuron imaging in the awake mouse.It provides technical support for the long-term stable recording of neural activity,and is of great significance for exploring the function of the neural circuit and understanding the neural mechanism behind the complex behavior of the brain.