In Vivo Labeling Of Active Neurons In Mice Medial Prefrontal Cortex Using CaMPARI Techology

Multi-channel in vivo recording techniques can monitor and record neuronal assemblies in free moving animals in real time,which is one of the main techniques for studying the neural coding mechanism of brain cognition.Benefit from it's extracellular recording characteristic,it can detect the firing sequence of the action potential,but it cannot further identify the cell-type and morphology of the recorded neurons.A new type of calcium(Ca2+)-indicating fluorescent protein tool,CaMPARI(calciummodulated photoactivatable ratiometric integrator)has been reported recently,which works as follow:CaMPARI undergoes efficient and irreversible green-to-red photoconvertion(PC)when two conditions of high intracellular Ca2+and ultraviolet light illumination coincide,allowing a temporally precise“activity snapshot" of the firing neuronal assemblies in vivo.According to the activation characteristics of the dual conditions(high Ca2+and UV light)of CaMPARI,we combined it with the multichannel in vivo technique to moniter the actived neuronal assemblies in the medial prefrontal cortex(mPFC)of the mice,providing a potential utility for studying of the active neurons and circuits in vivo.First,we inspected the feasibility of using CaMPARI technique to label the active neurons of mPFC during different fear behavior.Results show that due to different fear behavior activated the number of mPFC neurons is different,so observed that photonconversion rate of CaMPARI in mPFC is different also,the stronger the fear behavior activity,the higher the photonconversion rate of CaMPARI protein,specifically,the photonconversion rate of CaMPARI transfection neurons in shock group(75.1%)was greater than the photonconversion rate of platform group(30.5%),and the photonconversion rate of the non-fear group(18.7%)was the lowest.These results indicate that CaMPARI can specifically mark active neurons in the brain region under ultraviolet light.Secondly,in vitro patch clamp technique was used to explore the changes of light parameters in CaMPARI photonconversion of individual cortical neurons.According to the results on neurons recording by patch clamp,with frequency of 50 Hz induce cells to produce action potential or in a random frequency induced cells clusters of discharge after interval of 2 milliseconds apart ultraviolet light to illuminate(photoelectric paired stimulus),with the increasing of stimulus numbers,neurons expressing the CaMPARI protein,it' s green fluorescent intensity will gradually weaken,and the red fluorescence intensity will gradually increased.This provides the basis for the subsequent combination of multi-channel in vivo recording technology to in vivo label active neurons.Further,reference on neurons of CaMPARI protein in vitro photoconversion parameter change pattern,in mice mPFC combining multichannel recording technique in vivo,according to the recorded neurons action potential in vivo discharge sequence,give the action potential matching ultraviolet light illumination,a total of three mice in mPFC brain regions to carry out in vivo optical labeling active neurons using CaMPARI technology,in which a mouse mPFC brain regions near electrode recording site,confocal microscope imaging found that the one and only one neuron of CaMPARI protein happened photonconversion,prompt the neuron may be recorded by multichannel in vivo recording technology.More in-depth experiments are needed to further verify and improve the new method of labeling active neurons in vivo using CaMPARI technology.In summary,using the double-activated photonconversion property of CaMPARI,we explored a new method for in vivo labeling of active neurons recorded by the in vivo multi-channel recording technique,in order to provide a new research route for the analysis of the relationship between the structure and function of neurons.