Dual-color Fluorescence Reactivation Of Mouse Neurons And The Whole Mount MRNA In Situ Hybridization

Neurons are the basic units of the brain structure and function.Numerous neurons extend neurites from their somas to form neural circuits and to communicate each other.The molecular events like genes expression and proteins action occur constantly in neurons and neural circuits.The projections and the molecular activities of neurons underlie the brain structure and function together.The combination of genes expression analysis and the single neuron morphology,is better for the understanding of neuronal types and function.However,for intact tissues there is no report about the simultaneous acquisition of the single neuron morphology and the genes expression information.The development of fluorescence micro-optical sectioning tomography(fMOST)sets the foundation for acquisition of the brain connectivity maps and the molecular maps at whole mount scale.The chemical sectioning of GFP and its pH-sensitive variants,makes fMOST have both the speed of wide-field imaging and the resolution of confocal imaging.However,two-color chemical sectioning is both a need and a challenge.For the whole mount biological tissues such as the intact mouse brain,when the neuron shape and projections are acquired via chemical sectioning,there are also methodological difficulties to obtain the genes expression information at the same time.Focusing on the need for simultaneous acquisition of the single neuron morphology and the genes expression information across the whole brain,this thesis aims at the specific problems on the methods of the two aspects research.The main contributions of this thesis are as following:(1)The red fluorescent protein,pHuji,is verified to be compatible with resin embedding and chemical sectioning imaging.The simultaneous two-color chemical sectioning is achieved via pHuji plus EGFP or EYFP.According to the demands of resin embedding and chemical sectioning to fluorescent proteins,pHuji,a pH-sensitive red fluorescent protein which may meet these demands,is selected and cloned into the adeno-associated virus(AAV)vector.And the sparse and high fluorescence intensity labeling of neurons morphology comes true.Thus the two-color chemical sectioning is realized through dual-color labeling by pHuji plus EGFP.(2)The dual-color labeling based on pHuji and EGFP,makes the simultaneous high fluorescence intensity labeling of neurons morphology and synapses structures come true.It sets the basis for simultaneous acquisition of both the single neuron morphology and the synapses distribution via fMOST and two-color chemical sectioning.In this thesis the neurons morphology is labeled by AAV-mediated pHuji expression,and the presynaptic structures are labeled by the fusion protein of complete EGFP and Synapsin,while the postsynaptic structures are labeled by the fusion protein of complete EGFP and PSD95.Eventually,the synapses are labeled with high fluorescence intensity and high accuracy.(3)Based on the tissue permeabilization techniques of iDISCO and the signal amplification approach termed hybridization chain reaction,the whole mount fluorescence in situ hybridization to intact mouse brain is developed.To target endogenous or exogenous mRNAs in medium or high abundance,many oligonucleotide probes are devised to act as the primary probes and the hairpin amplifiers are labeled by fluorescent dyes to act as the secondary probes.Thus the specific mRNA is labeled precisely.Through the combining of whole mount in situ hybridization and the real time counterstaining by nucleic acid fluorescent dyes(propidium iodide or DAPI)during fMOST imaging,the distribution of mRNAs and the cytoarchitecture reference of the same sample are acquired simultaneously.Therefore,a simple,rapid way to precisely locate mRNAs in the whole brain has been developed.(4)The intact mouse brain of Thy1-EGFP-M is permeabilized via non-methanol dependent iDISCO and then the whole mount in situ hybridization is performed.This results in the obtaining of both the genes expression information and the preservation of neurons morphology labeled by fluorescent proteins.Thus it proves the good compatibility of the labeling of neurons morphology via fluorescent proteins and the whole mount in situ hybridization based on iDISCO.