Integrating Neurotropic Virus Tracing And Magnetic Resonance Imaging To Develop New Approaches For Dissecting Neural Networks

The connection of different neurons in the brain constitutes complex and sophisticated neural networks.The elucidation of the structure and function of the neural networks is essential to understanding the mechanisms of brain functions and disorders.Neurotropic virus based circuit tracing and magnetic resonance imaging(MRI)are two important approaches for analyzing neural networks.Their principal advantages are enabling precise targeting in neural circuit and in vivo observation,respectively.By combining these two technologies,powerful new tools have been developed for resolving neural networks,such as optogenetic functional magnetic resonance imaging(ofMRI)and chemogenetic functional magnetic resonance imaging(chemo-fMRI).This dissertation mainly focused on neural networks resolving methods that combining neurotropic virus tracing and MRI in two aspects,and aims to develop new approaches to analyze brain networks in vivo precisely and specifically.On the one hand,we attempted to integrate MRI reporter gene and neural circuit tracing virus tools so that the virus labeled neural connections could be observed in vivo by MRI instead of in vitro fluorescent imaging.First,we constructed a recombinant vesicular stomatitis virus(rVSV),rVSV-Ferritin-EGFP,which encoding the MRI reporter Ferritin and green fluorescent protein(EGFP).By injecting it into the mouse somatosensory cortex(SC)and imaging with ex vivo MRI,we observed significant T2 weighted MRI hypointensity in the brain regions of SC,motor cortex(MC),caudate putamen(CPu)and thalamus(TH),etc.The location of MRI hypo-signals highly overlap with EGFP signals.Our method enables ex vivo MRI and fluorescent imaging on the same subjects,and more importantly achieves the direct correlation between the whole brain-wide macroscopic neural networks observed by MRI and the mesoscopic neural networks observed by fluorescent imaging.However,mice injected with rVSV-Ferritin-EGFP virus were not allowed for in vivo MRI experiments due to VSV related biosafety limitations.In order to observe the neural connections in vivo,we constructed the non-pathogenic recombinant adeno-associated virus rAAV2-retro,rAAV-retro-CAG-Ferritin-WPRE-pA,which carrying the Ferritin gene.The recombinant virus was injected into the CPu brain region of mice.Then,we observed T2 weighted hypointense contrast in the brain regions of lasolateral amygdaloid nucleus(BLA),hippocampus(Hipp),and MC at different time points by in vivo MRI experiments.In conclusion,the Ferritin-EGFP encoding anterogradely trans-multi-synaptic rVSV can reveal the downstream brain regions that are connected to the injection site by ex vivo MRI.The Ferritin encoding rAAV2-retro can longitudinally display the upstream brain regions which projects neural fibers to the injection site by in vivo MRI.The method we proposed here makes it possible to monitor neurotropic virus labeled structure neural circuit by MRI in living animals.On the other hand,through molecular genetics strategies,chemogenetic element carried by rAAV was expressed in specific type of neurons in transgenic animals so that chemo-fMRI could be utilized to resolve downstream functional networks of specific type of neurons.For example,we evaluated the effectiveness of cell-type specific chemo-fMRI in analyzing the downstream functional networks of dopaminergic neurons in ventral tegmental area(VTA).We specifically activated dopaminergic neurons or CamKII-positive neurons(comprising of glutamatergic neurons and dopaminergic neurons)in VTA,meanwhile recording the BOLD-fMRI signal response in the whole brain.The results showed that after the activation of the dopaminergic neurons of VTA in right hemisphere,besides VTA,only medial prefrontal cortex(mPFC),cingulate gyrus(Cg)and septum showed minor BOLD signal response.No BOLD signal changes were observed in ventral striatum regions(including nucleus accumbens and olfactory tubercle)that directly receive substantial VTA dopaminergic innervation.After activating CamKII-positive neurons of VTA in right hemisphere,multiple brain regions showed significant BOLD signal changes,including VTA,mPFC,Cg,septum,Hipp,right insular,right TH,right MC,right parietal association cortex(PtA)and right visual cortex(ViC).Thus,we believe that dopamine release from dopaminergic neurons in VTA and concurrent downstream BOLD signal changes may not be corresponding.As a neuromodulator,the effect of dopamine on downstream BOLD signals is more complicated.Therefore,the downstream functional networks of dopaminergic neurons obtained by the ofMRI or chemo-fMRI methods need to be cautiously interpreted.