Whole Brain Mapping of Long-Range Direct Input to Glutamatergic and GABAergic Neurons in Motor Cortex

In this study, we used a monosynaptic rabies tracing technique to label the whole-brain inputs to specific cell types in the MOp and MOs simultaneously in a same transgenic mouse. First, 150 nl AAV helper mixtures were injected into the ipsilateral MOp (AP:1.54 mm, ML:1.70 mm, DV:-1.50 mm) and MOs (AP:1.54 mm, ML:0.50 mm, DV:-1.35 mm) in Thy1-cre or Vgat-cre mice respectively, mixed with rAAV2/9-Ef1α-DIO-BFP-2a-TVA-WPRE-pA and rAAV2/9-Ef1α-DIO-RG-WPRE-pA as the ratio of 1:2. Three weeks later, 300 nl RV-ΔG-EnVA-EGFP and RV-ΔG-EnVA-Dsred were injected into the two subregions of the MC respectively. One week later, the mice were perfused. The titer of both AAVs is 2.00E+12 vg/ml, while the titer of RV is 2.00E+8 IFU/ml. The virus used was produced by BrainVTA. The amplification origins of RVs were from SADΔG-EGFP (EnvA) (Wickersham et al., 2007b; Osakada et al., 2011). The AAV virus vectors were constructed by BrainVTA. The coding region of the TVA element and RG element were obtained from the AAV-EF1a-FLEX-GT plasmid (Addgene plasmid 26198) and pAAV-EF1a-FLEX-RG plasmid (Addgene plasmid 98221) respectively, and were separately constructed into the DIO cassette of the plasmid pAAV-EF1a-DIO-hChR2 (H134R)-EYFP (Addgene plasmid 20298) (Hu et al., 2016). During the stereotaxic injection, we set the Bregma as the zero point of the stereotactic coordinate. Briefly, Bregma is the junction of the coronal suture and the sagittal suture of the skull, a...
Source: Frontiers in Neuroanatomy - Category: Neurology Source Type: research