Fig. 6: Spatial and molecular organization of PFC projection to the major subcortical targets.
a, Schematics of the strategy for inferring neuronal projection of MERFISH clusters. The MERFISH and scRNA-seq data are integrated into a reduced dimensional space. An SVM is used to predict neuronal projection of the MERFISH neuron subtypes (Methods). b, UMAP visualization of cells derived from MERFISH (9,544 cells) and scRNA-seq (4,294 cells) data after integration. c, The ROC curves show the prediction powers of six projection targets; w/o represents the cells without projection information. d, A coronal slice showing in silico retrograde tracing from six injection sites, labeled by different colors as indicated. e, The inferred projection targets of molecularly defined excitatory neuron subtypes, represented by an alluvial diagram. f, PFC to PAG projection validation. Retrograde mCherry-expressing AAV was injected in PAG—injection scheme cartoon and injection site in PAG are shown (scale bar = 0.5 mm); brain slice of PFC was used for smFISH. mCherry (red) labeled neurons coexpressing the L5 ET 1 marker Pou3f1 (green), arrows in the enlarged image indicate double-labeled neurons. Co-immunostaining for mCherry protein with Pou3f1 RNA-FISH further confirmed extensive colocalization. (scale bars = 20 µm). Bar graph shows the percentage of mCherry positive cells that also express Pou3f1 (mean ± s.e.m., two-tailed t test, n = 4 biologically independent adult male mice, P < 0.001). Majority of mCherry+ neurons are Pou3f1+. Hypo, hypothalamus.
