Fig. 4: VP Glu neurons mono-synaptically innervate the locus coeruleus.

a Schematic diagram for injection of AAV-EF1α-DIO-eYFP into the VP in Vglut2-Cre mice to label VP Glu neurons. b eYFP-labeled axonal fibers (green) of VP Glu neurons and noradrenergic (NA) neurons in the locus coeruleus (LC), labeled with tyrosine hydroxylase (TH)-antibody (red). c Diagram and typical images showing injection of AAV-CaMKII-ChR2-eYFP into the VP and AAV-EF1α-DIO-mCherry into the LC in DBH-Cre mice. d Blue light-evoked EPSCs at a holding potential of − 50 mV and spikes in an NA neuron (Vm = − 46 mV). e Representative traces and summary showing photo-EPSCs in LC NA neurons at baseline (black trace) and in the presence of 1 µM TTX + 100 µM 4-AP (red trace) or 20 µM CNQX (blue trace). n = 6 neurons. f Diagram of Cre-dependent retrograde tracing with rabies virus. g Example images of viral expression in the LC. Starter cells (white arrows) co-expressing AAV-DIO-TVA-eYFP (green) and RV-CVS-EnvA-ΔG-tdTomato (red). h–j Example images of tdTomato-labeled neurons in the VP Glu neurons. Most tdTomato (red) neurons were stained by CaMKII-antibody (green). n = 3 mice. k Schematic diagram of fiber photometry recording from LC NA neurons innervated by VP Glu neurons. l, m Example images and summary showing that most GCaMP6(+) LC neurons were stained by the TH-antibody. n = 3 mice. n–t Heat maps, averaged traces, peak z-score, and AUC of z-score of fluorescent signals in the VP of GCaMP6 and eYFP mice when the mice were rearing, recognizing novel object, encountering unfamiliar mice, and receiving an air puff. Two-tailed t tests for (s, t). GCaMP6: n = 15 trials from 5 GCaMP6 mice. eYFP: n = 8 trials from 4 eYFP mice. P-values are two-sided. Mean ± SEM in (j, m, r–t). The source data are in the Supplementary Dataset, Source Data.xlsx. Statistical results are in Supplementary Table 1.