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Viral-genetic tracing of the input–output organization of a central noradrenaline circuit



Deciphering how neural circuits are anatomically organized with regard to input and output is instrumental in understanding how the brain processes information. For example, locus coeruleus noradrenaline (also known as norepinephrine) (LC-NE) neurons receive input from and send output to broad regions of the brain and spinal cord, and regulate diverse functions including arousal, attention, mood and sensory gating1,2,3,4,5,6,7,8. However, it is unclear how LC-NE neurons divide up their brain-wide projection patterns and whether different LC-NE neurons receive differential input. Here we developed a set of viral-genetic tools to quantitatively analyse the input–output relationship of neural circuits, and applied these tools to dissect the LC-NE circuit in mice. Rabies-virus-based input mapping indicated that LC-NE neurons receive convergent synaptic input from many regions previously identified as sending axons to the locus coeruleus, as well as from newly identified presynaptic partners, including cerebellar Purkinje cells. The ‘tracing the relationship between input and output’ method (or TRIO method) enables trans-synaptic input tracing from specific subsets of neurons based on their projection and cell type. We found that LC-NE neurons projecting to diverse output regions receive mostly similar input. Projection-based viral labelling revealed that LC-NE neurons projecting to one output region also project to all brain regions we examined. Thus, the LC-NE circuit overall integrates information from, and broadcasts to, many brain regions, consistent with its primary role in regulating brain states. At the same time, we uncovered several levels of specificity in certain LC-NE sub-circuits. These tools for mapping output architecture and input–output relationship are applicable to other neuronal circuits and organisms. More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

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Figure 1: Strategy and proof-of-principle of TRIO and cTRIO.
Figure 2: Presynaptic input to LC-NE neurons revealed by rabies-mediated trans-synaptic tracing.
Figure 3: Input–output relationship of LC-NE neurons revealed by TRIO and cTRIO.
Figure 4: Broad output divergence of LC-NE neurons revealed by projection-based viral-genetic labelling.


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We thank N. Makki for initiating the locus coeruleus project, Stanford and UNC Viral Cores for producing AAVs, K. Touhara for support, D. Berns for suggesting the TRIO acronym, and members of the Luo laboratory, L. de Lecea and S. Hestrin for critiques. L.A.S. is supported by a Ruth L. Kirschstein National Service Research Award from NIMH, X.J.G. is supported by a Stanford Bio-X Enlight Foundation Interdisciplinary Fellowship, B.W. is supported by a Stanford Graduate Fellowship and an NSF Graduate Research Fellowship, E.J.K. is supported by EU FP7 BrainVector (no. 286071), K.M. was a Research Specialist and L.L. is an investigator of HHMI. This work is supported by an HHMI Collaborative Innovation Award.

Author information

Authors and Affiliations



L.A.S. performed all the experiments on locus coeruleus input, output and TRIO analysis, as well as motor cortex TRIO experiments and analysis. K.M. designed the TRIO and cTRIO methods, made all the constructs, performed proof-of-principle experiments for TRIO along with B.W. and performed rat TRIO and mitral cell experiments. X.J.G. performed statistical analysis together with L.A.S. K.T.B. participated in testing TRIO and cTRIO conditions and is co-supervised by R.C.M. and L.L. K.E.D. and J.R. provided technical support. S.I. and E.J.K. produced CAV-FLExloxP-Flp. L.L. supervised the project and wrote the paper together with L.A.S., with contributions from all authors, in particular K.M. and X.J.G.

Corresponding author

Correspondence to Liqun Luo.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Controls for TRIO and cTRIO at the motor cortex.

ac, Negative control experiments omitting CAV-Cre for TRIO (a), and omitting CAV-FLExloxP-Flp (b) or the Rpb4-Cre transgene (c) for cTRIO showed only local non-specific infection of RVdG. This background labelling is likely due to Cre- or Flp-independent leaky expression of a small amount of TVA–mCherry (TC), too low for mCherry to be detected but still capable of permitting infection by EnvA-pseudotyped RVdG due to the high sensitivity of TVA10. d, Quantification for three controls (n = 4, 4, 7 animals, respectively). By comparison, 672 GFP+ neurons were counted in the same region for an experimental brain that has the lowest starter cells among the 11 brains whose data were used for quantitative analysis of motor cortex TRIO input tracing. These background cells were restricted within 500 μm of the injection site. Because of these observations, GFP+ cells on sections within 600 μm of the injection site were excluded from the input analysis in Fig. 1g. Scale bar, 100 µm. Error bars, s.e.m.

Extended Data Figure 2 TRIO applied to rat primary motor cortex.

a, Schematic of injection sites used for TRIO in rat motor cortex (see Fig. 1c for details of the viruses). Two different C regions were tested: striatum or contralateral motor cortex (cMC). b, c, Coronal section of rat motor cortex stained with DAPI (blue). Starter pyramidal neurons projecting to contralateral motor cortex (b) or striatum (c) (yellow, a subset indicated by arrowheads) can be distinguished from neurons receiving CAV-Cre and AAV-FLExloxP-TC (red) or GFP from RVdG (green). Bottom insets, coronal sections showing representative presynaptic GFP+ cells in somatosensory cortex (SC) or thalamus (Th). These data indicate that callosal-projecting neurons and striatum-projecting neurons in rat motor cortex both receive direct synaptic input from somatosensory cortex and thalamus (n = 2 animals for cMC C region; n = 3 animals for striatum C region). d, e, Omitting CAV-Cre for TRIO in the rat also resulted in local non-specific infection of RVdG. On average 200 cells were observed (n = 4 animals) within 800 µm from the injection site in these control experiments. By comparison, 1,392 GFP+ neurons were counted in the same region of a TRIO sample that has the lowest starter cells among the 5 brains analysed. Scale bars, 100 µm. Error bars, s.e.m.

Extended Data Figure 3 Evaluation of CAV-Cre spread by using the OB→APC projection.

a, Four representative 60-μm coronal sections of the CAV-Cre injection site in the anterior piriform cortex (APC) of four Ai14 Cre-reporter mice. Red, tdTomato; green, retrobeads; blue, DAPI. The location of the injection site was readily visualized by concentrated retrobeads. D, dorsal; L, lateral; M, medial; V, ventral. In each mouse, we determined the minimal distance (D) between the injection site and layer 1a of the piriform cortex, where mitral cell axons terminate, or lateral olfactory tract, where mitral cell axon bundles are present. Dashed lines represent the boundary between layer 1a and layer 1b. For each sample, we counted the number of tdTomato-labelled mitral cells (numbers below each image) from serial olfactory bulb (OB) sections. b, An example 60-μm coronal section of the OB. Both tdTomato and retrobeads signals were found to be mostly restricted to the mitral cell layer (M) of the main olfactory bulb (MOB) and accessory olfactory bulb (AOB) with minor labelling in the granule cell layer (Gra). As AOB mitral cells do not form synapses in the APC, this observation indicates that CAV-Cre can infect axons-in-passage. c, Distribution of D among 26 injections (x axis) and relationship between D and the numbers of labelled cells in the MOB (y axis). d, Histogram based on c. Dense labelling (over 1,000) was obtained only when D < 100 μm. CAV-Cre injections with D > 800 μm rarely labelled the OB (2.8 ± 1.9 cells per bulb, n = 4 animals). e, Cumulative distribution plot of MOB cell counts. A sample of the ninth smallest D (D = 200 μm) reached 90% of the labelling (indicated by vertical dotted line) detected in all 26 samples, suggesting that given our sample distribution, 90% of axonal transduction occurred within 200 μm from the CAV-Cre injection site. Scale bars, 100 µm. Error bars, s.e.m.

Extended Data Figure 4 Evaluation of retrograde infection by CAV-Cre.

a, Representative coronal sections of the injection sites where CAV-Cre plus retrobeads were delivered into the olfactory bulb, dorsal hippocampus, auditory cortex, cerebellum or medulla of the Ai14 Cre-reporter mice (see Methods for coordinates). Red, tdTomato; green, retrobeads; blue, DAPI. tdTomato labelling was densest at the injection site, and corresponded with the presence of retrobeads. We did not observe dense tdTomato or retrobeads labelling in other brain regions adjacent to the injection site unless these sites sent direct projections to the injection site, indicating that for our experiments, CAV-Cre was efficiently and specifically delivered to the targeted brain regions. n = 4 animals per injection site. b, Representative coronal sections of brain regions that contained tdTomato+ labelling of specific cell populations known to project to CAV-Cre injection sites. The following is a partial list: neurons projecting to olfactory bulb (first column): ipsi- and contralateral anterior olfactory nucleus (AON), piriform cortex (Pir), nucleus of the lateral olfactory tract (nLOT), but not contralateral olfactory bulb; to dorsal hippocampus (second column): lateral and medial septum (LS, MS) and entorhinal cortex (Ent); to auditory cortex (third column): somatosensory cortex (SC), entorhinal cortex (Ent), and medial geniculate nucleus (MGN); to cerebellum (fourth column): contralateral pontine nuclei (PN) and inferior olive (IF); to medulla (fifth column): insular cortex (Ins), central amygdala (CeA), and paraventricular hypothalamic nucleus (PVH). Coronal images are composites generated from overlapping tiled images. Insets show high magnification images of boxed regions. Bottom, sagittal schematic of the CAV-Cre injection sites (a) and the approximate location of the two representative coronal sections above. Scale bars, 1 mm; inset, 100 µm.

Extended Data Figure 5 Controls for Dbh-Cre-based trans-synaptic tracing and TRIO analysis in locus coeruleus.

a, A Representative coronal section of the locus coeruleus from a mouse heterozygous for Dbh-Cre and Ai14 Cre-reporter transgenes. Sections were labelled with an antibody against tyrosine hydroxylase (TH), an enzyme in the biosynthetic pathway for noradrenaline (green), while cells expressing Cre recombinase are visible by expression of tdTomato (red). b, Quantification of the number of tdTomato+ neurons in the locus coeruleus that were also labelled by TH antibody (n = 3 animals). Every 50-µm section through the locus coeruleus was collected for quantification. Qualitatively, all TH+ cells expressed tdTomato; however, we cannot determine quantitatively because we could not accurately count TH+ cells due to dense process staining. c, Top, schematic for negative control where AAVs that express Cre-dependent TVA–mCherry fusion (TC) and rabies glycoprotein (G) were injected into the locus coeruleus of wild-type mice, followed by injection of RVdG. Middle, coronal section of the locus coeruleus stained with DAPI (blue) shows a small number of GFP+ neurons at the injection site. The dotted rectangle highlights GFP+ neurons magnified in the bottom panel. d, Top, in this negative control, Dbh-Cre mice received Cre-dependent TVA–mCherry fusion (without rabies glycoprotein) via AAV injection into the locus coeruleus, followed by RVdG. Middle, a coronal section of the locus coeruleus stained with DAPI (blue) shows infection of Cre+ locus coeruleus neurons with TC (red) or TC and RVdG (yellow) at the injection site. The dotted rectangle highlights infected locus coeruleus neurons magnified in the bottom panel. Most green cells are also red. No GFP+ cells were observed outside the region immediately adjacent to the injection site, indicating that trans-synaptic tracing depends on rabies glycoprotein. e, Quantification of the number of GFP+ cells (c), or GFP+ cells that did not colocalize with TC (d), that were observed in the experiments described in (c, n = 8 animals) and (d, n = 6 animals). By comparison, 1,381 GFP+ neurons were counted in the same region for an experimental brain that has the median number of starter cells among the 9 brains. For explanation of background labelling, see Extended Data Fig. 1a–c. In either case, no GFP+ neurons were visible >800 μm away from the injection site. f, Schematic of brain regions quantified for presynaptic GFP+ neurons. Regions approximately 800 μm anterior and posterior to the centre of the locus coeruleus were excluded from analysis due to local background labelling from TVA–mCherry fusion and GFP. Scale bars, 50 µm (a), 1 mm (c, d, middle panels), 100 µm (c, d, bottom panels). Error bars, s.e.m.

Extended Data Figure 6 Purkinje cell axons contact noradrenaline processes in the locus coeruleus.

a, Coronal sections counterstained with DAPI (blue) showing representative GFP+ Purkinje cells (green) from Dbh-Cre trans-synaptic tracing experiments described in Fig. 2. Labelled Purkinje cells span the anterior–posterior axis, but are enriched in the medial portion of the ipsilateral cerebellum. b, Sagittal section through the locus coeruleus of mice heterozygous for the transgenes Pcp2-Cre and Ai14, in which tdTomato (tdT) expression was restricted to cerebellar Purkinje cells and their processes (red). Sections were labelled with DAPI (blue) and anti-TH antibody (green) to label LC-NE neurons. The right panel is a maximum-projection confocal stack taken with a 40× objective of the boxed region in the left panel. Purkinje cell axons are intermingled with TH+ locus coeruleus neurons and their processes. c, Left, Representative image of a horizontal section collected through the locus coeruleus of a mouse heterozygous for the transgenes Pcp2-Cre and Ai14. Sections were stained with anti-TH antibody (green) to label LC-NE neurons and their processes, and anti-gephyrin (geph) antibody (white) to label inhibitory post-synaptic densities. Middle, maximum-projection confocal stack taken with a 40× objective of the dashed box of the left panel showing the overlap between tdTomato+ Purkinje cell axons and TH+ LC processes. Right, high magnification of the dashed box of the middle panel, showing that several of these contact points also contained gephyrin+ puncta (arrowheads) within green processes apposing the red processes, consistent with GABAergic Purkinje cell axons forming synapses onto dendrites of TH+ LC-NE neurons. Images in a were derived from larger composite images generated by a Leica Ariol Slide Scanner. A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral. Scale bars, 1 mm (a; b and c, left), 100 μm (a, inset; b, right), 10 μm (c, middle and right).

Extended Data Figure 7 Spatial distribution of LC-NE neurons projecting to distinct output brain regions.

a, Representative images of individual LC-NE neurons labelled within the locus coeruleus by injection of CAV-Cre at specific output sites (see Extended Data Fig. 4a) in Ai14 Cre-reporter mice. Coronal sections through the locus coeruleus were collected in order and stained with anti-TH antibody (pseudocoloured in green). All tdTomato (tdT)+ neurons within the locus coeruleus were also TH+, and many of these cells also contained retrobeads (green in inset). Injection of CAV-Cre into the olfactory bulb, hippocampus or auditory cortex resulted in high tdTomato expression in NE+ neurons within the locus coeruleus, whereas tdTomato labelling was almost completely absent in adjacent brain regions, indicating that regions next to the locus coeruleus contribute minimal projections to these output sites. However, CAV-Cre injected into the cerebellum or medulla labelled NE+ locus coeruleus neurons as well as adjacent, NE cell populations (a subset of which are highlighted by arrowheads). b, The locations of tdTomato+ LC-NE neurons from sequential 50-μm coronal sections collected through the entire locus coeruleus were transferred to corresponding sections of a digital locus coeruleus model and are represented by coloured dots (see Methods). c, Schematic of the dorsal/ventral and medial/lateral classifications used with tdTomato+ LC-NE neurons occurring from CAV-Cre injections into the olfactory bulb (left) or cerebellum (right) of Ai14 mice. These classifications were made by drawing horizontal and vertical lines through the cross (b) designating the middle of each locus coeruleus section. d, Quantification of the fraction of tdTomato+ LC-NE cells in each locus coeruleus section along the anterior–posterior axis of the locus coeruleus. No significant differences were observed for the anterior–posterior distribution of tdTomato+ LC-NE neurons projecting to different output sites. e, Quantification of the medial–lateral distribution of LC-NE neurons projecting to different output sites. LC-NE neurons showed no bias in the medial versus lateral portion of the locus coeruleus, regardless of where they sent projections. f, Quantification of the dorsal–ventral distribution of tdTomato+ LC-NE neurons projecting to different output sites. Although no bias was observed in the posterior locus coeruleus, significant differences were observed in the anterior and mid-LC. Specifically, LC-NE neurons projecting to the forebrain showed a dorsal bias for tdTomato+ cell labelling within the anterior locus coeruleus, whereas LC-NE neurons projecting to the cerebellum and medulla were located in more ventral portions of the anterior- and mid-LC. n = 4 animals per CAV injection site. Data in d was analysed with one-way ANOVA. Data in e, f were analysed by first performing two-way ANOVA, which did not uncover any significance in the medial/lateral bias of tdTomato+ LC-NE neurons. Two-way ANOVA determined that (1) the location of the CAV injection site contributes to the dorsal/ventral bias of tdTomato+ LC-NE neurons within the locus coeruleus (P < 0.0001), (2) there is interaction between the CAV injection site and the location (anterior, mid, posterior) of tdTomato+ NE neurons within the locus coeruleus (P = 0.0389), and (3) the locus coeruleus subdivisions themselves did not significantly contribute to the variance observed in tdTomato+ LC-NE neurons. One-way ANOVA and post hoc Tukey’s multiple comparison were then performed to test the significance of dorsal/ventral bias in each locus coeruleus region based on CAV injection sites. Scale bars, 50 µm. Error bars represent s.e.m. *P < 0.05; **P < 0.01, ***P < 0.001.

Extended Data Figure 8 Controls for locus coeruleus cTRIO.

a, Top, schematic for negative controls where AAVs expressing Flp-dependent TVA–mCherry fusion and rabies glycoprotein were injected into the locus coeruleus of Dbh-Cre mice, followed by RVdG injection into the locus coeruleus, but the CAV-FLExloxP-Flp injection was omitted. Middle, coronal section of the locus coeruleus stained with DAPI (blue) shows a small number of GFP+ neurons at the injection site. The dotted rectangle highlights GFP+ neurons magnified in the lower panel. b, Top, schematic for negative control where CAV-FLExloxP-Flp was injected into the olfactory bulb and AAVs expressing Flp-dependent TVA–mCherry fusion and rabies glycoprotein were injected into the locus coeruleus of wild-type mice, followed by RVdG injection; hence there was no Cre to mediate Flp expression in locus coeruleus cells. Middle, coronal section of the locus coeruleus stained with DAPI (blue) shows a small number of GFP+ neurons at the injection site. The dotted rectangle highlights GFP+ neurons magnified in the lower panel. c, Quantification of GFP+ background labelling in the locus coeruleus (n = 4 and 8 animals). This labelling is likely caused by leaky TVA expression as discussed in Extended Data Fig. 1. In none of these control experiments did we observe GFP+ or TC+ neurons >800 μm away from the injection site. Scale bars, 1 mm (middle panels), 100 µm (lower panels). Error bars represent s.e.m.

Extended Data Figure 9 Simulation of input convergence in Dbh-Cre tracing experiments.

In the sparsest Dbh-Cre trans-synaptic tracing brain, 4 starter cells received input from 43 distinct input regions (309 input neurons, see Supplementary Table 2, sample number 8). In the second sparsest sample, 22 starter cells received input from 66 distinct input regions (756 input neurons; see Supplementary Table 2, sample number 9). a, The relation between the number of input regions for each LC-NE starter cell and the probability of observing >42 (left) or >65 (right) input regions in simulation, assuming that each starter cell receives input from a given region with the same probability. As the number of input regions per starter cell increases, the probability of observing inputs from >42 or >65 regions also increases. Based on a threshold of P value <0.001, these simulations suggest that, to account for the total number of observed input areas in each brain sample, there must be individual LC-NE neurons that receive input from more than 15 regions for the sparsest sample (red dot, left) or more than 9 regions for second sparsest sample (red dot, right). b, Detailed view of the distribution of simulation results corresponding to the red dots in a. Assuming that each cell receives input from 15 (left) or 9 (right) distinct regions, only 5 (left) or 6 (right) out of 10,000 simulations label >42 (left) or >65 (right) input regions. Note that if the assumption that each starter cell receives input from the same number of regions does not apply, then there must be at least one cell receiving input from more regions than the number specified in the simulation.

Extended Data Figure 10 Representative images and distribution of individual samples for projection-based viral-genetic labelling experiments.

a, Representative images from sagittal sections of TC+ LC-NE axons in 8 brain regions indicated at the top of each column (the last column shows cell bodies for LC-NE neurons) resulting from CAV injections at four projection sites indicated on the left (top four rows), or AAV-FLExloxP-TC injection at the locus coeruleus of Dbh-Cre animals (bottom row). All TC+ processes were confirmed to contain noradrenaline transporter (NET, an NE neuron marker) by anti-NET immunostaining (not shown; see Fig. 4 inset). b, The normalized fraction of TC+ LC-NE axons for individual experiments for five conditions are colour coded on the top right. Filled symbols represent experiments where Dbh-Cre mice were used along with CAV-FLExloxP-Flp; open symbols represent experiments where wild-type mice were used along with CAV-Cre. The distribution of individual samples with regards to the fraction of TC+ axons observed at output sites was similar between wild-type and Dbh-Cre mice. Collectively, the samples for each condition were averaged to quantify the normalized fraction of TC+ LC-NE axons in each brain region as reported in Fig. 4d. Scale bar, 50 µm. Error bars represent s.e.m. Abbreviations: AC, auditory cortex; CC, cingulate cortex; Cb, cerebellum; Hi, hippocampus; Hy, hypothalamus; LC, locus coeruleus; Me, medulla; OB, olfactory bulb; SC, somatosensory cortex.

Supplementary information

Supplementary Information

This file contains Supplementary Notes 1-2 and additional references. (PDF 157 kb)

Supplementary Table 1

Distribution of presynaptic input to layer 5 motor cortex neurons for Rbp4-Cre input tracing and cTRIO. (XLSX 12 kb)

Supplementary Table 2

Distribution of presynaptic input to locus coeruleus norepinephrine neurons for Dbh-Cre input tracing, TRIO, and cTRIO. (XLSX 134 kb)

Supplementary Table 3

One-way ANOVA p-values for Dbh-Cre input tracing, TRIO, and cTRIO comparisons. (XLSX 17 kb)

Supplementary Table 4

Contingency table for testing input heterogeneity. (XLSX 14 kb)

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Schwarz, L., Miyamichi, K., Gao, X. et al. Viral-genetic tracing of the input–output organization of a central noradrenaline circuit. Nature 524, 88–92 (2015).

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