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

Schwarz LA, Miyamichi K, Gao XJ, Beier KT, Weissbourd B, DeLoach KE, Ren J, Ibanes S, Malenka RC, Kremer EJ, Luo L - Nature (2015)

Bottom Line: 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.More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, California 94305, USA.

ABSTRACT
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 gating. 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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Controls for LC cTRIOa, Top, schematic for negative controls where AAVs expressing Flp-dependent TC and G were injected into the LC of Dbh-Cre mice, followed by RVdG injection into the LC, but the CAV-FLExloxP-Flp injection was omitted. Middle, coronal section of the LC 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 TC and G were injected into the LC of wild-type mice, followed by RVdG injection; hence there was no Cre to mediate Flp expression in LC cells. Middle, coronal section of the LC 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 labeling in the LC (n=4 and 8). This labeling 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, 1 mm (middle panels), 100 μm (lower panels). Error bars, s.e.m.
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Figure 12: Controls for LC cTRIOa, Top, schematic for negative controls where AAVs expressing Flp-dependent TC and G were injected into the LC of Dbh-Cre mice, followed by RVdG injection into the LC, but the CAV-FLExloxP-Flp injection was omitted. Middle, coronal section of the LC 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 TC and G were injected into the LC of wild-type mice, followed by RVdG injection; hence there was no Cre to mediate Flp expression in LC cells. Middle, coronal section of the LC 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 labeling in the LC (n=4 and 8). This labeling 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, 1 mm (middle panels), 100 μm (lower panels). Error bars, s.e.m.

Mentions: We next applied TRIO and cTRIO to test if populations of LC-NE neurons, defined by their output targets, received distinct input. We selected five diverse brain regions known to receive LC-NE projections: the olfactory bulb, auditory cortex, hippocampus, cerebellum, and medulla (Fig. 3a). CAV-Cre injection into these regions in Ai14 mice confirmed labeling of NE neurons throughout the LC (Extended Data Fig. 7a). We did not observe significant differences in the spatial distribution along the anterior–posterior or medial–lateral axes for LC-NE neurons that projected to these brain regions. However, forebrain-projecting LC-NE neurons were more dorsally biased compared to the hindbrain-projecting ones (Extended Data Fig. 7b–f), consistent with a previous observation in the rat18. We applied TRIO to olfactory bulb, auditory cortex, and hippocampus, and cTRIO to cerebellum and medulla, since LC projections to the former group predominately came from TH+ neurons, whereas the latter group contained TH− neurons (Extended Data Fig. 7a). Control experiments indicated that the labeling of input neurons depended on CAV-Cre in the case of TRIO (Extended Data Fig. 5c, e), and on both Dbh-Cre and CAV-FLExloxP-Flp in the case of cTRIO (Extended Data Fig. 8).


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

Schwarz LA, Miyamichi K, Gao XJ, Beier KT, Weissbourd B, DeLoach KE, Ren J, Ibanes S, Malenka RC, Kremer EJ, Luo L - Nature (2015)

Controls for LC cTRIOa, Top, schematic for negative controls where AAVs expressing Flp-dependent TC and G were injected into the LC of Dbh-Cre mice, followed by RVdG injection into the LC, but the CAV-FLExloxP-Flp injection was omitted. Middle, coronal section of the LC 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 TC and G were injected into the LC of wild-type mice, followed by RVdG injection; hence there was no Cre to mediate Flp expression in LC cells. Middle, coronal section of the LC 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 labeling in the LC (n=4 and 8). This labeling 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, 1 mm (middle panels), 100 μm (lower panels). Error bars, s.e.m.
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Related In: Results  -  Collection

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Figure 12: Controls for LC cTRIOa, Top, schematic for negative controls where AAVs expressing Flp-dependent TC and G were injected into the LC of Dbh-Cre mice, followed by RVdG injection into the LC, but the CAV-FLExloxP-Flp injection was omitted. Middle, coronal section of the LC 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 TC and G were injected into the LC of wild-type mice, followed by RVdG injection; hence there was no Cre to mediate Flp expression in LC cells. Middle, coronal section of the LC 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 labeling in the LC (n=4 and 8). This labeling 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, 1 mm (middle panels), 100 μm (lower panels). Error bars, s.e.m.
Mentions: We next applied TRIO and cTRIO to test if populations of LC-NE neurons, defined by their output targets, received distinct input. We selected five diverse brain regions known to receive LC-NE projections: the olfactory bulb, auditory cortex, hippocampus, cerebellum, and medulla (Fig. 3a). CAV-Cre injection into these regions in Ai14 mice confirmed labeling of NE neurons throughout the LC (Extended Data Fig. 7a). We did not observe significant differences in the spatial distribution along the anterior–posterior or medial–lateral axes for LC-NE neurons that projected to these brain regions. However, forebrain-projecting LC-NE neurons were more dorsally biased compared to the hindbrain-projecting ones (Extended Data Fig. 7b–f), consistent with a previous observation in the rat18. We applied TRIO to olfactory bulb, auditory cortex, and hippocampus, and cTRIO to cerebellum and medulla, since LC projections to the former group predominately came from TH+ neurons, whereas the latter group contained TH− neurons (Extended Data Fig. 7a). Control experiments indicated that the labeling of input neurons depended on CAV-Cre in the case of TRIO (Extended Data Fig. 5c, e), and on both Dbh-Cre and CAV-FLExloxP-Flp in the case of cTRIO (Extended Data Fig. 8).

Bottom Line: 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.More broadly, our viral-genetic approaches provide an efficient intersectional means to target neuronal populations based on cell type and projection pattern.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, California 94305, USA.

ABSTRACT
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 gating. 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.

Show MeSH
Related in: MedlinePlus