Limits...
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.

Show MeSH

Related in: MedlinePlus

Strategy and proof-of-principle of TRIO and cTRIOa, b, Schematic of two extreme connection patterns of region B neurons with inputs from A regions and outputs to C regions. c, d, Strategies for trans-synaptic input tracing from B neurons based on their outputs. TRIO (c) does not distinguish between region B cell types projecting to the selected C region (two different cell types are outlined in grey and blue). cTRIO (d) avoids labeling promiscuous projections from Cre− cells (blue). Open and filled triangles, incompatible loxP sites; open and filled half circles, incompatible FRT sites. e, Schematic of TRIO and cTRIO in mouse motor cortex (MC). CAV was injected into contralateral MC (cMC) or medulla (Me) along with AAVs expressing Cre- or Flp-dependent TVA-mCherry (TC)/rabies glycoprotein (G) into MC, followed by RVdG. Experiments were performed in wt (TRIO) or Rbp4-Cre (cTRIO) mice. f, Example coronal sections of MC starter cells in TRIO and cTRIO. Cortical layers are separated by dotted lines based on the DAPI stain (blue). Starter cells (yellow, a subset indicated by arrowheads) can be distinguished from input cells labeled only with GFP from RVdG (green). TC+ cells in MC spanned layers 2/3 and 5 for TRIO (left), but were restricted to L5 with cTRIO. Bottom inset, example images of input neurons from the somatosensory cortex (SC) and ventral anterior thalamus (VA), derived from larger composites (see Methods). g, Average fraction of total input neurons in Rbp4-Cre-based input tracing and cTRIO of MC L5 pyramidal neurons. Values represent the average fraction of input in each category (n=4 for Rbp4-Cre input tracing and cMC cTRIO; n=3 for Me cTRIO). 2-way ANOVA determined that inputs to Rbp4-Cre+ MC starter cells generated by input tracing or cTRIO (C=cMC or C=Me) are significantly different in brain regions from which they receive input (interaction p<0.0001). 1-way ANOVA and post hoc Tukey’s multiple comparison tested the significance within each input region. Error bars, s.e.m. **, p<0.01; ***, p<0.001. Scale, 250 μm (f, middle row), 100 μm (f, bottom row).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4587569&req=5

Figure 1: Strategy and proof-of-principle of TRIO and cTRIOa, b, Schematic of two extreme connection patterns of region B neurons with inputs from A regions and outputs to C regions. c, d, Strategies for trans-synaptic input tracing from B neurons based on their outputs. TRIO (c) does not distinguish between region B cell types projecting to the selected C region (two different cell types are outlined in grey and blue). cTRIO (d) avoids labeling promiscuous projections from Cre− cells (blue). Open and filled triangles, incompatible loxP sites; open and filled half circles, incompatible FRT sites. e, Schematic of TRIO and cTRIO in mouse motor cortex (MC). CAV was injected into contralateral MC (cMC) or medulla (Me) along with AAVs expressing Cre- or Flp-dependent TVA-mCherry (TC)/rabies glycoprotein (G) into MC, followed by RVdG. Experiments were performed in wt (TRIO) or Rbp4-Cre (cTRIO) mice. f, Example coronal sections of MC starter cells in TRIO and cTRIO. Cortical layers are separated by dotted lines based on the DAPI stain (blue). Starter cells (yellow, a subset indicated by arrowheads) can be distinguished from input cells labeled only with GFP from RVdG (green). TC+ cells in MC spanned layers 2/3 and 5 for TRIO (left), but were restricted to L5 with cTRIO. Bottom inset, example images of input neurons from the somatosensory cortex (SC) and ventral anterior thalamus (VA), derived from larger composites (see Methods). g, Average fraction of total input neurons in Rbp4-Cre-based input tracing and cTRIO of MC L5 pyramidal neurons. Values represent the average fraction of input in each category (n=4 for Rbp4-Cre input tracing and cMC cTRIO; n=3 for Me cTRIO). 2-way ANOVA determined that inputs to Rbp4-Cre+ MC starter cells generated by input tracing or cTRIO (C=cMC or C=Me) are significantly different in brain regions from which they receive input (interaction p<0.0001). 1-way ANOVA and post hoc Tukey’s multiple comparison tested the significance within each input region. Error bars, s.e.m. **, p<0.01; ***, p<0.001. Scale, 250 μm (f, middle row), 100 μm (f, bottom row).

Mentions: Fig. 1a–b illustrates two extreme connectivity models for input–output relationships of projection neurons. At one extreme, neurons from each input A region connect to a subtype of B neurons that send output to a unique C region (Fig. 1a); this model segregates information into discrete pathways. At the other extreme, B neurons are homogeneous in receiving input from all A regions and sending output to all C regions with a common probability distribution (Fig. 1b); this model allows an overall indiscriminate integration and broadcast of information. The input–output relationship for most neural circuits is not known (Supplementary Note 1).


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)

Strategy and proof-of-principle of TRIO and cTRIOa, b, Schematic of two extreme connection patterns of region B neurons with inputs from A regions and outputs to C regions. c, d, Strategies for trans-synaptic input tracing from B neurons based on their outputs. TRIO (c) does not distinguish between region B cell types projecting to the selected C region (two different cell types are outlined in grey and blue). cTRIO (d) avoids labeling promiscuous projections from Cre− cells (blue). Open and filled triangles, incompatible loxP sites; open and filled half circles, incompatible FRT sites. e, Schematic of TRIO and cTRIO in mouse motor cortex (MC). CAV was injected into contralateral MC (cMC) or medulla (Me) along with AAVs expressing Cre- or Flp-dependent TVA-mCherry (TC)/rabies glycoprotein (G) into MC, followed by RVdG. Experiments were performed in wt (TRIO) or Rbp4-Cre (cTRIO) mice. f, Example coronal sections of MC starter cells in TRIO and cTRIO. Cortical layers are separated by dotted lines based on the DAPI stain (blue). Starter cells (yellow, a subset indicated by arrowheads) can be distinguished from input cells labeled only with GFP from RVdG (green). TC+ cells in MC spanned layers 2/3 and 5 for TRIO (left), but were restricted to L5 with cTRIO. Bottom inset, example images of input neurons from the somatosensory cortex (SC) and ventral anterior thalamus (VA), derived from larger composites (see Methods). g, Average fraction of total input neurons in Rbp4-Cre-based input tracing and cTRIO of MC L5 pyramidal neurons. Values represent the average fraction of input in each category (n=4 for Rbp4-Cre input tracing and cMC cTRIO; n=3 for Me cTRIO). 2-way ANOVA determined that inputs to Rbp4-Cre+ MC starter cells generated by input tracing or cTRIO (C=cMC or C=Me) are significantly different in brain regions from which they receive input (interaction p<0.0001). 1-way ANOVA and post hoc Tukey’s multiple comparison tested the significance within each input region. Error bars, s.e.m. **, p<0.01; ***, p<0.001. Scale, 250 μm (f, middle row), 100 μm (f, bottom row).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4587569&req=5

Figure 1: Strategy and proof-of-principle of TRIO and cTRIOa, b, Schematic of two extreme connection patterns of region B neurons with inputs from A regions and outputs to C regions. c, d, Strategies for trans-synaptic input tracing from B neurons based on their outputs. TRIO (c) does not distinguish between region B cell types projecting to the selected C region (two different cell types are outlined in grey and blue). cTRIO (d) avoids labeling promiscuous projections from Cre− cells (blue). Open and filled triangles, incompatible loxP sites; open and filled half circles, incompatible FRT sites. e, Schematic of TRIO and cTRIO in mouse motor cortex (MC). CAV was injected into contralateral MC (cMC) or medulla (Me) along with AAVs expressing Cre- or Flp-dependent TVA-mCherry (TC)/rabies glycoprotein (G) into MC, followed by RVdG. Experiments were performed in wt (TRIO) or Rbp4-Cre (cTRIO) mice. f, Example coronal sections of MC starter cells in TRIO and cTRIO. Cortical layers are separated by dotted lines based on the DAPI stain (blue). Starter cells (yellow, a subset indicated by arrowheads) can be distinguished from input cells labeled only with GFP from RVdG (green). TC+ cells in MC spanned layers 2/3 and 5 for TRIO (left), but were restricted to L5 with cTRIO. Bottom inset, example images of input neurons from the somatosensory cortex (SC) and ventral anterior thalamus (VA), derived from larger composites (see Methods). g, Average fraction of total input neurons in Rbp4-Cre-based input tracing and cTRIO of MC L5 pyramidal neurons. Values represent the average fraction of input in each category (n=4 for Rbp4-Cre input tracing and cMC cTRIO; n=3 for Me cTRIO). 2-way ANOVA determined that inputs to Rbp4-Cre+ MC starter cells generated by input tracing or cTRIO (C=cMC or C=Me) are significantly different in brain regions from which they receive input (interaction p<0.0001). 1-way ANOVA and post hoc Tukey’s multiple comparison tested the significance within each input region. Error bars, s.e.m. **, p<0.01; ***, p<0.001. Scale, 250 μm (f, middle row), 100 μm (f, bottom row).
Mentions: Fig. 1a–b illustrates two extreme connectivity models for input–output relationships of projection neurons. At one extreme, neurons from each input A region connect to a subtype of B neurons that send output to a unique C region (Fig. 1a); this model segregates information into discrete pathways. At the other extreme, B neurons are homogeneous in receiving input from all A regions and sending output to all C regions with a common probability distribution (Fig. 1b); this model allows an overall indiscriminate integration and broadcast of information. The input–output relationship for most neural circuits is not known (Supplementary Note 1).

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