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A genetically specified connectomics approach applied to long-range feeding regulatory circuits

Nature Neuroscience volume 17, pages 1830–1839 (2014)Cite this article

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Abstract

Synaptic connectivity and molecular composition provide a blueprint for information processing in neural circuits. Detailed structural analysis of neural circuits requires nanometer resolution, which can be obtained with serial-section electron microscopy. However, this technique remains challenging for reconstructing molecularly defined synapses. We used a genetically encoded synaptic marker for electron microscopy (GESEM) based on intra-vesicular generation of electron-dense labeling in axonal boutons. This approach allowed the identification of synapses from Cre recombinase–expressing or GAL4-expressing neurons in the mouse and fly with excellent preservation of ultrastructure. We applied this tool to visualize long-range connectivity of AGRP and POMC neurons in the mouse, two molecularly defined hypothalamic populations that are important for feeding behavior. Combining selective ultrastructural reconstruction of neuropil with functional and viral circuit mapping, we characterized some basic features of circuit organization for axon projections of these cell types. Our findings demonstrate that GESEM labeling enables long-range connectomics with molecularly defined cell types.

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Figure 1: A genetically encoded synaptic EM marker for cell type–specific connectomics.
Figure 2: GESEM labeling for identification and reconstruction of ARCPOMC→PVH circuits.
Figure 3: GESEM labeling for identification and reconstruction of ARCAGRP→PVH circuits.
Figure 4: Penetrance for VAMP2:HRP DAB-labeling of synaptic vesicles.
Figure 5: Quantitative intra-axonal organelle profile of ARCAGRP→PVH and ARCPOMC→PVH boutons.
Figure 6: Postsynaptic profile of molecularly defined ARC→PVH projections.

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Acknowledgements

We thank A. Wardlaw for mouse breeding and genotyping, K. Morris for HSV stereotaxic injections, A. Hu and M. Copeland for histology, and L. Lo, D. Anderson and R. Gong with advice on HSV129 anterograde tracing. This research was funded by the Howard Hughes Medical Institute. The HSV129ΔTK-TT anterograde trans-synaptic tracer virus was provided by the Center for Neuroanatomy with Neurotropic Viruses (P40RR018604).

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  1. Deniz Atasoy & Sinem M Sertel

    Present address: Present addresses: Research Center for Restorative and Regenerative Medicine, Istanbul Medipol University, Istanbul, Turkey (D.A.), Department of Physiology, School of Medicine, Istanbul Medipol University, Istanbul, Turkey (D.A. and S.M. Sirtel).,

Authors and Affiliations

  1. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA

    Deniz Atasoy, J Nicholas Betley, Wei-Ping Li, Helen H Su, Sinem M Sertel, Louis K Scheffer, Julie H Simpson, Richard D Fetter & Scott M Sternson

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Contributions

D.A. and S.M. Sternson designed the experiments and wrote the paper. D.A. and S.M. Sertel analyzed EM images and reconstructed synapses. J.H.S. suggested VAMP2:HRP labeling and generated the UAS-nSyb:HRP fly line. J.N.B. performed immunohistochemistry and image analysis. H.H.S. performed molecular cloning. R.D.F. developed tissue preparation and staining protocols. W.-P.L. and R.D.F. performed sectioning and transmission EM imaging. R.D.F. and L.K.S. performed electron micrograph registration and alignment.

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Integrated supplementary information

Supplementary Figure 1 Vesicle pool profile with respect to presence or absence of a postsynaptic contact site.

a,b, Distribution of small vesicle (a) or large vesicle (b) quantity in ARCAGRP→PVH boutons (n = 51 boutons from one mouse). Profile for synaptic contacts (green) and non-synaptic (black) are plotted separately. c,d, Distributions for ARCPOMC→PVH boutons (n = 51 boutons from one mouse).

Supplementary Figure 2 Docked vesicles in GESEM-labeled synapses.

Example electron micrographs showing docked vesicles (red arrows) at synapses for ARCAGRP→PVH and ARCPOMC→PVH, in physical contact with the plasma membrane. Scale, 0.5 μm. b, Mean number and s.e.m. of docked vesicles at synapses for ARCAGRP→PVH and ARCPOMC→PVH (n = 15 AGRP boutons from one mouse, n = 28 POMC boutons from one mouse, U-test, U = 179.5, P = 0.44). n.s. P > 0.05.

Supplementary Figure 3 Evidence for vesicle exocytosis activity in predominantly LV-containing non-synaptic boutons from ARCAGRP→PVH projections.

a,b Extracellular membrane localization of electron-dense DAB reaction product. Fusion of LVs expressing VAMP2:HRP results in extracellularly oriented HRP, which leads to membrane labeling after exposure to DAB/H2O2 (red arrows). Scale, 0.5 μm. c–h, Labeled non-synaptic, LV-containing boutons with evidence of vesicle fusion based on Ω–structures at the extracellular membrane (arrowheads). Scale, 0.5 μm. For the bouton in f, DAB vesicle labeling is in a different section of the bouton than the Ω–structure (inset).

Supplementary Figure 4 Subcellular targeting of molecularly defined ARC→PVH projections and molecular identity of ARC→PVH axonal boutons

a, Distribution of postsynaptic dendrite diameter at contact sites as well as somatic and spine targeting synapses from GESEM-labeled AGRP and POMC neuron projections to the PVH. b, In brain slices from Agrp-IRES-Cre;Ai9(tdtomato) mice, immunoreactivity for NPY, AGRP, and vGat in tdtomato-labeled ARCAGRP→PVH axons. Top row and bottom left, triple colocalization of markers. Bottom center and right, colocalization of a subset of markers. c, Quantification of marker colocalization with tdtomato. d, Venn diagram for molecular identity of ARCAGRP→PVH boutons. We observed extensive colocalization of AGRP and NPY with tdtomato-filled AGRP varicosities (AGRP+/tdtomato+: 91%; NPY+/tdtomato+: 96%, n = 54 boutons from 2 mice). A large fraction of AGRP varicosities showed vGat-ir (69%, n = 108 boutons from 2 mice). Not shown: There was scant or no colocalization in boutons of markers for glutamate (vGlut2-ir: 1/27 from one mouse) or acetylcholine (vAcht-ir: 0/54 from 2 mice) release. e, In Pomc-topazFP mice, immunoreactivity for POMC, vGlut1, vGlut2, vGat and vAcht in topazFP-labeled ARCPOMC→PVH axons. Top left and middle, triple colocalization of markers. Rest of images, colocalization of a subset or no markers. f, Quantification of marker colocalization with topazFP. g, Venn diagram for molecular identity of ARCPOMC→PVH boutons. We found that 75% of boutons contained POMC-ir (n = 108 boutons from 2 mice). The presence of topazFP boutons that lack POMC are consistent with the presence of GESEM-labeled boutons that show very low LV counts (Fig. 5a), for which immunodetection of POMC may be limited. Many boutons were glutamatergic, based on vGlut2-ir (50%, n = 108 boutons from 2 mice). Conversely, there was only limited colocalization with vGat-ir (9%, n = 54 boutons from 2 mice). vMat2-ir was observed in only 1/27 boutons (not shown, from one mouse) and none of the boutons showed vGlut1-ir (0/54 boutons from 2 mice) or vAcht-ir (0/54 boutons from 2 mice).

Supplementary Figure 5 Functional connectivity of ARCAGRP→PVH and ARCPOMC→PVH circuits.

a, Schematic of Cre-dependent ChR2:tdtomato virus infection of AGRP neurons and POMC neurons. PVH-containing coronal brain slices (red plane) with cut ChR2-expressing AGRP or POMC axons. b, Optically evoked AGRP and POMC neuron-mediated synaptic currents recorded from PVH neurons. Excitatory and inhibitory currents were pharmacologically isolated using picrotoxin and CNQX/AP5, respectively. c, Connectivity profile and location of postsynaptic neuron for ARCAGRP→PVH (circles, left) and ARCPOMC→PVH (squares, right) projections (green/red: evoked synaptic current detected/not detected). Data on left from ref. 24. d, Representative IPSC traces from ARCAGRP→PVH projections show mix of success and failure events, ideally representing release from a single axon. The IPSC amplitude is smaller than for saturating light intensity, indicating that PVH neurons receive input from multiple AGRP axons. (Right) To quantify quantal size from ARCAGRP→PVH synapses, Ca2+ was replaced with Sr2+ to desynchronize neurotransmitter release events. Representative traces are shown below, with individual desynchronized ARCAGRP→PVH release events aligned to IPSC onset. Note that quantal amplitudes tend to be smaller than minimal stimulation amplitudes, indicating multiple synaptic release sites from single axons. e, A summary plot for amplitudes of minimal stimulation protocol; a representative recording with increasing light power(1× – 10× light power). The success rate for synaptic release events also increases with light power.

Supplementary Figure 6 Convergence of AGRP and POMC axon projections onto PVH neurons.

a, Schematic for anterograde transsynaptic labeling of ARCPOMC→PVH. HSV129-ΔTK-TT (HSV129-loxSTOPlox-tdtomato-2a-TK) was injected into the ARC of Pomc-Cre mice. a–c, Tdtomato-expression in POMC neurons and also in postsynaptic neurons in the (b) ARC and (c) PVH. Scale bar, 100 µm. c, (left) AGRP axon projections to the PVH and ARCPOMC→PVH tdtomato-expressing cells. Scale bar, 100 µm. (right) POMC boutons, but not AGRP boutons, in the PVH are labeled by tdtomato. Scale bar, 20 µm. Lack of colocalization with AGRP indicates specificity of HSV recombination in infected POMC neurons and the absence of retrograde transfer. d, Projection image (proj.) for 7 µm z-stack and confocal images (Z1–Z7) separated by 1 µm with AGRP-immunoreactive boutons on an ARCPOMC→PVH tdtomato-expressing neuron (see Methods). AGRP boutons juxtaposed to the soma are numbered in the maximal projection. These are sites of putative AGRP synapses with ARCPOMC→PVH neurons. Scale bar, 10 µm. e, AGRP-immunoreactive boutons abutting the membrane of tdtomato-labeled PVH neurons were also confirmed in the XZ and YZ planes; shown for bouton #10 in optical section Z7. Red line denotes YZ-plane, green line denotes XZ-plane. f–h, Additional example of image analysis from transsynaptic viral tracing and AGRP immunohistochemistry. (f), Maximal projection of an ARCPOMC→PVH neuron with AGRP boutons mostly on the bottom surface of the neuron. AGRP boutons juxtaposed to the neuron are numbered, other AGRP boutons that are not adjacent to the soma are labeled with letters. (g), Single optical section from near the bottom of the neuron reveals some AGRP bouton contact points (numbering as in f). (h), Array of YZ image planes along the x-axis at 1 µm intervals showing AGRP bouton juxtaposition. Numbering and lettering as in f. These images reveal that all abutting boutons, except 3, 9 and 12, are on the bottom of the imaged neuron. Scale bar, 10 µm. i,j, Cumulative probability plot showing the fraction (i) and histogram showing the number (j) of tdtomato-labeled PVH neuron somata (n = 19 neurons from 2 mice) juxtaposed to different numbers of AGRP boutons.

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Atasoy, D., Betley, J., Li, WP. et al. A genetically specified connectomics approach applied to long-range feeding regulatory circuits. Nat Neurosci 17, 1830–1839 (2014). https://doi.org/10.1038/nn.3854

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