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

Nature Neuroscience volume 17, pages 18301839 (2014) | Download Citation

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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References

  1. 1.

    , & Genetic dissection of neural circuits. Neuron 57, 634–660 (2008).

  2. 2.

    et al. Ultrastructural analysis of hippocampal neuropil from the connectomics perspective. Neuron 67, 1009–1020 (2010).

  3. 3.

    , , , & Superresolution imaging of chemical synapses in the brain. Neuron 68, 843–856 (2010).

  4. 4.

    et al. GFP reconstitution across synaptic partners (GRASP) defines cell contacts and synapses in living nervous systems. Neuron 57, 353–363 (2008).

  5. 5.

    , , , & Monosynaptic circuit tracing in vivo through Cre-dependent targeting and complementation of modified rabies virus. Proc. Natl. Acad. Sci. USA 107, 21848–21853 (2010).

  6. 6.

    , , & Channelrhodopsin-2–assisted circuit mapping of long-range callosal projections. Nat. Neurosci. 10, 663–668 (2007).

  7. 7.

    , , & FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range circuit mapping. J. Neurosci. 28, 7025–7030 (2008).

  8. 8.

    et al. Network anatomy and in vivo physiology of visual cortical neurons. Nature 471, 177–182 (2011).

  9. 9.

    , & Wiring specificity in the direction-selectivity circuit of the retina. Nature 471, 183–188 (2011).

  10. 10.

    et al. A visual motion detection circuit suggested by Drosophila connectomics. Nature 500, 175–181 (2013).

  11. 11.

    , , , & A protocol for preparing GFP-labeled neurons previously imaged in vivo and in slice preparations for light and electron microscopic analysis. Nat. Protoc. 4, 1145–1156 (2009).

  12. 12.

    , , , & Control of dopamine release in the retina: a transgenic approach to neural networks. Neuron 18, 723–736 (1997).

  13. 13.

    , , , & Regulated endocytic routing modulates wingless signaling in Drosophila embryos. Cell 105, 613–624 (2001).

  14. 14.

    , , & Membrane-targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies. Front. Neural Circuits 4, 6 (2010).

  15. 15.

    et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).

  16. 16.

    et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143–1148 (2012).

  17. 17.

    , , , & Glia engulf degenerating axons during developmental axon pruning. Curr. Biol. 14, 678–684 (2004).

  18. 18.

    , , & Chimeric molecules employing horseradish peroxidase as reporter enzyme for protein localization in the electron microscope. Methods Enzymol. 327, 35–45 (2000).

  19. 19.

    , & Horseradish peroxidase cDNA as a marker for electron microscopy in neurons. J. Neurosci. Methods 165, 210–215 (2007).

  20. 20.

    & Electron Microscopy. Principles and Techniques for Biologists 2nd edn. (Jones and Bartlett Publishers, 1999).

  21. 21.

    & Neurobiology of feeding and energy expenditure. Annu. Rev. Neurosci. 30, 367–398 (2007).

  22. 22.

    , & AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training. Nat. Neurosci. 14, 351–355 (2011).

  23. 23.

    et al. Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. J. Clin. Invest. 121, 1424–1428 (2011).

  24. 24.

    , , & Deconstruction of a neural circuit for hunger. Nature 488, 172–177 (2012).

  25. 25.

    et al. Divergence of melanocortin pathways in the control of food intake and energy expenditure. Cell 123, 493–505 (2005).

  26. 26.

    , , , & Monosynaptic pathway between the arcuate nucleus expressing glial type II iodothyronine 5′-deiodinase mRNA and the median eminence-projective TRH cells of the rat paraventricular nucleus. J. Neuroendocrinol. 10, 731–742 (1998).

  27. 27.

    et al. Piccolo modulation of Synapsin1a dynamics regulates synaptic vesicle exocytosis. J. Cell Biol. 181, 831–846 (2008).

  28. 28.

    , , & Axonal transport and distribution of synaptobrevin I and II in the rat peripheral nervous system. J. Neurosci. 16, 137–147 (1996).

  29. 29.

    et al. Leptin activates anorexigenic POMC neurons through a neural network in the arcuate nucleus. Nature 411, 480–484 (2001).

  30. 30.

    Characterization of luteinizing hormone-releasing hormone fibres in the mesencephalic central grey substance of the rat. Neuroendocrinology 49, 623–630 (1989).

  31. 31.

    & Morphological correlates of functionally defined synaptic vesicle populations. Nat. Neurosci. 4, 391–395 (2001).

  32. 32.

    & Ultrastructural demonstration of nonsynaptic release sites in the central nervous system of the snail Lymnaea stagnalis, the insect Periplaneta americana, and the rat. Neuroscience 17, 867–879 (1986).

  33. 33.

    , & Synaptic mitochondria in synaptic transmission and organization of vesicle pools in health and disease. Front. Synaptic Neurosci 2, 139 (2010).

  34. 34.

    & Role of dendritic synapse location in the control of action potential output. Trends Neurosci. 26, 147–154 (2003).

  35. 35.

    The magnocellular and parvocellular paraventricular nucleus of rat: intrinsic organization. J. Comp. Neurol. 206, 317–345 (1982).

  36. 36.

    & Agouti-related protein containing nerve terminals innervate thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus. Endocrinology 140, 3643–3652 (1999).

  37. 37.

    & Dendritic spines of CA 1 pyramidal cells in the rat hippocampus: serial electron microscopy with reference to their biophysical characteristics. J. Neurosci. 9, 2982–2997 (1989).

  38. 38.

    , , & Pathway-specific feedforward circuits between thalamus and neocortex revealed by selective optical stimulation of axons. Neuron 65, 230–245 (2010).

  39. 39.

    & Cre-dependent, anterograde transsynaptic viral tracer for mapping output pathways of genetically marked neurons. Neuron 72, 938–950 (2011).

  40. 40.

    , & Neuropeptide-Y and ACTH-immunoreactive innervation of corticotropin releasing factor (CRF)-synthesizing neurons in the hypothalamus of the rat. An immunocytochemical analysis at the light and electron microscopic levels. Histochemistry 88, 227–234 (1988).

  41. 41.

    et al. Anterograde or retrograde transsynaptic labeling of CNS neurons with vesicular stomatitis virus vectors. Proc. Natl. Acad. Sci. USA 108, 15414–15419 (2011).

  42. 42.

    et al. alpha-Melanocyte–stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression. J. Neurosci. 20, 1550–1558 (2000).

  43. 43.

    , & Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

  44. 44.

    , , & Anterograde and retrograde traffic between the rough endoplasmic reticulum and the Golgi complex. J. Cell Biol. 131, 1387–1401 (1995).

  45. 45.

    et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).

  46. 46.

    , & Synaptic vesicles interchange their membrane proteins with a large surface reservoir during recycling. Neuron 51, 179–186 (2006).

  47. 47.

    , , , & Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 385, 165–168 (1997).

  48. 48.

    , , & Rapid versus delayed stimulation of feeding by the endogenously released AgRP neuron mediators GABA NPY, and AgRP. Cell Metab. 18, 588–595 (2013).

  49. 49.

    & Release of LHRH is linearly related to the time integral of presynaptic Ca2+ elevation above a threshold level in bullfrog sympathetic ganglia. Neuron 10, 465–473 (1993).

  50. 50.

    et al. Nonlinear dendritic integration of sensory and motor input during an active sensing task. Nature 492, 247–251 (2012).

  51. 51.

    , , & Transcriptional regulation of agouti-related protein (Agrp) in transgenic mice. Endocrinology 145, 5798–5806 (2004).

  52. 52.

    et al. Leptin receptor signaling in POMC neurons is required for normal body weight homeostasis. Neuron 42, 983–991 (2004).

  53. 53.

    , , , & Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance. Nat. Neurosci. 11, 998–1000 (2008).

  54. 54.

    et al. A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010).

  55. 55.

    et al. Rapid rewiring of arcuate nucleus feeding circuits by leptin. Science 304, 110–115 (2004).

  56. 56.

    & Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation. J. Cell Biol. 96, 51–62 (1983).

  57. 57.

    & The use of osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods to enhance membrane contrast and preservation in cultured cells. J. Histochem. Cytochem. 32, 455–460 (1984).

  58. 58.

    et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

  59. 59.

    , & Automated alignment of imperfect EM images for neural reconstruction. Preprint at (2013).

  60. 60.

    et al. TrakEM2 software for neural circuit reconstruction. PLoS ONE 7, e38011 (2012).

  61. 61.

    et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).

  62. 62.

    et al. Disruption of the gene encoding SF-1 alters the distribution of hypothalamic neuronal phenotypes. J. Comp. Neurol. 423, 579–589 (2000).

  63. 63.

    Cao, Zhen Fang H., Ritola, Kimberly D. & Sternson, Scott M. Parallel, redundant circuit organization for homeostatic control of feeding behavior. Cell 155, 1337–1350 (2013).

  64. 64.

    et al. An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size. Proc. Natl. Acad. Sci. USA 101, 7158–7163 (2004).

  65. 65.

    et al. Vesicular glutamate transporter VGLUT2 expression levels control quantal size and neuropathic pain. J. Neurosci. 26, 12055–12066 (2006).

  66. 66.

    et al. Stringent specificity in the construction of a GABAergic presynaptic inhibitory circuit. Cell 139, 161–174 (2009).

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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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Author notes

    • Deniz Atasoy
    •  & Sinem M Sertel

    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).

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.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Deniz Atasoy or Richard D Fetter or Scott M Sternson.

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https://doi.org/10.1038/nn.3854

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