Sonic hedgehog regulates presynaptic terminal size, ultrastructure and function in hippocampal neurons

Summary Sonic hedgehog (Shh) signaling is essential to the patterning of the embryonic neural tube, but its presence and function in the postmitotic differentiated neurons in the brain remain largely uncharacterized. We recently showed that Shh and its signaling components, Patched and Smoothened, are expressed in postnatal and adult hippocampal neurons. We have now examined whether Shh signaling has a function in these neurons. Using cultured hippocampal neurons as a model system, we found that presynaptic terminals become significantly larger in response to the application of Shh. Ultrastructural examination confirmed the enlarged presynaptic profiles and also revealed variable increases in the size of synaptic vesicles, with a resulting loss of uniformity. Furthermore, electrophysiological analyses showed significant increases in the frequency, but not the amplitude, of spontaneous miniature excitatory postsynaptic currents (mEPSCs) in response to Shh, providing functional evidence of the selective role of Shh in presynaptic terminals. Thus, we conclude that Shh signaling regulates the structure and functional properties of presynaptic terminals of hippocampal neurons.

[1]  Michael D. Cole,et al.  Nmyc upregulation by sonic hedgehog signaling promotes proliferation in developing cerebellar granule neuron precursors , 2003, Development.

[2]  M. Scott,et al.  Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. , 1996, Genes & development.

[3]  Min Jiang,et al.  High Ca2+-phosphate transfection efficiency in low-density neuronal cultures , 2006, Nature Protocols.

[4]  M. Scott,et al.  Control of Neuronal Precursor Proliferation in the Cerebellum by Sonic Hedgehog , 1999, Neuron.

[5]  Junko Kurisu,et al.  Sonic hedgehog signaling regulates actin cytoskeleton via Tiam1–Rac1 cascade during spine formation , 2010, Molecular and Cellular Neuroscience.

[6]  S. Kaech,et al.  Culturing hippocampal neurons , 2006, Nature Protocols.

[7]  Q. Gu,et al.  Activating Smoothened mutations in sporadic basal-cell carcinoma , 1998, Nature.

[8]  Johan Ericson,et al.  Two Critical Periods of Sonic Hedgehog Signaling Required for the Specification of Motor Neuron Identity , 1996, Cell.

[9]  J. Dittman,et al.  Molecular circuitry of endocytosis at nerve terminals. , 2009, Annual review of cell and developmental biology.

[10]  D. Jenkins.  Hedgehog signalling: emerging evidence for non-canonical pathways. , 2009, Cellular signalling.

[11]  P. Ingham,et al.  Hedgehog signaling in animal development: paradigms and principles. , 2001, Genes & development.

[12]  Jussi Taipale,et al.  Small molecule modulation of Smoothened activity , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[13]  G. Bishop,et al.  Type III adenylyl cyclase localizes to primary cilia throughout the adult mouse brain , 2007, The Journal of comparative neurology.

[14]  A. Hall,et al.  Axonal Remodeling and Synaptic Differentiation in the Cerebellum Is Regulated by WNT-7a Signaling , 2000, Cell.

[15]  Anatol C. Kreitzer,et al.  Sonic Hedgehog Expression in Corticofugal Projection Neurons Directs Cortical Microcircuit Formation , 2012, Neuron.

[16]  V. Budnik,et al.  Wnt signaling during synaptic development and plasticity , 2011, Current Opinion in Neurobiology.

[17]  Heike Hering,et al.  Activity-Dependent Redistribution and Essential Role of Cortactin in Dendritic Spine Morphogenesis , 2003, The Journal of Neuroscience.

[18]  M. Mattson,et al.  Clathrin Assembly Protein AP180 and CALM Differentially Control Axogenesis and Dendrite Outgrowth in Embryonic Hippocampal Neurons , 2008, The Journal of Neuroscience.

[19]  M. Mattson,et al.  Subcellular Distribution of Patched and Smoothened in the Cerebellar Neurons , 2012, The Cerebellum.

[20]  M. Scott,et al.  Patched 1 Regulates Hedgehog Signaling at the Primary Cilium , 2022 .

[21]  Chi-Chung Hui,et al.  Hedgehog signaling in development and cancer. , 2008, Developmental cell.

[22]  V. Wallace.  Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum , 1999, Current Biology.

[23]  R. Petralia,et al.  Immunocytochemistry of NMDA receptors. , 1999, Methods in molecular biology.

[24]  P. Beachy,et al.  Gli2 trafficking links Hedgehog-dependent activation of Smoothened in the primary cilium to transcriptional activation in the nucleus , 2009, Proceedings of the National Academy of Sciences.

[25]  P. De Camilli,et al.  Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[26]  Christian Rosenmund,et al.  Unique Luminal Localization of VGAT-C Terminus Allows for Selective Labeling of Active Cortical GABAergic Synapses , 2008, The Journal of Neuroscience.

[27]  M. Muenke,et al.  A previously unidentified amino-terminal domain regulates transcriptional activity of wild-type and disease-associated human GLI2. , 2005, Human molecular genetics.

[28]  M. Mattson,et al.  Subcellular localization of patched and smoothened, the receptors for sonic hedgehog signaling, in the hippocampal neuron , 2011, The Journal of comparative neurology.

[29]  Robert J. Wechsler-Reya,et al.  Transcriptional profiling of the Sonic hedgehog response: A critical role for N-myc in proliferation of neuronal precursors , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  M. Scott,et al.  Neuropilins are positive regulators of Hedgehog signal transduction. , 2011, Genes & development.

[31]  A. Ruiz i Altaba,et al.  Sonic hedgehog regulates the growth and patterning of the cerebellum. , 1999, Development.

[32]  P. De Camilli,et al.  Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons , 1992, The Journal of cell biology.

[33]  M. Ruat,et al.  Discrete localizations of hedgehog signalling components in the developing and adult rat nervous system , 1999, The European journal of neuroscience.

[34]  M. Scott,et al.  Effects of oncogenic mutations in Smoothened and Patched can be reversed by cyclopamine , 2000, Nature.

[35]  M. Mattson,et al.  Sonic hedgehog distribution within mature hippocampal neurons , 2011, Communicative & integrative biology.

[36]  Jussi Taipale,et al.  Inhibition of Hedgehog signaling by direct binding of cyclopamine to Smoothened. , 2002, Genes & development.

[37]  W. Betz,et al.  Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. , 1992, Science.

[38]  H. Lester,et al.  Enhancement of Neurotransmitter Release Induced by Brain-Derived Neurotrophic Factor in Cultured Hippocampal Neurons , 1998, The Journal of Neuroscience.

[39]  Kristina D. Micheva,et al.  Single-Synapse Analysis of a Diverse Synapse Population: Proteomic Imaging Methods and Markers , 2010, Neuron.

[40]  R. Petralia,et al.  Organization of NMDA receptors at extrasynaptic locations , 2010, Neuroscience.

[41]  H. Urlaub,et al.  Quantitative Comparison of Glutamatergic and GABAergic Synaptic Vesicles Unveils Selectivity for Few Proteins Including MAL2, a Novel Synaptic Vesicle Protein , 2010, The Journal of Neuroscience.

[42]  Surajit Sinha,et al.  Purmorphamine activates the Hedgehog pathway by targeting Smoothened , 2006, Nature chemical biology.

[43]  Jussi Taipale,et al.  Hedgehog: functions and mechanisms. , 2008, Genes & development.

[44]  C. Garner,et al.  Bassoon, a Novel Zinc-finger CAG/Glutamine-repeat Protein Selectively Localized at the Active Zone of Presynaptic Nerve Terminals , 1998, The Journal of cell biology.

[45]  Alan Carleton,et al.  Sonic hedgehog controls stem cell behavior in the postnatal and adult brain , 2005, Development.

[46]  David M. Berman,et al.  Tissue repair and stem cell renewal in carcinogenesis , 2004, Nature.

[47]  J. García-Verdugo,et al.  Hedgehog signaling and primary cilia are required for the formation of adult neural stem cells , 2008, Nature Neuroscience.

[48]  D. Lo,et al.  Brain-Derived Neurotrophic Factor Differentially Regulates Excitatory and Inhibitory Synaptic Transmission in Hippocampal Cultures , 2000, The Journal of Neuroscience.

[49]  S. Ge,et al.  A role for primary cilia in glutamatergic synaptic integration of adult-born neurons , 2012, Nature Neuroscience.

[50]  Fred H. Gage,et al.  Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo , 2003, Nature Neuroscience.

[51]  R. Palmiter,et al.  ZnT-3, a putative transporter of zinc into synaptic vesicles. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[52]  J. Arellano,et al.  Primary cilia regulate hippocampal neurogenesis by mediating sonic hedgehog signaling , 2008, Proceedings of the National Academy of Sciences.

[53]  B. Sakmann,et al.  Imaging Synaptic Activity in Intact Brain and Slices with FM1-43 in C. elegans, Lamprey, and Rat , 1999, Neuron.

[54]  S. Kater,et al.  Fibroblast growth factor and glutamate: opposing roles in the generation and degeneration of hippocampal neuroarchitecture , 1989, Journal of Neuroscience.

[55]  P. Scheiffele,et al.  Genetics and cell biology of building specific synaptic connectivity. , 2010, Annual review of neuroscience.