Chemogenetic Synaptic Silencing of Neural Circuits Localizes a Hypothalamus→Midbrain Pathway for Feeding Behavior

Brain function is mediated by neural circuit connectivity, and elucidating the role of connections is aided by techniques to block their output. We developed cell-type-selective, reversible synaptic inhibition tools for mammalian neural circuits by leveraging G protein signaling pathways to suppress synaptic vesicle release. Here, we find that the pharmacologically selective designer Gi-protein-coupled receptor hM4D is a presynaptic silencer in the presence of its cognate ligand clozapine-N-oxide (CNO). Activation of hM4D signaling sharply reduced synaptic release probability and synaptic current amplitude. To demonstrate the utility of this tool for neural circuit perturbations, we developed an axon-selective hM4D-neurexin variant and used spatially targeted intracranial CNO injections to localize circuit connections from the hypothalamus to the midbrain responsible for feeding behavior. This synaptic silencing approach is broadly applicable for cell-type-specific and axon projection-selective functional analysis of diverse neural circuits.

[1]  B. Lowell,et al.  Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance , 2008, Nature Neuroscience.

[2]  V. Doležal,et al.  The effects of brucine and alcuronium on the inhibition of [3H]acetylcholine release from rat striatum by muscarinic receptor agonists , 1998, British journal of pharmacology.

[3]  L. Looger,et al.  Chemical and Genetic Engineering of Selective Ion Channel–Ligand Interactions , 2011, Science.

[4]  R. Palmiter,et al.  Deciphering a neuronal circuit that mediates appetite , 2012, Nature.

[5]  Hanchuan Peng,et al.  mGRASP enables mapping mammalian synaptic connectivity with light microscopy , 2011, Nature Methods.

[6]  C. Gremel,et al.  Strengthening the accumbal indirect pathway promotes resilience to compulsive cocaine use , 2013, Nature Neuroscience.

[7]  B. Roth,et al.  Rapid, reversible activation of AgRP neurons drives feeding behavior in mice. , 2011, The Journal of clinical investigation.

[8]  Alice M Stamatakis,et al.  The Inhibitory Circuit Architecture of the Lateral Hypothalamus Orchestrates Feeding , 2013, Science.

[9]  J. Betley,et al.  Deconstruction of a neural circuit for hunger , 2012, Nature.

[10]  J. Sandkühler,et al.  Multiple Targets of μ-Opioid Receptor-Mediated Presynaptic Inhibition at Primary Afferent Aδ- and C-Fibers , 2011, The Journal of Neuroscience.

[11]  S. Alford,et al.  G protein βγ-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties , 2006 .

[12]  M. Missler,et al.  Polarized Targeting of Neurexins to Synapses Is Regulated by their C-Terminal Sequences , 2008, The Journal of Neuroscience.

[13]  D. Wirtshafter,et al.  Comparative studies of the ingestive behaviors produced by microinjections of muscimol into the midbrain raphe nuclei of the ventral tegmental area of the rat. , 1988, Life sciences.

[14]  Karel Svoboda,et al.  Rapid and Reversible Chemical Inactivation of Synaptic Transmission in Genetically Targeted Neurons , 2005, Neuron.

[15]  S. Sternson,et al.  AGRP neurons are sufficient to orchestrate feeding behavior rapidly and without training , 2010, Nature Neuroscience.

[16]  G. Schultz,et al.  The GTP-binding protein, Go9 regulates neuronal calcium channels , 1987, Nature.

[17]  S. Leibowitz,et al.  Efferent projections from the paraventricular nucleus mediating α2-noradrenergic feeding , 1985, Brain Research.

[18]  L. Goutebroze,et al.  Axonal targeting of Caspr2 in hippocampal neurons via selective somatodendritic endocytosis , 2009, Journal of Cell Science.

[19]  B. Lowell,et al.  Glutamate mediates the function of melanocortin receptor 4 on Sim1 neurons in body weight regulation. , 2013, Cell metabolism.

[20]  Tianyi Mao,et al.  A Role for Myosin VI in the Localization of Axonal Proteins , 2011, PLoS biology.

[21]  W. Regehr,et al.  Short-term synaptic plasticity. , 2002, Annual review of physiology.

[22]  Sadegh Nabavi,et al.  Optogenetic Inhibition of Synaptic Release with Chromophore-Assisted Light Inactivation (CALI) , 2013, Neuron.

[23]  C. Stevens,et al.  Heterogeneity of Release Probability, Facilitation, and Depletion at Central Synapses , 1997, Neuron.

[24]  S. Alford,et al.  G protein betagamma subunit-mediated presynaptic inhibition: regulation of exocytotic fusion downstream of Ca2+ entry. , 2001, Science.

[25]  S. Leibowitz,et al.  Bombesin-induced anorexia: Sites of action in the rat brain , 1987, Peptides.

[26]  Alice M Stamatakis,et al.  Excitatory transmission from the amygdala to nucleus accumbens facilitates reward seeking. , 2011, Nature.

[27]  Lief E. Fenno,et al.  Amygdala circuitry mediating reversible and bidirectional control of anxiety , 2011, Nature.

[28]  S. Leibowitz,et al.  Efferent projections from the paraventricular nucleus mediating alpha 2-noradrenergic feeding. , 1985, Brain research.

[29]  WG Regehr,et al.  Contributions of calcium-dependent and calcium-independent mechanisms to presynaptic inhibition at a cerebellar synapse , 1996, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[30]  S. Sternson,et al.  A FLEX Switch Targets Channelrhodopsin-2 to Multiple Cell Types for Imaging and Long-Range Circuit Mapping , 2008, The Journal of Neuroscience.

[31]  J. Wess,et al.  Characterization of Central Inhibitory Muscarinic Autoreceptors by the Use of Muscarinic Acetylcholine Receptor Knock-Out Mice , 2002, The Journal of Neuroscience.

[32]  Steven P. Wilson,et al.  Designer Receptors Show Role for Ventral Pallidum Input to Ventral Tegmental Area in Cocaine Seeking , 2014, Nature Neuroscience.

[33]  Dai Watanabe,et al.  Reversible Suppression of Glutamatergic Neurotransmission of Cerebellar Granule Cells In Vivo by Genetically Manipulated Expression of Tetanus Neurotoxin Light Chain , 2003, The Journal of Neuroscience.

[34]  R. Wise,et al.  Opioid receptor subtypes associated with ventral tegmental facilitation and periaqueductal gray inhibition of feeding , 1987, Brain Research.

[35]  B. Roth,et al.  Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand , 2007, Proceedings of the National Academy of Sciences.

[36]  Allan R. Jones,et al.  A robust and high-throughput Cre reporting and characterization system for the whole mouse brain , 2009, Nature Neuroscience.

[37]  A. Sclafani,et al.  PVN-hindbrain pathway involved in the hypothalamic hyperphagia-obesity syndrome , 1988, Physiology & Behavior.

[38]  T. Kitamoto.  TARGETED EXPRESSION OF TEMPERATURE-SENSITIVE DYNAMIN TO STUDY NEURAL MECHANISMS OF COMPLEX BEHAVIOR IN Drosophila , 2002, Journal of neurogenetics.

[39]  P. Fletcher,et al.  Injecting 5-HT into the PVN does not prevent feeding induced by injecting 8-OH-DPAT into the raphe , 1993, Pharmacology Biochemistry and Behavior.

[40]  Gordon M G Shepherd,et al.  Topographic mapping of VMH → arcuate nucleus microcircuits and their reorganization by fasting , 2005, Nature Neuroscience.

[41]  A. Levey,et al.  Differential presynaptic and postsynaptic expression of m1–m4 muscarinic acetylcholine receptors at the perforant pathway/granule cell synapse , 1998, Neuroscience.

[42]  S. Alford,et al.  G Protein βγ Subunit-Mediated Presynaptic Inhibition: Regulation of Exocytotic Fusion Downstream of Ca2+ Entry , 2001, Science.

[43]  A. Loewy,et al.  Paraventricular hypothalamic nucleus: Axonal projections to the brainstem , 2010, The Journal of comparative neurology.

[44]  M. Verhage,et al.  Presynaptic signal transduction pathways that modulate synaptic transmission , 2009, Current Opinion in Neurobiology.

[45]  E. ten Dam,et al.  The 'cleavage' activities of foot-and-mouth disease virus 2A site-directed mutants and naturally occurring '2A-like' sequences. , 2001, The Journal of general virology.

[46]  K. Svoboda,et al.  Channelrhodopsin-2–assisted circuit mapping of long-range callosal projections , 2007, Nature Neuroscience.

[47]  M. Seki,et al.  Descending projections from the paraventricular hypothalamic nucleus to the spinal cord: anterograde tracing study in the rat. , 1995, Okajimas folia anatomica Japonica.

[48]  Robert A. McGovern,et al.  Divergence of Melanocortin Pathways in the Control of Food Intake and Energy Expenditure , 2005, Cell.

[49]  Y. Kajikawa,et al.  G-Protein-Coupled Modulation of Presynaptic Calcium Currents and Transmitter Release by a GABAB Receptor , 1998, The Journal of Neuroscience.

[50]  J. McCabe,et al.  Clonidine-induced feeding: Analysis of central sites of action and fiber projections mediating this response , 1984, Brain Research.