title: P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate Nucleus.
abstract: P2X4 receptors are ATP-gated cation channels that are widely expressed in the nervous system. To identify P2X4 receptor-expressing cells, we generated BAC transgenic mice expressing tdTomato under the control of the P2X4 receptor gene (P2rx4). We found sparse populations of tdTomato-positive neurons in most brain areas with patterns that matched P2X4 mRNA distribution. tdTomato expression within microglia was low but was increased by an experimental manipulation that triggered microglial activation. We found surprisingly high tdTomato expression in the hypothalamic arcuate nucleus (Arc) (i.e., within parts of the neural circuitry controlling feeding). Immunohistochemistry and genetic crosses of P2rx4 tdTomato mice with cell-specific GFP reporter lines showed that the tdTomato-expressing cells were mainly AgRP-NPY neurons and tanycytes. There was no electrophysiological evidence for functional expression of P2X4 receptors on AgRP-NPY neuron somata, but instead, we found clear evidence for functional presynaptic P2X4 receptor-mediated responses in terminals of AgRP-NPY neurons onto two of their postsynaptic targets (Arc POMC and paraventricular nucleus neurons), where ATP dramatically facilitated GABA release. The presynaptic responses onto POMC neurons, and the expression of tdTomato in AgRP-NPY neurons and tanycytes, were significantly decreased by food deprivation in male mice in a manner that was partially reversed by the satiety-related peptide leptin. Overall, we provide well-characterized tdTomato reporter mice to study P2X4-expressing cells in the brain, new insights on feeding-related regulation of presynaptic P2X4 receptor responses, and the rationale to explore extracellular ATP signaling in the control of feeding behaviors.
  
  
  
  Cells expressing ATP-gated P2X4 receptors have proven problematic to identify and study in brain slice preparations because P2X4 expression is sparse. To address this limitation, we generated and characterized BAC transgenic P2rx4 tdTomato reporter mice. We report the distribution of tdTomato-expressing cells throughout the brain and particularly strong expression in the hypothalamic arcuate nucleus. Together, our studies provide a new, well-characterized tool with which to study P2X4 receptor-expressing cells. The electrophysiological studies enabled by this mouse suggest previously unanticipated roles for ATP and P2X4 receptors in the neural circuitry controlling feeding.
authors: Departments of Physiology and.
authors: Ji
authors: J
authors: Xu
authors: Neurobiology, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095.
authors: Alexander M
authors: AM
authors: Bernstein
authors: Neurobiology, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095.
authors: Angela
authors: 0000-0003-2530-5727
authors: A
authors: Wong
authors: Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California-Los Angeles, Los Angeles, California 90095.
authors: Xiao-Hong
authors: 0000-0002-2363-1708
authors: XH
authors: Lu
authors: Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089.
authors: Sheraz
authors: S
authors: Khoja
authors: Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California-Los Angeles, Los Angeles, California 90095, Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095, and.
authors: X William
authors: XW
authors: Yang
authors: Titus Family Department of Clinical Pharmacy, School of Pharmacy, University of Southern California, Los Angeles, California 90089.
authors: Daryl L
authors: 0000-0003-2940-7739
authors: DL
authors: Davies
authors: Neurobiology, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095.
authors: Paul
authors: 0000-0002-9577-5874
authors: P
authors: Micevych
authors: Neurobiology, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095.
authors: Michael V
authors: 0000-0001-6075-0178
authors: MV
authors: Sofroniew
authors: Departments of Physiology and Neurobiology, David Geffen School of Medicine, University of California-Los Angeles, Los Angeles, California 90095, bkhakh@mednet.ucla.edu.
authors: Baljit S
authors: BS
authors: Khakh
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D054369:Agouti-Related Protein / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001111:Arcuate Nucleus of Hypothalamus / Q000378:metabolism; D001921:Brain / Q000166:cytology* / Q000187:drug effects; D004435:Eating / Q000187:drug effects / Q000502:physiology*; D019706:Excitatory Postsynaptic Potentials / Q000187:drug effects / Q000235:genetics; D005508:Food Deprivation / Q000502:physiology; D054439:Ghrelin / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D020738:Leptin / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009478:Neuropeptide Y / Q000378:metabolism; D018377:Neurotransmitter Agents / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011333:Pro-Opiomelanocortin / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D058848:Receptors, Purinergic P2X4 / Q000235:genetics / Q000378:metabolism*; D018709:Statistics, Nonparametric; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D054369:Agouti-Related Protein; C515199:Agrp protein, mouse; D054439:Ghrelin; D005904:Glial Fibrillary Acidic Protein; D020738:Leptin; D008070:Lipopolysaccharides; D008164:Luminescent Proteins; D009478:Neuropeptide Y; D018377:Neurotransmitter Agents; D010975:Platelet Aggregation Inhibitors; D012333:RNA, Messenger; D058848:Receptors, Purinergic P2X4; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D005680:gamma-Aminobutyric Acid; D011333:Pro-Opiomelanocortin; D000255:Adenosine Triphosphate
keywords: ATP; P2X; arcuate; ion channel; mouse model; receptor