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P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate Nucleus

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P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate Nucleus 8902 • The Journal of Neuroscience, August 24, 2016 • 36(34):8902–8920 Cellular/Molecular P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate Nucleus Ji Xu,1 Alexander M. Bernstein,2* XAngela Wong,2* X Xiao-Hong Lu,4 Sheraz Khoja,6 X. William Yang,4,5 X Daryl L. Davies,3 XPaul Micevych,2 XMichael V. Sofroniew,2 and Baljit S. Khakh1,2 Departments of 1Physiology and 2Neurobiology, David Geffen School of Medicine, University of California–Los Angeles, Los Angeles, California 90095, 3Titus Family Department of Clinical Pharmacy, School of Pharmacy, University of Southern California, Los Angeles, California 90089, 4Center for Neurobehavioral Genetics, Semel Institute for Neuroscience and Human Behavior, University of California–Los Angeles, Los Angeles, California 90095, 5Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine, University of California–Los Angeles, Los Angeles, California 90095, and 6Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, California 90089 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 expres- sion 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. Key words: arcuate; ATP; ion channel; mouse model; P2X; receptor Significance Statement 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. Introduction (Burnstock, 1972; Khakh and North, 2006). P2X receptors are ATP- Extracellular ATP functions as a signaling molecule in the nervous gated cation channels comprising seven subunits (P2X1-P2X7), six system by activating plasma membrane P2X and P2Y receptors homomeric receptors, and several heteromeric assemblies (Khakh et Received May 7, 2016; revised June 17, 2016; accepted June 20, 2016. This work was supported by National Institutes of Health Grant NS073980, University of California–Los Angeles Author contributions: J.X., P.M., and B.S.K. designed research; J.X., A.M.B., A.W., and B.S.K. performed research; unrestrictedfunds,andinpartbyGrantNS060677toB.S.K.ThebreedingoftheP2X4knock-outmicewassupported X.-H.L., S.K., X.W.Y., D.L.D., P.M., and M.V.S. contributed unpublished reagents/analytic tools; J.X. and B.S.K. ana- by The National Institute of Alcohol Abuse and Alcoholism Grant AA022448 to D.L.D. The in vivo feeding experi- lyzed data; J.X. and B.S.K. wrote the paper. ments were supported by The National Institute of Drug Abuse Grant DA013185 to P.M. X.W.Y. and Xu et al. • P2X4 Receptor Reporter Mice J. Neurosci., August 24, 2016 • 36(34):8902–8920 • 8903 al., 2001; North, 2002). We focused on P2X4, which was cloned pression in the hypothalamic arcuate nucleus (Arc), an area ϳ20 years ago (Bo et al., 1995; Buell et al., 1996; Se´gue´la et al., where P2X responses had not been described in detail (Stojilkovic, 1996; Soto et al., 1996; Wang et al., 1996). Important progress has 2009). Immunohistochemistry (IHC) and breeding of P2rx4 been made in understanding how P2X4 receptors work (Gonza- tdTomato mice with cell-specific GFP reporter lines revealed that les et al., 2009; Kawate et al., 2009; Coddou et al., 2011; Hattori P2X4 receptors were expressed in the AgRP-NPY neurons. Electro- and Gouaux, 2012; Jiang et al., 2013; Samways et al., 2014; Hab- physiological evaluations showed that P2X4 receptors mediated pre- ermacher et al., 2016), but less is known about their expression synaptic facilitation of GABA release onto POMC neurons. These and functions in the brain. responses were strongly regulated by food deprivation and leptin, P2X4 receptors are expressed on the cell surface and also which engage Arc satiety-related signaling. Together, our studies within acidic intracellular organelles (Bobanovic´ et al., 2002; provide a new well-characterized tool with which to study P2X4 Royle et al., 2002, 2005; Bowler et al., 2003; Royle and Murrell- receptor-expressing cells and suggest unanticipated roles for ATP Lagnado, 2003; Qureshi et al., 2007; Murrell-Lagnado and signaling in the neural circuitry controlling feeding. Qureshi, 2008; Stokes and Surprenant, 2009; Toulme et al., 2010; Miklavc et al., 2011). Important roles for P2X4 receptors are Materials and Methods emerging in a variety of pathophysiological processes. For exam- All animal experiments were conducted in accordance with the National ple, P2X4 receptors are upregulated in microglia following neu- Institute of Health Guide for the care and use of laboratory animals and were approved by the Chancellor’s Animal Research Committee at the ropathic pain and epilepsy (Tsuda et al., 2003; Coull et al., 2005; University of California–Los Angeles. Ulmann et al., 2013). They are also implicated in inflammatory Generation of P2rx4 tdTomato BAC transgenic mice. A 191 kb mouse pain (Ulmann et al., 2010), lung surfactant secretion (Miklavc et bacterial artificial chromosome (BAC #RP23-448O6) containing the 21 al., 2011), alcohol intake (Wyatt et al., 2014), alcohol preference kb P2X ligand-gated ion channel 4 (P2X4) coding region, ϳ100 kb 5Ј (Ostrovskaya et al., 2011; Yardley et al., 2012), morphine- flanking region, and ϳ70 kb 3Ј flanking region was identified through a induced hyperalgesia (Ferrini et al., 2013), cardiac function database search and obtained from the BACPAC Resource Center (Oak- (Yang et al., 2004), and neurodevelopmental disorders (Bortolato land Children’s Hospital, Oakland, CA). The cDNA of the orange/red et al., 2013; Wyatt et al., 2013). These diverse roles for P2X4 fluorescent protein, tdTomato with a PolyA sequence, was inserted into receptors likely reflect their distribution in several brain regions exon 1 of the P2rx4 gene to replace the endogenous initiation of transla- and other organs, which was first reported by in situ hybridiza- tion codon, according to an established protocol (Yang and Gong, 2005). The construction of the recombination cassette, subcloning into a shuttle tion studies (Buell et al., 1996). However, identifying native P2X4 vector (pLD53.SC-AB), homologous BAC recombination, and purifica- receptor responses within intact preparations, such as brain tion of intact BAC DNA for pronuclear injections were all done following slices, has proven extremely challenging. First, there are no selec- described procedures (Yang and Gong, 2005).A10␮l sample of the fully tive P2X4 receptor agonists or antagonists for use in rodents, modified and verified BAC DNA was analyzed by pulse-field gel electro- making it difficult to pinpoint P2X4 receptor-expressing cells phoresis to confirm BAC band integrity, quality, and size. Circular BAC based on function (Ase et al., 2015). Moreover, although iver- DNA concentration was measured using a Nanodrop spectrophotometer mectin is a potent allosteric regulator of P2X4 receptors (Khakh before being diluted to a final concentration of ϳ2 ng/␮l by the trans- et al., 1999), it has actions at other receptors (Zemkova´ et al., genic core facility (University of California–Los Angeles) and used for 2014) that limit its usefulness as a selective P2X4 receptor probe embryo injections. The incorporation of the transgene into the mouse in multicellular preparations. Second, despite Ͼ15 years of work, genome was identified through PCR. The genotyping primers for the transgene were as follows: 5Ј-AGT CAG GGG
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