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.

The actions of ghrelin in the paraventricular nucleus: energy balance and neuroendocrine implications

“…The ARC is a point of integration between the periphery and the brain for metabolic regulation. The ARC contains orexigenic dopamine and neuropeptide Y (NPY)/agouti‐related peptide (AgRP) neurons that inhibit PVN neurons. The ARC also contains anorexigenic proopiomelanocortin (POMC) neurons that project to the PVN releasing α‐melanocyte−stimulating hormone (α‐MSH) that activates melanocortin receptors 3/4 (MC3/4) and increases the activity of PVN neurons .…”

“…Ghrelin directly affects PVN neurons and acts presynaptically on afferent neurons either decreasing inhibition, excitation, or both . The presynaptic effects may be reversed by CB1 receptor antagonists and ATP . ATP is possibly released by astrocytes after retrograde signaling mediated by the dendritic release of AVP .…”

Activation of basal forebrain purinergic P2 receptors promotes wakefulness in mice

“…2d ; 4.8 ± 1.4 mV, n = 16; p < 0.0001 compared to TTX control; 6 cholinergic neurons, 5 large Ih and 5 small Ih GABAergic neurons tested). Activation of P2XRs on presynaptic terminals 8 , 9 , 28 or astrocytes 7 can induce neurotransmitter release. Thus, we tested the ATP-γ-S response in a cocktail of TTX, glutamate receptor antagonists (20 µM CNQX + 50 µM D-AP5), and a GABA A receptor antagonist (10 µM GABAzine).…”

“…Activation of P2YR by ATP triggers G-protein coupled pathways and induces Ca 2+ release from intracellular stores 6 . Furthermore, activation of P2R can lead to the release of neurotransmitters or neuromodulators from presynaptic terminals or from astrocytes 7 – 9 . While the cellular effects of P2R have been studied in many tissues, the physiological and pharmacological roles of P2Rs in vivo are still being uncovered.…”

Amino acid sensing in hypothalamic tanycytes via umami taste receptors

“…Our discovery that tanycytes also sense amino acids in the CSF via at least two receptors is an important advance that suggests: 1) that tanycytes are anorexigenic; and 2) that they might act with the neural networks in the hypothalamus to regulate food intake. This could potentially be via the wave of ATP release that travels down the tanycyte processes into ARC, as ARC neurons express P2 receptors [57] , [58] . The ATP from tanycytes could potentially excite the anorexigenic POMC neurons in the ARC or activate NPY/AgRP neurons via mTOR (tanycytes have been shown to have a close anatomical association with NPY neurons [59] ) to inhibit NPY secretion and suppress appetite this way.…”