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P2X4 Receptor Reporter Mice: Sparse Brain Expression and Feeding-Related Presynaptic Facilitation in the Arcuate
Ji Xu1, Alexander M Bernstein2, Angela Wong2
1Departments of Physiology and.
Unlabelled:
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.
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.
Insights
We developed new P2X4 receptor reporter mice to identify brain cells expressing these ATP-gated channels. These mice reveal P2X4 receptor roles in feeding control circuitry, particularly in the hypothalamus.
Area of Science:
- Neuroscience
- Molecular Biology
- Endocrinology
Background:
- P2X4 receptors are ATP-gated cation channels crucial for nervous system function.
- Identifying P2X4 receptor-expressing cells is challenging due to sparse expression patterns.
- Understanding P2X4 receptor roles in neural circuits, including feeding regulation, requires precise cell identification.
Purpose of the Study:
- To generate and characterize BAC transgenic P2rx4 tdTomato reporter mice for visualizing P2X4 receptor-expressing cells.
- To investigate the distribution and function of P2X4 receptors in the brain, with a focus on feeding control circuits.
- To explore the regulation of P2X4 receptor activity by physiological states like feeding and satiety.
Main Methods:
- Generation of BAC transgenic mice with tdTomato reporter under the P2rx4 promoter.
- Distribution analysis of tdTomato expression in various brain regions using immunohistochemistry.
- Electrophysiological recordings to assess functional P2X4 receptor activity in specific neuronal populations.
- Genetic crosses with cell-specific reporter lines to identify tdTomato-expressing cell types.
- Experimental manipulation involving food deprivation and leptin administration to study regulatory effects.
Main Results:
- Established well-characterized P2rx4 tdTomato reporter mice showing P2X4 expression patterns in neurons and microglia.
- Identified significant P2X4 receptor expression in hypothalamic arcuate nucleus (Arc) neurons (AgRP-NPY neurons) and tanycytes.
- Demonstrated functional presynaptic P2X4 receptor-mediated facilitation of GABA release from AgRP-NPY neuron terminals onto POMC and PVN neurons.
- Observed decreased presynaptic responses and tdTomato expression in AgRP-NPY neurons during food deprivation, partially reversed by leptin.
Conclusions:
- The P2rx4 tdTomato reporter mice provide a valuable tool for studying P2X4 receptor-expressing cells in the brain.
- Presynaptic P2X4 receptors in AgRP-NPY neuron terminals play a significant role in modulating GABAergic signaling within feeding circuits.
- Extracellular ATP signaling via P2X4 receptors is implicated in the neural control of feeding behaviors and is sensitive to nutritional status and satiety signals.
