Development of a Mouse Reporter Strain for the Purinergic P2X₂Receptor

Abstract

The ATP-sensitive P2X 2 ionotropic receptor plays a critical role in a number of signal processes including taste and hearing, carotid body detection of hypoxia, the exercise pressor reflex and sensory transduction of mechanical stimuli in the airways and bladder. Elucidation of the role of P2X 2 has been hindered by the lack of selective tools. In particular, detection of P2X 2 using established pharmacological and biochemical techniques yields dramatically different expression patterns, particularly in the peripheral and central nervous systems. Here, we have developed a knockin P2X 2-cre mouse, which we crossed with a cre-sensitive tdTomato reporter mouse to determine P2X 2 expression. P2X 2 was found in more than 80% of nodose vagal afferent neurons, but not in jugular vagal afferent neurons. Reporter expression correlated in vagal neurons with sensitivity to mATP. P2X 2 was expressed in 75% of petrosal afferents, but only 12% and 4% of dorsal root ganglia and trigeminal afferents, respectively. P2X 2 expression was limited to very few cell types systemically. Together with the central terminals of P2X 2expressing afferents, reporter expression in the CNS was mainly found in brainstem neurons projecting mossy fibers to the cerebellum, with little expression in the hippocampus or cortex. The structure of peripheral terminals of P2X 2-expressing afferents was demonstrated in the tongue (taste buds), carotid body, trachea and esophagus. P2X 2 was observed in hair cells and support cells in the cochlear, but not in spiral afferent neurons. This mouse strain provides a novel approach to the identification and manipulation of P2X 2-expressing cell types. 3 Significance statement Inhibitor and knockout studies have demonstrated the critical role of P2X 2 in multiple sensory signaling pathways. Nevertheless, P2X 2 expression patterns are controversial, as biochemical studies suggest widespread expression whereas functional studies suggest restricted expression. Functional characterization is further complicated by heteromeric P2X 2/3 channels that have hybrid pharmacology and biophysical properties. We have developed a P2X 2-cre mouse to determine the expression pattern of P2X 2. In the periphery, P2X 2 expression is found in almost all nodose sensory afferents but is limited to only minor subsets of trigeminal and DRG afferents. Centrally, P2X 2 is mostly expressed in neurons projecting mossy fibers to the cerebellum. Thus we provide novel evidence for the specific expression of P2X 2 , which is more limited than previously thought.

BAC transgenic mice to study the expression of P2X2 and P2Y1 receptors

“…Another reporter mouse line developed as a knockin mouse that is expressing Cre recombinase in a P2X2dependent fashion and subsequently crossed with a cre-sensitive tdTomato mouse line [39]. Moreover, similar to our findings, this mouse line shows reporter expression in neurons in the dentate gyrus within the hippocampus.…”

“…In our C57BL/6 J BAC transgenic mice, we were able to demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. The expression pattern on DRG neurons and in the specified locations of the CNS, such as the hippocampus, was recently confirmed by other P2X2R transgenic mouse lines detailed below [39,40]. However, it should be reemphasized here that the weak fluorescence intensity in our P2X2R-TagRFP mouse and the therefore necessary use of anti-TagRFP immunostaining and subsequent signal amplification have precluded tracking of living cells or identifying them in sections for targeted examination, e.g., by patch-clamp electrophysiology.…”

Update of P2X receptor properties and their pharmacology: IUPHAR Review 30

“…A floxed humanized P2X7 receptor knock-in mouse (P2rx7 tm1.1(P2RX7)Jde ) showed an abnormal sleep pattern upon hP2X7R deletion. (Kim et al, 2020).…”

“…While both the BAC transgenic Tg( P2rx4 -tdTomato)1Khakh (expressing soluble tdTomato) and the P2rx4 mCherryIN knock-in mouse (conditionally expressing mCherry-tagged P2X4) reliably reported P2X4 receptor expression ( Bertin et al, 2020 ; Xu et al, 2016 ), two BAC transgenic P2X7 receptor reporter mice (Tg(P2rx7-EGFP)FY174Gsat and Tg(RP24–114E20-P2X7-His-StrepEGFP)Ani) (expressing soluble and P2X7-fused EGFP, respectively) show highly divergent expression patterns ( Kaczmarek-Hajek et al, 2018 ), further demonstrating the need for careful characterization of mouse models, in particular those generated in high-throughput approaches. Most recently, a knock-in P2X2 Cre mouse was described, which upon crossing with Cre-sensitive reporter mice reveals P2X2 receptor expression ( Kim et al, 2020 ).…”

The Role of Microglial Purinergic Receptors in Pain Signaling

“…Expression of these receptors has been demonstrated in rodent DRGs, but primate DRGs appear to only express P2X3 [ 48 ]. A recent study using P2X2 reporter mice showed that P2X2 expression is lower in DRGs and trigeminal ganglia than the level suggested by past studies [ 49 ].…”