Immunolocalization of the P2X4 receptor on neurons and glia in the mammalian retina

doi:10.1016/j.neuroscience.2014.06.055

Neuroscience

Volume 277, 26 September 2014, Pages 55-71

https://doi.org/10.1016/j.neuroscience.2014.06.055 Get rights and content

Highlights

•
P2X4-R-IR in the retina is conserved across the mouse, rat and cat retinae.
•
P2X4-Rs were expressed post-synaptically to photoreceptors and bipolar cells.
•
P2X4-R-IR was observed on Müller cells, astrocytes and microglia.
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P2X4-Rs may be involved in lateral modulation of retinal synaptic transmission.
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P2X4-Rs may also be involved in modulating tissue homeostasis of the retina.

Abstract

Extracellular adenosine 5′-triphosphate (eATP) acts as a neurotransmitter within the retina and brain, activating a range of ionotropic P2X and metabotropic P2Y receptors. In this study, the specific localization of the P2X4 receptor (P2X4-R) subunit was evaluated in the retina using fluorescence immunohistochemistry and pre-embedding immuno-electron microscopy. Punctate P2X4-R labeling was largely localized to the inner and outer plexiform layers of mouse, rat and cat retinae. In the mouse outer retina, double-labeling of P2X4-R with the horizontal cell marker, calbindin, revealed P2X4-R immunoreactivity (P2X4-R-IR) on horizontal cell somata and processes. In the inner retina, P2X4-R expression was found closely associated with rod and cone bipolar cell terminals, and the punctate labeling was observed on calretinin-positive amacrine cells. Using immuno-electron microscopy, P2X4-Rs were observed on processes post-synaptic to photoreceptor and bipolar cell terminals, likely representing horizontal, amacrine and ganglion cells, respectively. Furthermore, P2X4-R expression was also observed on Müller cells, astrocytes and microglia. These data suggest a role for P2X4-Rs in the lateral inhibitory pathways of the retina, modulating neuronal function of photoreceptors and bipolar cells. The expression on macro- and microglial cells implicates a role for P2X4-Rs in glial signaling, tissue homeostasis and immunosurveillance within the mammalian retina.

Graphical abstract

By using immunohistochemistry combined with confocal and electron microscopy, the authors demonstrate P2X4 purinergic receptor expression on horizontal, amacrine and ganglion cells, and on macroglia and microglia in the mouse retina. This suggests a role for P2X4-R in the modulation of neuronal and glial signaling and tissue homeostasis in the retina.

Introduction

Extracellular adenosine 5′-triphosphate (eATP) acts as a neurotransmitter in the central and peripheral nervous systems and exerts its effects on two families of membrane-bound receptors: ligand-gated P2X cation channels (Khakh, 2001) and G-protein-coupled P2Y receptors (Lazarowski et al., 2003). Both P2X and P2Y receptors are further classified into different subtypes; seven P2X (P2X1-7) and eight P2Y (P2Y1, 2, 4, 6, 11–14) receptors have been characterized in mammals by molecular means (North and Barnard, 1997, Ralevic and Burnstock, 1998). eATP elicits a fast excitatory response via P2X receptors, resulting in non-selective cation permeability of Ca²⁺, Na⁺ and K⁺ (North and Surprenant, 2000). Additionally, each P2X receptor subtype has unique gating and pharmacological properties that are defined by the trimeric arrangement of subunits assembled as either homomeric or heteromeric complexes (Nicke et al., 1998, Torres et al., 1999, Aschrafi et al., 2004, Nicke, 2008, Kawate et al., 2009).

P2X4 receptor (P2X4-R) expression has been localized to neurons throughout the CNS. It has been implicated in physiological functions including the modulation of neurotransmission (Le et al., 1998, Rubio and Soto, 2001), contributing to synaptic strengthening (Baxter et al., 2011). In addition, immunohistochemical studies have identified abundant P2X4-R immunoreactivity (P2X4-R-IR) on microglia in the brain and spinal cord (Cavaliere et al., 2003, Tsuda et al., 2003, Ulmann et al., 2008), and a role in mediating neuroinflammatory events post-injury has been suggested (de Rivero Vaccari et al., 2012). As a result, activation of glial P2X4-Rs has been extensively studied in microglial responses to injuries and degeneration in the CNS (Guo and Schluesener, 2005, Ulmann et al., 2008, Domercq et al., 2013).

The localization of P2X4-Rs in the mammalian retina has been identified with a variety of techniques. Reverse transcriptase polymerase chain reaction (RT-PCR) detected P2X 2, 3, 4, 5 and 7 gene expression in the rat retina (Brändle et al., 1998a, Brändle et al., 1998b, Wheeler-Schilling et al., 2000, Wheeler-Schilling et al., 2001). At the protein level, P2X4-R expression has been identified in the rodent (Wheeler-Schilling et al., 2001, Kaneda et al., 2004) and macaque retinae (Ishii et al., 2003, Gu et al., 2013), where it was found to be localized to neuronal cell somata and microglia. However, a detailed analysis of P2X4-R expression on specific neuronal and glial cell populations in the retina has not been undertaken. Furthermore, although it has been implicated in synaptic signaling in other parts of the CNS, the synaptic characterization of P2X4-R in specific circuits of the retina remains to be determined. The aim of this study was to characterize P2X4-R expression in the mouse, rat and cat retinas, and to identify the specific retinal cell types expressing P2X4-R in the mouse retina so as to gain further insight into the potential physiological function of the purinergic system in retinal signaling.

Section snippets

Animals

All experimental procedures using animals were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23) and The University of Melbourne Animal Ethics Committee (Ethics #: 1112260). All efforts were made to minimize the number of animals used and their suffering. Adult C57BL/6J, wildtype mice and dark agouti rats were obtained from the Animal Resource Centre (WA, Australia), and were used at 6–8 weeks of age. All

P2X4-R is expressed in the mouse, rat and cat retinae

P2X4-R protein expression has been identified using immunohistochemistry in the rodent (Wheeler-Schilling et al., 2001, Kaneda et al., 2004) and macaque retinae (Ishii et al., 2003, Gu et al., 2013). However, the specific neuronal and glial cell classes that express P2X4-R-IR remains elusive. To confirm the specificity of the P2X4-R antibody (Alomone, #APR-002) used in the current study, Western blotting and immunofluorescence histochemistry with peptide block were completed using mouse retinal

Discussion

This study demonstrates that P2X4-R is localized to the synaptic layers of the adult mouse retina and that this expression characteristic is conserved across different mammalian species. In the outer retina, P2X4-R expression was observed on horizontal cells at the light and ultrastructural levels. In the inner retina, P2X4-R was observed on elements post-synaptic to the bipolar cell terminals, amacrine and ganglion cells. In addition, Müller cells, astrocytes and microglia were also P2X4-R

Conflict of interest

None.

Roles of authors

All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: ELF, KAV. Acquisition of data: TH, KAV, ELF. Analysis and interpretation of data: TH, KAV, ELF. Drafting of the manuscript: TH, KAV, ELF. Critical revision of the manuscript for important intellectual content: TH, KAV, ELF. Statistical analysis: TH, KAV. Obtained funding: ELF. Administrative, technical, and material

Funding

This work was funded by NHMRC – Australia (P2X7-grant #APP1061419) to ELF and the Macular Disease Foundation of Australia, 2013 to ELF.

Acknowledgments

The authors would like to thank Prof. Paul McMennamin (Monash University, VIC, Australia) for providing breeding pairs of the CX3CR1^GFP/+ mice, for A/Prof Anthony Hannan (Howard Florey Institute, VIC, Australia) for providing breeding pairs of the Thy-1-HYFP mice, and Robert Shepherd (The Bionics Institute, VIC, Australia) for providing adult cat retinas to this project.

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Whether P2X4 receptors are expressed in neurons or not have caused great controversy. With the continuous exploration of the function of P2X4 receptors in nervous system, it has been confirmed that P2X4 receptors are also expressed in neurons, which induces abnormal discharges of neurons and enhances the transmission of sensory information (Ho et al., 2014; Kuroda et al., 2012). Another important P2X receptors is the P2X7 receptor.
The development of cerebral ischemia involves brain damage and abnormal changes in brain function, which can cause neurosensory and motor dysfunction, and bring serious consequences to patients. P2X purinergic receptors are expressed in nerve cells and immune cells, and are mainly expressed in microglia. The P2X4 and P2X7 receptors in the P2X purinergic receptors play a significant role in regulating the activity of microglia. Moreover, ATP-P2X purine information transmission is involved in the progression of neurological diseases, including the release of pro-inflammatory factors, driving factors and cytokines after cerebral ischemia injury, inducing inflammation, and aggravating cerebral ischemia injury. P2X receptors activation can mediate the information exchange between microglia and neurons, induce neuronal apoptosis, and aggravate neurological dysfunction after cerebral ischemia. However, inhibiting the activation of P2X receptors, reducing their expression, inhibiting the activation of microglia, and has the effect of protecting nerve function. In this paper, we discussed the relationship between P2X receptors and nervous system function and the role of microglia activation inducing cerebral ischemia injury. Additionally, we explored the potential role of P2X receptors in the progression of cerebral ischemic injury and their potential pharmacological targets for the treatment of cerebral ischemic injury.
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Recently, choline transport through P2X2 channel in OFF cholinergic amacrine cells was observed in the mouse retina, raising the possibility that these receptors can work as a new choline transport system in the retina (Ishii et al., 2017). While high immunoreactivity for P2X3 receptor subtype was detected in GABAergic amacrine cells, over the ganglion cell layer and plexiform layers in the rodent retina (Shigematsu et al., 2007; Puthussery and Fletcher, 2007), immunoreactivity against P2X4 receptors was found over horizontal cell bodies and processes, amacrine and ganglion cell processes in the mammalian retina (Kaneda et al., 2004; Ho et al., 2014). P2X4 immunoreactivity was also observed over Müller cell processes in the mouse retina (Ho et al., 2014).
Retinal injuries and diseases are major causes of human disability involving vision impairment by the progressive and permanent loss of retinal neurons. During development, assembly of this tissue entails a successive and overlapping, signal-regulated engagement of complex events that include proliferation of progenitors, neurogenesis, cell death, neurochemical differentiation and synaptogenesis. During retinal damage, several of these events are re-activated with both protective and detrimental consequences. Purines and pyrimidines, along with their metabolites are emerging as important molecules regulating both retinal development and the tissue’s responses to damage. The present review provides an overview of the purinergic signaling in the developing and injured retina. Recent findings on the presence of vesicular and channel-mediated ATP release by retinal and retinal pigment epithelial cells, adenosine synthesis and release, expression of receptors and intracellular signaling pathways activated by purinergic signaling in retinal cells are reported. The pathways by which purinergic receptors modulate retinal cell proliferation, migration and death of retinal cells during development and injury are summarized. The contribution of nucleotides to the self-repair of the injured zebrafish retina is also discussed.
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Immunohistochemical studies in the retina have revealed the precise distribution of individual subtypes (P2X1-P2X7) of P2X receptors (Jabs et al., 2000; Kaneda et al., 2004; Puthussery and Fletcher, 2007; Shigematsu et al., 2007; Wheeler-Schilling et al., 2000). In the mouse retina, precise distribution of P2X receptors has been reported: P2X5 and P2X7 in the outer and inner segments of photoreceptors (IS/OS), P2X3, P2X4, and P2X7 in the outer plexiform layer (OPL), P2X2, P2X3, P2X5, and P2X7 in the inner nuclear layer (INL), P2X1, P2X2, P2X3, P2X4, and P2X7 in the inner plexiform layer (IPL), and P2X3, P2X4, P2X5, and P2X7 in the ganglion cell layer (GCL) (Ho et al., 2014; Kaneda et al., 2004; Shigematsu et al., 2007; Vessey and Fletcher, 2012). However, there have been few studies focusing on the physiological role of P2X receptors in visual information processing (P2X7 (Puthussery et al., 2006); P2X2 (Kaneda et al., 2008)).
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Immunolocalization of the P2X4 receptor on neurons and glia in the mammalian retina

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Animals

P2X4-R is expressed in the mouse, rat and cat retinae

Discussion

Conflict of interest

Roles of authors

Funding

Acknowledgments

J Mol Biol

Brain Res

Brain Res

Prog Retin Eye Res

Neurochem Int

Neuroscience

Neuron

Neuroscience

Exp Eye Res

Prog Retin Eye Res

Brain Res

Neuroscience

Trends Neurosci

Biochem Biophys Res Commun

Curr Opin Neurobiol

Neuron

Neuroscience

Neuron

J Neuroimmunol

J Biol Chem

Neurosci Lett

Neuroscience

Role of P2X4 receptors in synaptic strengthening in mouse CA1 hippocampal neurons

Eur J Neurosci

Müller cells express the cannabinoid CB2 receptor in the vervet monkey retina

J Comp Neurol

Selective synaptic distribution of kainate receptor subunits in the two plexiform layers of the rat retina

J Neurosci

Mechanisms of VEGF- and glutamate-induced inhibition of osmotic swelling of murine retinal glial (Müller) cells: indications for the involvement of vesicular glutamate release and connexin-mediated atp release

Neurochem Res

Functional interactions between P2X4 and P2X7 receptors from mouse salivary epithelia

J Physiol

Dicer inactivation leads to progressive functional and structural degeneration of the mouse retina

J Neurosci

P2X4 receptors influence inflammasome activation after spinal cord injury

J Neurosci

Monoclonal antibodies specific for glial fibrillary acidic (GFA) protein and for each of the neurofilament triplet polypeptides

Differentiation

Neurotransmitter signaling in the pathophysiology of microglia

Front Cell Neurosci

Organization of the primate retina: electron microscopy

Proc R Soc Lond B Biol Sci

Synaptic localization of NMDA receptor subunits in the rat retina

J Comp Neurol

Synaptotagmin I and II are present in distinct subsets of central synapses

J Comp Neurol

Colocalization of synaptic GABA(C)-receptors with GABA (A)-receptors and glycine-receptors in the rodent central nervous system

Cell Tissue Res

Identification of P2Y receptor subtypes in human müller glial cells by physiology, single cell RT-PCR, and immunohistochemistry

Investig Ophthalmol Visual Sci

Rod bipolar cells in the mammalian retina show protein kinase C-like immunoreactivity

J Comp Neurol

A rare functional haplotype of the P2RX4 and P2RX7 genes leads to loss of innate phagocytosis and confers increased risk of age-related macular degeneration

FASEB J

Evidence for functional P2X4/P2X7 heteromeric receptors

Mol Pharmacol

Localization and developmental expression of the NMDA receptor subunit NR2A in the mammalian retina

J Comp Neurol

Immunocytochemical analysis of the mouse retina

J Comp Neurol

Immunocytochemical description of five bipolar cell types of the mouse retina

J Comp Neurol

Corelease of dopamine and GABA by a retinal dopaminergic neuron

J Neurosci

Amyloid precursor protein is required for normal function of the rod and cone pathways in the mouse retina

PLoS One

Neuron-specific distribution of P2X7 purinergic receptors in the monkey retina