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Selective expression of Gi/o-coupled ATP receptor P2Y12 in microglia in rat brain
Yo Sasaki1, Masato Hoshi, Chihiro Akazawa
1Department of Neurochemistry, National institute of Neuroscience, Kodaira, Tokyo, Japan.
Abstract:
Extracellular nucleotides, including ATP, have been demonstrated to transmit important physiological signals in the brain through either G-protein-coupled P2Y receptors or P2X receptors, which are ligand-gated ion channels. In this study, we performed a detailed analysis of the expression of the Gi/o-coupled receptor P2Y12 in the brain. Northern blot analysis demonstrated that P2Y12 is expressed predominantly in the brain, and to a lesser extent in the spleen. The cellular localization of P2Y12 was investigated by in situ hybridization, and P2Y12 mRNA was detected in small cells distributed throughout the brain, including the hippocampus. Expression of P2Y12 was also observed in naive and axotomized facial nuclei, and the number of P2Y12-expressing cells increased following facial nerve axotomy. Selective expression of P2Y12 mRNA in microglia was confirmed by double-label in situ hybridization and immunohistochemistry with antibodies against NeuN and Iba1 as an immunohistochemical marker for neurons and microglia, respectively. Hardly any P2Y12 mRNA was detected in macrophages obtained from the spleen and abdominal cavity, which share many surface molecules with microglia.
Insights
Extracellular nucleotides signal in the brain via P2Y receptors. This study reveals that the P2Y12 receptor is primarily expressed in brain microglia, suggesting a key role in neuroinflammation.
Area of Science:
- Neuroscience
- Molecular Biology
- Immunology
Background:
- Extracellular nucleotides like ATP are crucial signaling molecules in the brain.
- These signals are mediated by G-protein-coupled P2Y receptors and ligand-gated P2X receptors.
- The specific role and distribution of the Gi/o-coupled P2Y12 receptor in the brain remain incompletely understood.
Purpose of the Study:
- To comprehensively analyze the expression and cellular localization of the P2Y12 receptor in the brain.
- To investigate changes in P2Y12 expression following neuronal injury.
Main Methods:
- Northern blot analysis to determine P2Y12 expression levels.
- In situ hybridization to identify the cellular distribution of P2Y12 mRNA.
- Double-label in situ hybridization and immunohistochemistry to confirm cell-specific expression, using markers for neurons (NeuN) and microglia (Iba1).
Main Results:
- P2Y12 is predominantly expressed in the brain, with lower levels in the spleen.
- P2Y12 mRNA is found in small cells throughout the brain, including the hippocampus.
- Expression is detected in naive and axotomized facial nuclei, with increased cell numbers post-axotomy.
- P2Y12 mRNA expression is selectively localized to microglia, not splenic or peritoneal macrophages.
Conclusions:
- The P2Y12 receptor is primarily expressed in brain microglia.
- Facial nerve axotomy leads to an increase in P2Y12-expressing cells in facial nuclei.
- These findings highlight the specific role of P2Y12 in microglia within the central nervous system, potentially in response to injury.
