Preliminary Study Of P2Y₁ Receptor-mediated Signal Transmission From Dorsal Horn Astrocyte To Neuron

The spinal dorsal horn plays an important role in nociceptive information transmission from the periphery to central nervous system. While neuronal functioning is altered, there is significant evidence showing that exaggerated pain is regulated by the activation of astrocytes and microglia. More and more evidences suggest that the activation of spinal cord glia is necessary for the development and maintenance of pathological pain and hyperalgesia. A variety of noxious stimulation, such as subcutaneous irritants, peripheral nerve trauma can activate astrocyte and microglia of the dorsal spinal cord. Activated glia can release a variety of proinflammatory cytokines and neuroactive substance to promote the transmission of nociceptive inputs. However, the mechanism of the activation of glia in spinal cord induced by nociceptive inputs remains to be further study.ATP is an important neurotransmitter in nervous system and plays an important role in the transmission of nociceptive information in the spinal cord through binding with and acting on ATP-gated ionotropic P2X receptor and the G protein-coupled P2Y receptor. P2Y receptors may modulate the nociception processes at peripheral and spinal cord levels. Astrocytes are endowed with a minor population of P2X and a major population of P2Y receptors. But it has not been reported whether P2Y₁ receptor contributes to the activation of astrocytes in cultured dorsal horn astrocytes.This study will use some methods include morphology, molecular biology and electrophysiology to investigate the relationship between P2Y₁ receptor and the activation of cultured rat dorsal horn astrocytes; to speculate the effects of P2Y₁ receptors activation on glutamate release from astrocytes and its signal transduction pathways; to investigate whether glutamate release from astrocytes acts on cultured dorsal horn neurons.Methods: Primary dissociated culture of dorsal spinal cord astrocytes were prepared from Sprague–Dawley rats. The expression of GFAP and P2Y₁ receptor in cultured dorsal horn astrocytes were observed by using immunohistochemical staining and western blot. A System of Image Prop–plus 5.0 was used to gain the AOD of the GFAP-positive astrocytes. The expression of GFAP were also detected with western blot. Changes of [Ca²⁺]i after ATP, ADP and ADPβS administration in astrocytes were detected with CLSM using fluo-4/AM as a calcium fluorescent indicator. ATP, ADP and ADPβS-evoked glutamate release were detected by using HPLC. CLSM and Patcn clamp recording were used to assay the effects of the astrocyte-conditioned medium (ACM) on cultured dorsal horn neurons. Results:Part one:1. There is direct immunohistochemical and western blot evidence that the P2Y₁ receptor protein is expressed in cultured dorsal horn astrocytes;2. ATP, ADP and ADPβS (0.001¹00μmol/L) can dose-dependently increase the [Ca²⁺]i of cultured rat dorsal horn astrocytes;3. MRS2179(0.001¹00μmol/L), P2Y₁-specific antagonist, can dose-dependently inhibit the [Ca²⁺]i responses induced by 100μM ATP, ADP and ADPβS;4. There is direct immunohistochemical and western blot evidence that P2Y₁ receptor participates in the 100μmol/L ATP, ADP and ADPβS-induced the increases of GFAP synthesis in cultured rat dorsal horn astrocytes.Part two:1. Using HPLC, we found that 100μmol/L ATP, ADP and ADPβS cause glutamate release with a time-dependent manner;2. 100μmol/L MRS2179 significantly inhibited the glutamate release induced by 100μmol/L ATP, ADP and ADPβS, which suggest that P2Y₁ receptor activation is involved in this process;3. P2Y₁ receptor-mediated [Ca²⁺]i increase and glutamate release do depend on the release of Ca²⁺ from IP3-sensitive intracellular stores into the cytosolic compartment;4. ADPβS–evoked glutamate release partly mediated by PKC, Na+-K+-Cl- anion cotransport and VSAC. PKA and gap junction are not related to glutamate release after P2Y₁ receptor activation;Part three:1. P2Y₁ receptor activation causes astrocytic glutamate release, which in turn induce primary cultured dorsl horn neuron [Ca²⁺]i increase that can be blocked by 50μmol/L AP-5 (NMDAR antagonist), but was not influrenced by suramin (unselective P2 receptor antagonist);2. Whole cell patch-clamp recordings shown that glutamate released from astrocyte can acts on dorsal horn neurons. Conditional culture medium causes inward current, which are blocked by AP-5 and non-NMDAR antagonist (CNQX),but not influrenced by suramin.Conclusions:1. P2Y₁ receptor protein is expressed in cultured dorsal horn astrocytes;2. P2Y₁ receptor mediate the [Ca²⁺]i responses induced by ATP, ADP and ADPβS;3. P2Y₁ receptor participate in the ATP, ADP and ADPβS-induced astrocytic activation;4. ATP, ADP and ADPβS can trigger glutamate release from cultured dorsal horn astrocytes, which are mediated via P2Y₁ receptor;5. P2Y₁ receptor-mediated [Ca²⁺]i increase and glutamate release do depend on the release of Ca²⁺ from intracellular stores into the cytosolic compartment;6. ADPβS-evoked glutamate release is partly mediated by PKC, anion cotransport and volume-sensitive chloride channel. PKA and gap junction are not related to glutamate release after P2Y₁ receptor activation;7. Activation of astrocytes causes glutamate release, which in turn induces primary cultured dorsl horn neuron [Ca²⁺]i increase and this effect is mediated by NMDA receptor;8. Whole cell patch-clamp recordings shown that glutamate released by astrocyte can acts on dorsal horn neurons. Conditional culture medium-induced membrane current is mediated by NMDA receptor and non- NMDA receptor.In summary, these results suggest that the cultured dorsal horn astrocytes express P2Y₁ purinoceptors, which participates in the ATP, ADP and ADPβS-evoked Ca²⁺ mobilization, astrocytic activation and glutamate release. We also provide evidence that glutamate released by astrocytes acts on cultured dorsal horn neurons. These results also suggested P2Y₁ receptor activation triggered calcium signal, which play an important role in bi-directional communication between neurons and astrocyte.