Effects Of General Anesthetics On The Excitability And Transmitter Release Of Cultured Rat Cortical Astrocytes

Objective: General anesthetics can induce reversible loss of consciousness, but the mechanism is still unknown. While, the synapse transmission in central nervous system(CNS) is closely related to consciousness. Astrocytes have been found to release various neurotransmitters to synaptic cleft after excitement. These neurotransmitters released from astrocytes was known as glial transmitters and they can modulate the function of synapse.Therefore, astrocytes are thought to play an important role in regulating synaptic transmission. This study aims to explore the effects of general anesthetics on the astrocyte calcium transients and glial neurotransmitter release, then analysis related mechanism. This study will explore a new way to study the mechanism of general anesthesia.Methods: Culturing, purifying rat cortical astrocytes and identifying the purity of culture astrocytes through immunofluorescence staining of glial fibrillary acidic protein(GFAP).Intracellular calcium transient([Ca2+]i)induced by adenosine diphosphate(ADP) under the use of general anesthetics(propofol, ketamine and dexmedetomidine) were observed by reflection fluorescence imaging system.ELISA method was used to detect the levels of intracellular three triphosphate inositol(IP3), and liquid chromatography technique was used to observe the release of glutamate(Glu), γ-amino acid(GABA)and serine( Ser) from astrocytes culture with different general anesthetics.Finally, muscarinic type acetylcholine receptor blocker(Atropine) and N-methyl-D-aspartic acid(NMDA) receptor antagonist(AP5) were used to test the target receptor of general anesthetics on astrocyte.Results: 1. The purity of cultured astrocytes was > 90% identified by immunofluorescence staining.2. 100 n M~100 μM ADP can increase the [Ca2+]i in cultured astrocytes in a dose-dependent manner.100 n M ADP can slightly upgrade [Ca2+]i without statistically significant, while 1 μM, 10 μM, 100 μM ADP can induce a statistical significance [Ca2+]i increase(P < 0.01) and the median effective concentration is 208.04 ± 14.94 n M.3. Ket inhibited the calcium transient induced by 10μM ADP in a concentration dependent manner, and the half inhibitory concentration is 236.23 ± 3.62μM;however,Propofol(100 μM) and Dex(50 μM, 100 μM) failed to inhibit astrocyte calcium transients(P > 0.05).4. 10 μM ADP significantly enhanced the level of intracellular IP3 in culture astrocytes(P < 0.01); Ket, Atropine and AP5 have no effect on the intracellular IP3 level in astrocytes under the influence of 10 μM ADP(P > 0.05).5. Ket does not affect the release of Ser and Glu from astrocytes at the presence of 10 μM ADP(P > 0.05).Conclusion: 1. Among Ket, Propofol and Dex, only Ket can effectively inhibit the astrocyte calcium transient in a concentration dependent manner.2. The application of ADP can significantly enhance the level of intracellular IP3 in astrocytes.3. The intracellular IP3 pathway may not be involved in the negative effect of Ket on the ADP induced astrocytes calcium transient.4. The inhibitory effect of ketamine on astrocytes calcium transient does not affect the release of Glu and Ser.