The Relationship Of Intrinsic Firing In Neonatal Auditory Central Neurons And Age-dependent Vulnerability Of Bilirubin Encephalopathy

Object: To explore spontaneous intrinsic firing in the first auditory central nucleus.To investigate biological developmental basis of age-dependent vulnerability of bilirubin encephalopathy.Method: SD or C57 mice between 0 and 19 postnatal days were used in this research.All recordings were made with the cell attach and whole cell patch-clamp technique using a patch-clamp amplifier.We recorded spontaneous firing,HCN current(Hyperpolarization-activated and cyclic nucleotide-gated channels,HCN)?s IPSCs(Spontaneous inhibitory postsynaptic currents,s IPSCs)?e EPSCs(Evoked excitatory postsynaptic currents,e EPSCs)etc.Bilirubin were used to explore its effect on the above index.We used pharmacological approach in WT and HCN1 KO mice.Electrophysiological data were analyzed offline using the Mini Analysis Program(Synaptosoft,NJ,USA)and Clampfit 10.2 software(Molecular Devices).Origin8 and Sigma Plot12 were used for graphic representation.Wilcoxon signed-ranks tests and Chi-squared test were ued to statistical analysees.All numerical results are presented as means ± SEMs.Values of P<0.05 were considered to be statistically significant.Results: CN neurons exhibited synapse-independent(glutamate,GABA and Glycine)intrinsic firing.Spontaneous firing were age-dependent increased(P1-4: 2.17 HZ,32%;P15-19: 9.46 HZ,66%).Percentage of neurons with intrinsic firing in HCN1 knockout mice was smaller than wild-type mice(34%VS17.5%).Blockers of HCN channels significantly decreased frequency of intrinsic firing(Cs Cl: 35.9 ±5.8% of control;ZD7288: 38.6±3.8% of control),indicating that HCN channels play a dominate role in triggering spontaneous intrinsic firing.Neurons with spontaneous firing exhibit wider half-width of action potential(1.14±0.35 mm VS 2.94±0.38mm),large number of synaptic terminals and high level of P2/P1(1.22±0.55 VS 1.64 ±0.1)than neurons without spontaneous firing.Enhancement of bilirubin on spontaneous firing were different in four age groups.Immature CN neurons were more sensitive to bilirubin(210±31% VS142±42%).Additionally,bilirubin can facilitate HCN current,the activation curve was shifted toward depolarized potentials compared to control.Bilirubin can significantly facilitate the frequency of s IPSCs(150±12.3% of control).Both fast calcium chelator BAPTA-AM and slow calcium chelator EGTA-AM can prevent bilirubin-induced enhancement of s IPSCs.In immature neurons,perfusion of bicuculline and strychnine significantly decreased bilirubin-induced facilitation of spontaneous intrinsic firing.Conclusion: Neonatal auditory central neurons exhibit synapse-independent spontaneous intrinsic firing,which play an important role in the development of central auditory network.Excitatory GABA/Glycinergic transmission and spontaneous intrinsic firing in early development contribute to age-dependent vulnerability of bilirubin encephalopathy.