The Role And Intervention Of Bilirubin On P/Q-type Calcium Channels And Neurotransmission In Newborn Neurons

Objective:Illuminate the role and intervention of bilirubin on P/Q-type calcium channels and neurotransmission in newborn neurons of ventral cochlear nucleus（VCN）.Methods:Prepare brain slices of SD rats in P4-17.Add bilirubin,antagonists of VGCCs,NAD+and other drugs with gravity perfusion system.We conducted patch clamp technology to record VGCC currents,excitatory postsynaptic currents,spontaneous spike currents and action potentials before and after drugs.We used t test,ANOVA and Kruskal-Wallis non-parametric test by SPSS17.0 for data analysis.Results:Series of calcium currents evoked in the VCN of SD rats at P4-17showed all types of calcium currents,and the distribution of P/Q subtype decreased from 20.8±1.4%at P4-6 to 11.0±1.9%at P15-17).Acute perfusion of bilirubin in newborn neurons at P4-12 increased the amplitude of voltage-gated calcium channels to 122.9±1.6%of the control condition,and caused a shift in steady-state activation of calcium currents to more negative potentials from-2.56±2.6 mV to-11.33±1.9mV.In the presence of blockers of P/Q-type calcium channels（ω-Aga IVA）,bilirubin failed to enhance VGCC currents with the mean current amplitude being 92.1±4.3%at co-application ofω-Aga IVA and bilirubin.The effect of bilirubin was attenuated by intracellular loading of Ca²⁺buffer EGTA or calmodulin inhibitory peptide.Calcium currents activated by pseudo-APs enhanced after exposure to bilirubin.After application ofω-Aga IVA,bilirubin induced enhancement in the area integral of calcium current recovered to control levels at 50 Hz（90.3±18.6%of the control condition）.Treatment with NAD+suppressed and prevented bilirubin-induced elevation of the voltage-gated calcium currents in VCN neurons（99.08±4.59%of the control condition and 100.39±6.7%of the control condition）.Bilirubin increased the charge of evoked excitatory postsynaptic current to 121.4%±9.6%of the control condition,reduced the paired-pulse ratio（PPR）from 0.97±0.05 to 0.74±0.03,enhanced the frequency of miniature excitatory postsynaptic currents from 11.3±7.2Hz to 17.7±9.9 Hz and increased the frequency of spontaneous spikes from 1.8±1.0Hz to 4.1±1.9 Hz.NAD+suppressed bilirubin-induced increase of the charge of evoked excitatory postsynaptic currents（93.6±6.6%of the control condition）,decrease of PPR（0.74±0.03 VS 0.97±0.03）,increase of the frequency of miniature excitatory postsynaptic currents（17.7±9.9 Hz to VS 6.1±3.4 Hz）and increase of spontaneous spike（4.1±1.9 Hz VS 1.8±1.1 Hz）.Conclusions:Bilirubin boots activity of P/Q-type calcium channels through Ca²⁺and calmodulin dependent mechanisms in auditory neurons of SD rats,and promotes neurotransmission.NAD+suppresses bilirubin-induced hyperexcitation by inhibiting P/Q-type calcium channels.