| 5-HT is one of the most important neurotransmitters in central nervous system.It comes from serotonin neurons which are responsible for 5-HT production and secretion located in brainstem.According to previous studies,5-HT and 5-HT neurons which innervate spinal cord play important roles in locomotion.But it is hard to investigate brainstem descending 5-HT neurons by electrophysiology directly by the limitation of technology.However,ePet-EYFP transgenic mice made it possible.Present study used this kind of animal model with whole cell patch clamp to investigate a special current ‘Persistent Inward Current(PIC)' in brainstem descending 5-HT neurons.PICs have been found in various vertebrates and have multiple functions including inducing plateau potential;bi-stable firing;facilitating repetitive firings;and modulating Rhythm Generator of Central Pattern Generator(CPG)to effect the generation of locomotion.Although PIC of spinal interneurons and motoneurons have been studied intensely and it is also proved that descending serotonin neurons express PIC,systematic characterization of PIC of descending serotonin neurons is lacked,nor to mention the report of acetylcholine modulation to PIC.Acetylcholine is an important transmitter in CNS and have versatile functions to excitability of brainstem 5-HT neurons.Its effects to 5-HT neurons from Midline Raphe Nuclei(MRN)and Parapyramidal Region(PPR)are different.But its effect to PIC of brainstem descending 5-HT neurons is unknown.For that reason,present study will use ePet-EYFP transgenic mice to investigate the electrophysiological characters of PIC in brainstem descending 5-HT neurons and the modulation to PIC by acetylcholine.Our study will reveal the ion channel mechanism of PIC which effects the generation of locomotion and the modulation to it by acetylcholine.Objective: Present study use ePet-EYFP transgenic mice to find fluormetric 5-HT neurons to investigate distribution of 5-HT neurons in caudal brainstem slices;to characterize electrophysical parameters of PIC and its characters relevant todistribution;to depict features of different kind of PIC and the effect of development to PIC;to reveal the functions of acetlycholine modulation to PIC.Methods: Experiments were carried out on neonatal(P3-P13)ePet-EYFP transgenic mice.Its brainstem was abstract and then cut to slices(200mm-250mm).We can find 5-HT neurons with florescence easily by the slices of this transgenic mice under fluorescence microscope.Then whole cell patch clamp recordings were made in voltage-clamp mode to record PIC.Some of the cells was applied with Ach to study the effects on PIC of brainstem descending serotonin neurons.Molecular Devices pClamp10.1 was used to record and process data,Excel was used to make data analysis,and CorelDRAW was used to make image processing of PIC and statistical graphics.In t-test,P-value less than 0.05 was considered statistically significant and was noted with ‘*';P-value less than 0.005 was considered highly significant and was noted with ‘**'.Results:(1)Using whole cell patch clamp(voltage clamp),we obtained data from 117 serotonin neurons.62 of the 117 cells were applied with acetylcholine,17 cells of the 62 cells were washed out.(2)109 of the 117 5-HT cells were marked positions in slices,50 of them were located in MRN,59 of them were located in PPR.(3)111 of the 117 recording cells expressed PIC which account of 94%,only 6of the 117 cells which account for 6% did not.(4)PIC of brainstem descending serotonin neurons have 1a,2a,1d,1a1 d,2a1d five types.(5)The amplitude of PIC of brainstem descending serotonin neurons is215.25±71.37 pA,onset(Vo)is-12.89±6.60 mV,peak voltage(Vp)is1.74±6.71mV(n=48).(6)The amplitude of blunt PIC(n=53)is 205.72±74.12 pA,onset is-12.34±6.60 mV,Vp is 2.20±6.74 mV,while the amplitude of sharp PIC(n=12)is398.5±100.77 pA,onset is-9.61±5.50 mV,Vp is-7.06±5.65 mV.Amplitude of sharp PIC is far more larger than that of blunt PIC(P<0.005),while Vp of sharp PIC is more hyperpolarized(P<0.005).(7)The amplitude of 1a type PIC is 199.04±63.60 pA,its onset and Vp is-12.38±7.37mV1.23±7.42 mV,respectively(n=25).The amplitude of 2a type PIC is234.37±76.55 pA,its onset and Vp is-13.19±5.72 mV,2.40±6.23 mV respectively(n=21).These two amplitudes showed statistically significance(t-test,P<0.05).(8)The amplitude,onset and Vp of PIC of serotonin neurons in MRN is190.6±50.6pA,-13.86±5.55 mV,0.69±6.32 mV respectively(n=19),those of PPN serotonin neurons is 228.32±80.58 pA,-12.77±6.82 mV,1.79±6.45 m V(n=27).There is no statistically differences between these two amplitudes,onsets and Vps(t-test).(9)The amplitude,onset,Vp of PIC of the serotonin neurons from 3-7d group is 191.62±62.90 pA,-12.09±7.45 mV,3.17±7.42mV(n=20).Those of 10-14 d group is 231.85±77.24 pA,-13.29±6.66 mV,0.99±6.47 mV respectively(n=22).There is no statistically differences between these two amplitudes,onsets and Vps(t-test).(10)In 46 cells which were applied with Ach,amplitude of 25 cells were amplified,13 were inhibited while 8 did not change.(11)The cells whose amplitude of PIC was larger after application with Ach(n=17),their amplitude of PIC,onset and Vp changed from 210.54±80.36 pA,-10.86±6.29 mV,0.56±6.25 mV to 300.89±145.51 pA,-14.70±6.19 mV,-4.85±7.36 mV respectively after application of Ach(P<0.005).(12)The cells whose amplitude of PIC was smaller after application with Ach,only amplitude of PIC of these neurons changed with statistical significance from267.15±120.12 pA to 185.87±90.24pA(P<0.005).(13)The cells whose amplitude of PIC did not change after application with Ach,their onset changed with statistical significance from-14.13±4.81 mV to-16.79±4.74mV(P<0.05).(14)The amplitude and Vp of PIC of cells of MRN did not change statistically after application with Ach,but their onset was hyperpolarized from-14.36±5.37mVto-17.32±3.94 m V(t-test,P<0.05).The amplitude,onset,Vp of PIC of cells in PPR all changed respectively from 212.19±82.42 pA,-12.01±5.62 mV,0.83±4.22 mV to280.97±161.66pA(P<0.05),-15.39±4.81 m V(P<0.005),-3.55±6.49mV(P<0.05)after application with Ach.Conclusion:(1)111 of 117(94.9%)investigated cells expressed PIC and this PIC has nothing to do with development and distribution about MRN(Midline Raphe Nuclei)and PPR(Parapyramidal Region).(2)PIC of descending 5-HT neurons in brainstem has different forms with different electrophysical characters(3)Acetylcholine has different effects on PIC of brainstem descending 5-HT neurons including increasing,decreasing and doing nothing to its amplitude.It showed different effects to PIC of brainstem descending 5-HT neurons in different locations. |
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