Effects Of NE On Synaptic Transmission And Its Long-Term Potentiation In Spinal Cord Motoneurons In Vitro

Objective:Norepinephrine（NE）has powerful modulatory effects on spinal motor neural networks,but the roles of NE on synaptic transmission of descending activation and afferent to spinal motoneuron（MN）and their synaptic plasticity remain unclear.To this end,we investigated the effects of NE on the synaptic transmission of spinal MN from afferents and descending activation,long-term potentiation（LTP）,and its possible receptor mechanisms,and analyzed the receptor kinetic changes of the synaptic transmission by the apparent receptor kinetics method.Methods:In this study,spinal cord slices（400-500 μm）were prepared from neonatal（814 d）SD rats,and the intracellular recording technique of spinal MN was used.The ipsilateral ventrolateral（iVLF）or ipsilateral dorsal root（iDR）was given single stimulation to evoke synaptic responses including excitatory postsynaptic potentials（EPSPs）,iVLF-EPSP or iDR-EPSP,respectively.In the LTP experiment,the tetanic stimulation of iVLF or iDR,was used to induce LTP of iVLF-EPSP or iDR-EPSP.NE bath was given to observe the modulation of EPSPs and LTP.The receptor kinetic process and the possible mechanism of the modulation of EPSP by NE were analyzed by administering α1-adrenergic receptor（α1-AR）selective antagonist prazosin and α₂adrenergic receptor（α2-AR）selective antagonist yohimbine.Results:1.In 7 stably recorded MN,after 1,5 and 25μmol/L NE were each perfused for 15min,NE induced concentration-dependent depolarization（P<0.01）,increased membrane resistance and firing frequency of action potential（I-F curve shifted up）（all P<0.01）.2.In 6 MN with iVLF-EPSP,the amplitude（P<0.01）,area under curve（AUC,P<0.05）and duration（P<0.01）of iVLF-EPSP were concentration-dependently decreased after perfused in turn with 1,5 and 25 μmol/L NE for 15 min.In the 7 MN with iDR-EPSP,the amplitude（P<0.01）,AUC（P<0.05）and duration（P<0.01）of iDR-EPSP were concentration-dependently reduced by perfusion in turn with 1,5 and 25 μmol/L NE for 15 min.In addition,the IC50 values of NE action on iVLF-EPSP and iDR-EPSP amplitudes were 7.9 μmol/L and 7.6μmol/L,respectively.3.In 4 MN with both iVLF-EPSP and iDR-EPSP,the comparative experiment of perfusion in turn with 1,5 and 25 μmol/L NE for 15 min in the same cell showed that the inhibition rate of NE on iDR-EPSP amplitude was higher than that on iVLF-EPSP（P<0.05）.4.Among the 55 tested MN with iVLF-EPSP,NE perfusion（0.2～1 μ mol/L）for 15 min in 4 MN increased the amplitude,AUC and duration of iVLF-EPSP（all P>0.05）.In addition,among the 74 tested MN with iDR-EPSP,NE bath（0.2～1 μ mol/L）for 15 min in 3 MN enhanced the amplitude,AUC and duration of iDR-EPSP（all P>0.05）.5.Among the 9 NE-perfused MN with iVLF-IPSP,the amplitude,AUC and duration of iVLF-IPSP was inhibited by NE（0.2～1μ mol/L）for 15 min only in 3 MN（all P>0.05）.Among the 5 test MN with iDR-IPSP,the amplitude,AUC and duration of iDR-IPSP in 2 MN was inhibited by NE treatment（all P>0.05）,while iDR-IPSP in 1 MN was completely inhibited and recovered after washing.6.In 11 MN with iVLF-EPSP,perfusion with 5 μmol/L NE for 15 min inhibited the amplitude（P<0.01）,AUC（P<0.05）and duration（P<0.05）of EPSP,and apparent receptor kinetic analysis of the iVLF-EPSPs showed that NE decreased the apparent maximum response（Vmax）（P<0.01）and the apparent association rate constant（K1）（P<0.05）,while increased the apparent equilibrium dissociation constant（KT）（P<0.05）and tended to increase the apparent dissociation rate constant（K2）.In addition,in 12 MN with iDR-EPSP,perfusion with 5 μmol/LNE for 15 minutes inhibited the amplitude（P<0.01）,AUC（P<0.01）and duration of EPSP,and apparent receptor kinetic analysis of iDREPSPs in 10 of the 12 cells tested showed that NE decreased the apparent maximum response（Vmax）and the apparent association rate constant（K1）（both P<0.05）,while increased the apparent equilibrium dissociation constant（KT）（P<0.05）and tended to increase the apparent dissociation rate constant（K2）.Moreover,in the 4 MN with iVLFEPSP and iDR-EPSP,the receptor kinetic changes were analyzed after perfusion of 5μmol/LNE.Compared with iVLF-EPSPs,the apparent association rate constant（K1）and the apparent maximum response（Vmax）decreased in iDR-EPSPs（all P>0.05）,while the apparent dissociation rate constant（K2）（P>0.05）and the apparent equilibrium dissociation constant（KT）（P<0.05）increased.7.The inhibitory effects of NE on iVLF-EPSP amplitude,AUC,and duration were partially abolished by administration of the α1-AR selective antagonist prazosin（n=6）or the α2-AR selective antagonist yohimbine（n=5）（all P<0.05）.The inhibitory effects of NE on iDR-EPSP amplitude,AUC,and duration were partially abolished by administration of the α1-AR selective antagonist prazosin（n=5）or the α2-AR selective antagonist yohimbine（n=6）（all P<0.05）.8.Tetanic stimulation was performed on 9 MN with iVLF-EPSPs,and iVLF-LTP phenomenon was induced in 3 MN with the induction rate 33.33%.NE（5 μmol/L and/or 25 μmol/L）was given for 15 min each in 3 MN with iVLF-LTP,the amplitude,AUC,and duration of iVLF-EPSPs decreased to below the baseline.In two MN with ACSF washout,the amplitude,AUC,and duration of iVLF-EPSPs only recovered to baseline in the 25μmol/L NE perfused MN,while the amplitude,AUC,and duration of iVLF-EPSPs could recover to more than 120%of baseline in the 5 μmol/L NE perfused MN.Conclusion1.NE enhances the activity of MN itself in spinal cord in vitro,but inhibits afferent and descending excitatory synaptic transmission to MN and the inhibitory effect probably by activating α1-AR and α2-AR.2.NE bath may decrease the apparent maximum response（Vmax）and apparent association rate constant（K1）,while increase the apparent equilibrium dissociation constant（KT）of EPSP,suggesting that NE inhibition of afferent and descending EPSP may involve a mechanism of postsynaptic receptor kinetics change or decrease of postsynaptic glutamate receptor affinity.3.The distinct inhibitory effect of NE on excitatory synaptic transmission in the afferent and descending activation pathways by NE and its apparent receptor kinetics analyses in the same cell with more obvious decrease in postsynaptic receptor affinity in the afferent pathway compared to the descending activation,suggest a pathway-distinct effect of NE.4.Medium concentration of NE can inhibit and cancel iVLF-LTP in spinal cord MN in vitro,suggesting that high concentration of NE may have a mechanism that affects the maintenance of iVLF-LTP.