| Locomotion is a fundamental movement in vertebrates,which is characterized as the body displacement produced by periodic and rhythmic movement of the limbs.Locomotion is induced and controlled by the Mesencephalic Locomotor Region?MLR?located in the midbrain,performed and operated by the neural networks distributed in the spinal cord that is called Central Pattern Generator?CPG?.It has been found that 5-HT neurons in brainstem play an important role in initiation and generation of locomotion.The reason is that 5-HT neurotransmitter released by 5-HT neurons plays an important regulatory role during locomotion.Persistent Inward Current?PIC?,a voltage-gated ion current,is present in many types of neurons and has multiple functions.The systematic study of the properties of PIC in 5-HT neurons is of great importance for revealing the role of 5-HT neurons in motor activity.Previous studies about PIC focused mostly on motor-related motoneurons and interneurons in the spinal cord,a systematic study is rarely reported.on the electrophysiological properties of PIC in medulla 5-HT neurons of the brainstem.In view of this,we use ePet-EYFP transgenic mouse model and whole-cell patch clamp technique to systematically study electrophysiological properties of PIC in medulla5-HT neurons of brainstem.Further,we investigate the modulatory effect of neurotransmitter 5-HT on the PIC of brainstem 5-HT neurons.The results showed thatPersistent Inward Current of 5-HT neurons in the Parapyramidal Region and Midline Raphe Nuclei contain a tetrodotoxin-sensitive sodium ion current and a dihydropyridine-sensitive calcium ion current.PIC and its different ionic components can be enhanced by 5-HT with different extent.This study can help us to understand the mechanism of the ion channel of PIC responsible for,regulation of excitability of brainstem medulla 5-HT neurons and deepen our understanding of the inherent mechanism underlying movement.In this study we investigated the properties of PIC of 5-HT neurons in the brainstem PPR and MRN regions,explored the relationship between the characteristics of PIC and their location distribution,and tested the pharmacological properties and neurotransmitter regulatory properties of PIC.This study could improve our understanding of mechanisms underlying generation,coordination and control of limb movement for locomotionObjective:With usage of ePet-EYFP transgenic mouse model and whole-cell patch clamp technique,theobjectivesofthisstudyaretoinvestigatethe electrophysiological properties of persistent inward current of 5-HT neurons in the PPR and MRN regions of the medulla;to explore the relationship between distribution of 5-HT neurons and electrophysiological properties of PIC in brainstem;to test the changes in PIC after bath application of antagonists TTX or DHP;and to study the modulatory effect of 5-HT on PIC,Ca-PIC and Na-PIC in 5-HT neurons of brainstem.This study could enhance our understanding of physlogical mechanisms underlying generation,coordination and control of limb movement.Methods:Experiments were carried out neonatal?P1-P15?ePet-EYFP mice,male or female.The brainstem was dissected to prepare brainstem slices?250?M?and 5-HT neurons were searched by a fluorescence microscope.PIC was recorded in voltage clamp mode of whole-cell patch clamp technique,to analyse activation voltage?Vo?,peak voltage?Vp?,PIC amplitude.To investigate the biophysical characteristics of PIC after bath application of sodium channel blocker?TTX?or calcium channel blocker?DHP?,including Vo,Vp and amplitude,as well as the neurotransmitter 5-HT adjust different types of electrophysiology characteristics of PIC.Results:?1?The medulla 5-HT neurons in the brainstem are distributed in the PPR and MRN regions.?2?A total of 195 5-HT neurons were recorded in this experiment,of which 169 cells expressed PIC?86.7%?.There were 83 5-HT neurons in the PPR region,of which 70cells expressed PIC?84.3%?.There were 112 5-HT neurons in MRN and 99 cells expressing PIC?88.4%?.?3?Five types of PIC were observed in 5-HT neurons:a-PIC?65.5%??2a-PIC?20.2%??d-PIC?4.8%??ad-PIC?7.1%??3a-PIC?2.%???4?Biophysical parameters of PIC?n=51?:Vo?-19.8±6mV?,Vp?-0.5±3.3m V?,Amplitude?220.8±81.9pA?.PIC of 5-HT neurons in the PPR region?n=19?:Vo?-17.4±3.7mV?,Vp?1.3±2.5mV?,Amplitude?210.6±68.4pA?.PIC of 5-HT neurons in the MRN region?n=32?:Vo?-19.5±7.3mV?,Vp?-0.7±4.2m V?,Amplitude?213.4±86pA?.PIC of 5-HT neurons in PPR and MRN regions showed that there was no significant difference in Vo,Vp and Amplitude?P>0.05?.?5?It is generally accepted that PIC is mediated by voltage-sensitive sodium current?Na-PIC?and voltage-sensitive calcium current?Ca-PIC?.In this paper,Ca-PIC was recorded with 1-2?M TTX in the solution for recording PIC.10 5-HT neurons were measured,PIC was activated at-15.7±5.8 mV,Vp?3.2±6.5 mV?,Amplitude?268.2±123.8pA?,Ca-PIC was activated at-15.5±2.9 mV,Vp?0.8±7mV?,Amplitude?225±102 pA?.Na-PIC was recorded after bath application of 20-30?M DHP.15 5-HT neurons were measured,PIC was activated at-17.7±5.4 mV,Vp?-0.8±4.3m V?,Amplitude?270.8±75.4pA?.Adding TTX or DHP can reduce the current amplitude and depolarize activation voltage.However,these results showed that there was significant difference in current amplitude between PIC and Ca-PIC,Na-PIC of the same cell?P<0.05?.?6?5-HT modulation of PIC properties?n=9?:The concentration of 5-HT used in the experiment was 15-20?M.5-HT increased the amplitude of PIC from 185.2±71.5 pA to 271.4±91.5pA?P<0.05?and hyperpolarization of activation voltage was from-19.7±7.5 mV to-23.6±7.2 mV?P<0.05?.5-HT modulated the PIC of serotonergic neurons in the PPR more significantly?n=4?,which was mainly reflected by activation voltage and current amplitude?P<0.05?.5-HT had no significant change in the electrophysiological of PIC in the MRN?P>0.05,n=5?.?7?5-HT modulation of Na-PIC?n=9?:5-HT increased the amplitude of Na-PIC from 201.6±62pA to 287±64.1pA,but there was no significant difference compared with the control group?p>0.05?.The activation voltage ranged from-17.8±3.2mV to-22.3±1.9mV,and there was a significant difference compared with the control group?p<0.05?.5-HT modulation of Ca-PIC?n=14?:5-HT increased the amplitude of Ca-PIC from194.3±91.2 pA to 258.3±149 pA?p<0.05?,with activation voltage from-20.8±5mV to-24.3±8.9 m V?p>0.05?.Conclusions:?1?PIC is universally expressed and has no relation with the distribution location in the brainstem 5-HT neurons of ePet-EYFP mice.Persistent Inward Current contains a tetrodotoxin-sensitive sodium ion current and a dihydropyridine-sensitive calcium ion current.?2?Electrophysiological properties of PIC in the Midline Raphe Nuclei and Parapyramidal Region are not significantly different,but the 5-HT neurons in the Parapyramidal Region are more sensitive to the regulation of neurotransmitter 5-HT,which may be related to the important role of 5-HT neurons in the PPR in intiating locomotion.?3?5-HT is an important neurotransmitter in the central nervous system.This study showed that 5-HT can increase the PIC amplitude and hyperpolarize the activation voltage,indicating that 5-HT can increase neuronal excitability.This neuromodulatory mechanism may play an important role during locomotion.?4?5-HT enhances the Ca-PIC and Na-PIC of 5-HT neurons in the brainstem medulla with different regulatory mechanisms.?5?The study of persistent inward current in brainstem 5-HT neurons explains the potential mechanismof locomotion at the level of neurons and ion channels. |
PDF Full Text Request