The Regulation Of Serotonin On NMDA Current In Hippocampal CA1Pyramidal Neurons

Serotonin（5-hydroxytryptamine;5-HT） is an important monoamineneurotransmitter in the central nervous system（CNS）, almost throughout thecentral nervous system. Serotonin neurotransmitter can combine with differentreceptor subtypes, activate second messengers and ion channels, and involvein the multiple physiological processes and pathological processes, includingthe regulations of the cardiovascular function, body temperature, feeding,sexual behavior, depression, anxiety, sleep, pain, emotional and so on. A largenumber of studies have found that a variety of mental diseases associated withcognitive disorder, such as Alzheimer’s disease, depression, anxiety, and otherdiseases, there are5-HT nervous system disorders.N-methyl-D-aspartate （NMDA） receptor is a special type of ion channelproteins, belonging to the voltage and ligand double-gated ion channels, whichpossesses Ca2+permeability, but can be blocked by Mg2+in avoltage-dependent manner. NMDA receptors are mainly distributed in thepostsynaptic membrane of hippocampus, hypothalamus, cortex, corpusstriatum, dentate gyrus and so on. The NMDA currents were induced by theexogenous application of NMDA to activate NMDA receptors. The miancomponents of the NMDA currents are Na+and Ca2+influx. In the centralnervous system, NMDA receptors are involved in the regulation ofphysiological functions such as synaptic plasticity, learning and memory, andso on. Moreover, NMDA receptors in mood disorders, brain function disorders（such as the cerebral ischemia, neurodegenerative diseases, epilepsy）,schizophrenia and other diseases also play an important role in regulating.5-HT nervous system and NMDA receptors are involved in cognitive andemotional regulation in normal and pathological conditions. The interaction of5-HT and NMDA receptor is complicated, and can affect hippocampus-dependent long term memory. In the absence of Mg²⁺reduced NMDA-mediated depolarization; whereas in the presence of Mg²⁺5-HT increased NMDA-mediated depolarization. In addition,5-HT inhibitedNMDA receptor-mediated response in pyramidal neurons of the rat prefrontalcortex. In contrast,5-HT enhanced NMDA receptor-mediated depolarizationin frog motor neurons. Due to the inconsistency of the above studies, thepresent study used whole-cell patch-clamp technique and the NMDA currentsas an experimental index to observe the regulation of5-HT on the NMDAcurrent in rat hippocampal CA1pyramidal neurons and to further explore thereceptor subtypes involved in the regulation of5-HT. A large number ofliterature reported that5-HT₂R and5-HT₃R can affect the long-termpotentiation formation, and involved in the regulation of learning and memoryin physiological functions, and mainly distributed in the hippocampal layersand subregions. For these reasons, our experiments will mainly confirm theinvolvement of5-HT₂R and5-HT₃R whether to participate in the regulation of5-HT on NMDA current.Objective: To observe the regulation of5-HT on NMDA current in rathippocampal CA1pyramidal neurons; whether5-HT₂R and5-HT₃R involve inthis regulation, and to explore the mechanism.Methods: We used the Sprague-Dawley rats that were born about twoweeks. Transverse300μm-thick hippocampal slices containing CA1neuronswere obtained by cutting with a vibroslice MA752in ice-cold artificialcerebrospinal fluid （ACSF） well-saturated with95%O₂and5%CO₂（PH7.3-7.4）, and transferred to oxygenated ACSF at30-32℃for1h incubation.The hippocampal slice containing CA1neuron was transferred to a perfusionchamber and fixed with a nylon net, continuously superfused with oxygenatedACSF at a constant rate of3ml/min at room temperature. Using of the patchclamp systems with infrared differential interference contrast （IR-DIC） optics,in the whole-cell mode, setting the holding potential of-60mV, using ofperfusion administration, to record NMDA currents in rat hippocampal CA1pyramidal neurons; to observe the regulation of NMDA receptor antagonist AP-V on NMDA current; to observe the regulation of different concentrationsof5-HT on the NMDA current; to study effects of5-HT₂R antagonistKetanserin or5-HT₃R antagonist ondansetron on the regulation of5-HT onNMDA currents. The amplitudes of NMDA currents were measured in thesame neurons by the superfusion application of NMDA in the absence （ascontrol value） and the presence of AP-V or the different concentrations of5-HT （as experimental values）, and the experimental values were normalizedusing respective control value.Results:（1）Identification of neurons and glial cells. In the Whole-cell mode,giving a-60mV--0mV---60mV depolarizing stimulation, we recorded aninward sodium current. This indicated that the cells we selected were neuronsrather than glial cells.（2）Identification of pyramidal neurons and interneurons. In thewhole-cell current-clamp recording mode, giving a0PA--100PA--0PA currentpulses stimulation, the action potential （AP） that we recored waslow-frequency spikes with frequency adaptation phenomenon in a neuron.This indicated that the neurons we recorded were pyramidal neurons ratherthan interneurons.（3）Identification of NMDA current in rat hippocampal CA1pyramidalneurons. In the present study, if the amplitude of the inward current inducedby the superfusion application of NMDA in control was standardized （100%）,perfusion application of100μM AP-V, the NMDA receptor antagonist,significantly inhibited NMDA-induced inward current （2.08±0.81%, n=7, p<0.01）, and the inhibitory rate of97.92%.The result suggested that the inwardcurrent we recorded was surely NMDA current.（4）The stability of NMDA current in hippocampal pyramidal neurons.In hippocampal pyramidal neurons, repeated to give the same concentrationdrugs of NMDA （30μM）, the NMDA currents recorded repeatedly within1hwere basically the same. Within the record time of50min, the NMDAcurrents did not appear to receptor desensitization or rundown phenomenon. This indicated that the NMDA current was stable at least within50min inhippocampal pyramidal neurons.（5）5-HT significantly enhanced NMDA current in hippocampal CA1pyramidal neurons. In the present study, if the amplitude of the currentinduced by the superfusion application of NMDA in control was standardized（100%）,5-HT （0.01,0.1,0.3,1mM）-mediated NMDA current were105.87±5.44%（n=19, p>0.05）,117.74±4.18%（n=8, p <0.01）,193.56±19.63%（n=14, p <0.01）,164.03±12.06%（n=18, p <0.01）. These data showed that5-HT （0.1,0.3,1mM） significantly enhanced NMDA current, and0.01mM5-HT had not the significant potentitation of NMDA current.（6）Effect of ketanserin （20μM,5-HT₂R antagonist） on theenhancement of5-HT on the NMDA current. In the present study, ketanserin（20μM） can be significantly reduced the enhancement of5-HT on the NMDAcurrents, which changed from179.79±25.17%to121.22±7.23%（n=8, p<0.05）. These results indicate that5-HT₂R is involved in the enhancement of5-HT on the NMDA current in hippocampal CA1pyramidal neurons.（7）Effect of ondansetron（20μM,5-HT₃R antagonist） on theenhancement of5-HT on the NMDA current. In the present study,ondansetron（20μM） did not significantly affect the enhancement of5-HT onthe NMDA currents, which changed from149.98±15.03%to138.21±11.08%（n=9, p>0.05）. These results show that5-HT₃R is not involved in theenhancement of5-HT on the NMDA current in hippocampal CA1pyramidalneurons.Conclusion: Serotonin significantly enhances NMDA currents throughactivation of5-HT2receptor in rat hippocampal CA1pyramidal neurons, butthe5-HT3receptor does not involve in this regulation.