Effects Of Interleukin-2 On Function Of Ionotropic Glutamate Receptors, And On Dendritic Development And Synaptogenesis In Cultured Hippocampal Neurons Of Rats

Interleukin-2(IL-2)is one of the most actively studied cytokines,and it has multiple immunoregulatory functions and biological properties not only related to T-cells.In addition to its immune activities,neurobiological and neuroendocrine data indicated that IL-2 plays roles in the central nervous systems(CNS).IL-2 and IL-2 receptors(IL-2R) are widely distributed in the brain regions,including frontal cortex,striatum, hippocampus,hypothalamus,locus coeruleus,cerebellum,pituitary and callose,and are potentially produced by neurons and astrocytes;Besides,it has been reported that IL-2 is able to cross the blood-brain barrier(BBB)by saturable and nonsaturable transport mechanisms,which makes it a likely candidate for humoral communication between the immune,endocrine and CNS,suggesting that IL-2 could act as a neuroregulatory factor.IL-2 may prove of therapeutic value in a wide range of pathological conditions,but the significant anticancer effects obtained with IL-2 immunotherapy are often associated with side effects in the CNS,like toxic complications,moderate or severe neurological and mental disorders as well as cognitive impairements.In rodents,exogenous treatment of IL-2 could induce biological and behavioral alterations related to pathogenesis of some degenerative diseases,including Parkinson's disease and Alzheimer's disease.It has reported that IL-2 suppresses the induction and maintenance of long-term potentiation(LTP),a long-lasting increase in synaptic transmission,suggesting that IL-2 may be a modulatory factor in synaptic plasticity.Hippocampus is a region enriched of brain-derived IL-2 and IL-2R proteins,and also closed associated with learning and memory.Thus,side effects in nervous system like cognitive impairments and memory loss induced by IL-2 may be related to direct effects of IL-2 on hippocampus.It is well known that LTP and long term depression(LTD)are the two forms of neuronal plasticity, and are thought to be the substrate of learning and memory.Glutamatic acid is the main excitatory neurotransmitter in hippcampus,and it can initiate synaptic excitatory transmission through binding to and then activating postsynaptic glutamate receptors, which play important roles in regulation of nervous system as major excitatory receptors, especially in synaptic plasticity.There are two major postsynaptic ionotropic glutamate receptor subtypes,that is,N-methyl-D-aspartate(NMDA)receptors andα-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate(AMPA)receptors,which are crucial for mediating hippocampal synaptic plasticity.Activation of NMDA receptors within the CNS represents a major signal for persistent alterations in glutamatergic signaling,like LTP and LTD,which may underlie higher order processes such as learning and memory in the hippocampus and are involved in pathogenesis of several types of neurological disorders such as epilepsy,ischemia-induced brain injury,and neurodegenerative disorders.These studies have implied that NMDA receptors may be a probable target of IL-2 in the regulation of synaptic plasticity and learning and memory. A previous study showed that IL-2 can modulate NMDA receptor-mediated currents (I_NMDA)in native mesolimbic neurons.However,the specific molecular targets of IL-2 on NMDA receptors remain unknown.In addition,AMPA receptors are also important to regulate alterations in synaptic plasticity and their activities are closely linked to the enhancement of synaptic transmission that occurs during LTP.Since ionotropic glutamate receptors and IL-2 are closely related to hippocampal synaptic plasticity,it is possible that there is relationship among IL-2,ionotropic glutamate receptors and synaptic plasticity.NMDA receptors and AMPA receptors may contribute to the possible effects of IL-2 on synaptic plasticity and learning and memory.However,there is no related report at present,we hypothesized that IL-2 may modulate excitability through NMDA receptors and/or AMPA receptors in the hippocampus.Furthermore,it is known that the biological effects of IL-2 are generally exerted through IL-2R.However,not all its effects are initiated by combination with its receptors, for instance,IL-2 can bind toδ-opioid receptors.So,IL-2 may also directly interact with either NMDA receptors or AMPA receptors.It was of interest to study the effects of IL-2, at physiologically relevant concentrations,on these two kinds of receptors.Moreover,IL-2 plays a role in development and regulation of brain neurons.IL-2 can promote development and survival of neurons from various brain regions.For example, it has multiple effects on morphology of hippocampal neurons,including significantly promoting the elongation and branching of neurites in both normal and damaged primary cultures.It is considered that dendrite is the important structure for receiving input signal and the location for integrating and exchanging synaptic information,and the length, diameter and arborazation of dendrites are all crucial to reflect situation of a neuron.It has been reported that many nutrients and stresses affected the formation and function of the whole neuronal circuit by interfering the developmental progress of dendrites.The studies suggested that IL-2 may have impact on dendritic development and synaptogenesis of cultured hippocampal neurons.Therefore,in order to understand effects of IL-2 on NMDA receptors and AMPA receptors as well as dendritic development and synaptogenesis of hippocampal neurons, we performed the following studies by molecular construction,primary neuronal cultures, electrophysiological technique and living cell images.Partâ… :Interleukin-2 inhibits NMDA receptor-mediated currents directly and may differentially affect subtypesUsing whole-cell patch-clamp recordings,this study investigated the effects of interleukin-2(IL-2)on N-methyl-D-aspartate(NMDA)receptor-mediated currents (I_NMDA)in rat cultured hippocampal neurons and human embryonic kidney(HEK)293 cells expressing recombinant NMDA receptors.We found that IL-2(0.01-1 ng/ml) immediately and significantly decreased peak I_NMDAin cultured neurons,causing a 47±3%inhibition at 0.1 ng/ml(p＜0.001).The effect of IL-2 on peak I_NMDAamplitude was depended on NMDA concentrations,and the value of EC₅₀for NMDA in the presence of IL-2 was 79±4μM(n=7),markedly greater than 42±7μM in controls.Interestingly, the peak I_NMDAinduced in HEK 293 cells was also inhibited by IL-2.We also found that IL-2 differentially decreased the peak amplitudes of NR2A- and NR2B-containing NMDA receptor-mediated currents(I_NR2Aand I_NR2B)by 54±5%(P＜0.001)and 30±4% (P＜0.001),respectively.These results provide new evidence that IL-2 induces rapid inhibition of peak currents of NMDA receptor-mediated responses with possible NR1/NR2A and NR1/NR2B subtype-differentiation,and suggest that the inhibition is possibly mediated by direct interaction between IL-2 and NMDA receptors. Partâ…¡:Interleukin-2 inhibits AMPA receptor function in cultured hippocampal neurons of ratsIL-2 is an important neuroregulatory molecule in the CNS.The present study investigated the effects of IL-2 onα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)receptors in cultured hippocampal neurons of rats using whole cell patch-clamp analysis.We found that IL-2(0.01-1 ng/ml)immediately and significantly decreased peak I_AMPAin cultured hippocampal neurons in a concentration-dependent manner,causing a 55±6%inhibition at 0.1 ng/ml(p＜0.001),similar to the effect on NMDA receptors.The effect of IL-2 on peak amplitude of I_AMPAwas also depended on glutamatic acid concentrations,and the value of EC₅₀for glutamatic acid in the presence of IL-2 was 144±7μM,markedly greater than 56±8μM in controls(n=6).In addition,we also found that the amplitudes of AMPA miniature excitatory postsynaptic currents(mEPSCs)were significant decreased by 24±6%(p＜0.05)after 0.1 ng/ml IL-2 application,whereas their frequency and decay times were not altered.These results demonstrate that IL-2 induces rapid inhibition of AMPA receptor-mediated responses, indicating that AMPA receptors may be involved in modulation of IL-2 in excitatory synaptic activity in cultured rat hippocampal neurons.Partâ…¢:Effects of interleukin-2 on dendritic development and synaptogenesis of cultured hippocampai neurons of ratsThis study has investigated the effects of IL-2 on dendritic filopodia,dendritic arborization,and spine maturation during development in cultured hippocampal neurons of rats.The cultured hippocampal neurons were transfected with F-GFP on days in vitro 5(DIV5)to display the subtle structures of dendrites,and then were treated with IL-2 at various concentrations,respectively,for different time before observation of living cell images.We found that both the dendritic arborization and the length of dendrites per neuron at DIV7,DIV10 and DIV14 were increased by IL-2 treatment in a dose-dependent manner.After treatment for 48 hours,10 ng/ml IL-2 caused 48.21±10.74%(P＜0.001),17.79±5.64%(P＜0.01)and 15.28±4.89%(P＜0.05)increases in neurons at DIV7,DIV10 and DIV14,respectively;while for the total length of branches, causing 43.01±9.43%(P＜0.001),35.04±7.01%(P＜0.05)and 24.48±5.83%(P＜0.01)increase at DIV7,DIV10 and DIV14,respectively.The most strongest effects on both dendritic number and length were observed in neurons at DIV7.And there was also a significant increase in the mobility of dendritic filopodia in the neurons at DIV7 treated with 10 ng/ml IL-2 for 48 h from DIV5 to DIV7,causing a 30.93±12.83%increase in mobile filopodia/100μm(P＜0.001)and 44.18±7.81%increase in ration of mobile/total filopodia(P＜0.001),but no significant change was observed in filopodia density and length.In addition,IL-2 also caused an increase in the spine density of neurons at DIV14 either treated with IL-2 from DIV5 to DIV7 or from DIV5 to DIV14,causing 18.48±6.22%increase in the former group(P＜0.05)and 20.57±10.89%increase in the latter group(P＜0.01),respectively,but did not affect neurons with IL-2 treatment from DIV12 to DIV14.These results indicate that IL-2 may affect the dendritic development and formation of synapses of cultured hippocampal neurons,especially during the early developmental stage of neurons.