EPA Impaired Learning And Memory Via Activating 5-HT6 Receptor

The brain is composed of two main types of cells:neurons and glial cells.Nervous tissue has the highest lipid concentration,immediately after adipose tissue.These fatty acids participate directly in the architecture and therefore in the functioning of cerebral cell membranes,including those which ensure compartmentalization of the cell and individualization of its organelles.Fatty acids can be classified into two categories:non-essential fatty acids that are synthesized by all organs,and essential fatty acids that must be provided in the diet.The vast majority of these essential fatty acids are polyunsaturated fatty acids(PUFAs)that contain more than one double bond in their backbone.Among the significant components of cell membranes are the phospholipids,which contain fatty acids.The types of fatty acids in the diet determine the types of fatty acids that are available to the composition of cell membranes.A phospholipid made from a saturated fat has a different structure and is less fluid than the one that incorporates an essential fatty acid.In addition,linoleic and alpha-linolenic acids per se have an effect on the neuronal membrane fluidity index.They are able to decrease the cholesterol level in the neuronal membrane,which would otherwise decrease membrane fluidity,which in turn would make it difficult for the cell to carry out its normal functions and increase the cell’s susceptibility to injury and death.These consequences for cell function are not restricted to absolute levels of Fas alone,rather it appears that the relative amounts of omega-3 fatty acids and omega-6 fatty acids in the cell membranes are responsible for affecting cellular function.These PUFA include omega 3 and omega 6 and others.Linoleic acid is a member of the family of omega-6(n-6)fatty acids,while alpha-linolenic acid is an omega-3(n-3)fatty acid.These terms refer to characteristics in the chemical structure of the fatty acids.Other omega-6 fatty acids,such as gamma-linolenic acid(GLA),dihomo-gamma-linolenic acid(DHGLA)and arachidonic acid(AA),can be manufactured in the body using linoleic acid as a starting point.Similarly,other omega-3 fatty acids that are manufactured in the body,using alpha linolenic acid as a starting point,include eicosapentaenoic acid(EPA)and docosahexaenoic acid(DHA).The effects of PUFA on brain function can be divided into at least five categories:(1)modification of neuronal membrane fluidity;(2)modification of membrane activity-bound enzymes;(3)modification of the number and affinity of receptors;(4)modification of the function of neuronal membrane ionic channels,and(5)modification of the production of neurotransmitters and brain peptides.Many studies have demonstrated that various PUFA mediate,or are associated with,several aspects of brain activity,ranging from the role of EFA in neuronal structure and functions,long-term potentiation(LTP),specific brain activation,prostaglandin activity,to neurological and mental disorders,to mood control.Synaptic plasticity is the remodelling and reinforcement of connections between neurons.Long-term potentiation(LTP)in the hippocampus is an experimental model of activity-dependent synaptic plasticity used to study synaptic efficiency in learning memory formation.LTP is triggered by activation of postsynaptic N-methyl-D-aspartate(NMDA)receptors via strong postsynaptic depolarization followed by activation of calcium-calmodulin-dependent protein kinases II(CaMKII)and cyclic adenosine monophosphate(cAMP)response element binding protein(CREB)in postsynaptic dendritic spines,resulting in an increased strength of synaptic transmission.It is triggered by tetanic electric stimulation,which causes a long-lasting potential recorded as excitatory postsynaptic potential in the activated synapse that is maintained for hours in in vitro hippocampal slice preparations and for days for the maintenance of memory in animals.It is worth stressing the role that the autoradiographic localization of 5-HT receptors has played in the elucidation of the complexities of 5-HT receptor families.Now we know that up to 14 different serotonergic receptors exist,belonging to 6 distinct classes of G protein-coupled receptor(GPCR)populations,namely 5-HT1,5-HT2,5-HT4,5-HT5,5-HT6,and 5-HT7,and a family of ligand gated ion channels,5-HT3.The 5-HT6 receptor is expressed almost exclusively within the CNS,offering the possibility that pro-cognitive compounds operating through this mechanism could have only a few peripheral side effects.Particularly high levels of 5-HT6-receptor mRNA occur in the striatum,nucleus accumbens and olfactory tubercles and in limbic and forebrain regions,including the hippocampus and cortex.The NMDA glutamate receptor,a principal subtype of excitatory ligand-gated ion channel,has been implicated in multiple neuronal functions ranging from synapse formation to synaptic plasticity to learning and memory Systemic administration of noncompetitive NMDA receptor(NMDAR)antagonists or knock-down the expression of NMDA receptors produces schizophrenia-like behavioral symptoms;hence,dysfunction of NMDA receptors is strongly linked to the pathophysiology of mental disorders.It prompts us to hypothesize that one important target of 5-HT1A receptors could be the NMD A receptor,and dysregulation of glutamatergic transmission and plasticity by altered serotonin system may contribute to the progress of neuropsychiatric disorders.These observations raise an interesting hypothesis that EPA regulates synaptic plasticity at excitatory synapses directly by regulating 5-HT6 receptor.It is likely that acutely application EPA activate the 5-HT6 receptor in hippocampus,resulting in the inhibited LTP induction in the hippocampal CA1 region.To explore this question,the studies described below using electrophysiological studies to analyze whether this impairment of LTP in the hippocampal CA1 region in mediated by the action of EPA on 5-HT6 receptor.In our present research we show that this suppressed LTP may be caused by EPA-induced activating of 5-HT6 receptor-mediated inhibition in CA1 region.Firstly,To verify whether acute treatment of EPA can also modulate the tetanus-induced LTP at Schaffer collateral-CA1 synapse in hippocampal slices in adult mice,we recorded fEPSP from young adult mice hippocampal slices to examined the effects of acute application of the extracellar 50μM EPA on Schaffer collateral-CA1 synapes LTP induced by high frequency stimulation(HFS)and theta burst stimulation(TBS).We found that both of the two paradigams severly suppressed the LTP.We also treated the slices with different concentration of the fatty acid.We analyzed the effects of 5μM,25μM;50μM EPA on the LTP induced by tetanus stimulation at Schaffer collateral-CA1 synapse in hippocampal slices in adult mice in 6-8 weeks old mice,the fatty acid was perfused by 20 min before tetanus and was continued after tetanus.This treatment strongly suppressed LTP of synaptic transmission Compared with control slices,50μM and 25pμM EPA inhibited the LTP significantly,and there was no significant difference between control and 5μM EPA slices groups.So,in our studies,we confirmed that acute treatment of EPA can also impair the HFS and TBS-induced LTP at Schaffer collateral-CA1 synapse in hippocampal slices in adult mice.To examine the effects of EPA on synaptic transmission and plasticity,we recorded the fEPSPs in the dentritic region of hippocampal CA1 from young adult mice hippocampal slices(6-8 weeks)acute perfused with EPA.50 μM EPA had no effects on baseline synaptic transmission.We also performed complete input-output(I-O)curves at a series of increasing stimulation intensities,after 20 min of EPA superfusion additional I-O curves performed at the same stimulation intensities as under control conditions also showed no detectable changes.These data suggested that acute EPA treatment did not affect normal synaptic transmission.To determine whether EPA treatment might affect the presynaptic neurotransmission,we studied paired-pulse facillitation at 25,50,100,150,200 and 250 ms interstmulus intervals.No significant difference was detected between the two groups,indicating that EPA treatment do not have presynaptic effects.We also applied the DHA to conform its role on the tetanus-induced LTP.This treatment had no effect on the LTP.The above results demonstrate that acute treatment of EPA can impair the HFS and TBS-induced LTP at Schaffer collateral-CAl synapse in hippocampal slices in adult mice and this effects exhibit a concentration-dependent,which means that high concentration exhibit stronger impairment effects.Acute treatment of EPA did not affect normal synaptic transmission and I-O curve.And acute treatment of EPA did riot have presynaptic effects.Also,acute treatment of DHA had no effect on the LTP induced by tetanus stimulation at Schaffer collateral-CA1 synapse in hippocampal slices.Secondly,to further investigate the role of EPA in the modulating learning and memory,we gave the mice intragastric administration of EPA and analyzed the fatty acid level in their brain regions.By using the gas chromatography,we found that the EPA fatty acid level can be acutely increased by intragastric administration of EPA,and there had a rapid increase in the 2 hours after i.g.EPA and a slight increase in the 1 hour and 3 hours after i.g.EPA.This gave us a effective method to investigate the role of EPA.To verify this,we made the mice intragastric(i.g.)administration of EPA and recorded their fEPSP on Schaffer collateral-CA1 synapes 1-3 hours later induced by HFS.This paradigam severly suppressed the LTP.Compared with control group,EPA-treated slice showed a significantly decreased LTP.We also analyzed the effects of different concentratin of intragastric(i.g.)administration of EPA on the LTP induced by tetanus stimulation at Schaffer collateral-CA1 synapse in hippocampal slices in adult mice after intragastric(i.g.)administration of EPA 1-3 hours later.This treatment strongly suppressed LTP of synaptic transmission.Compared with control slices,50mg/kg,75mg/kg and 150mg/kg intragastric(i.g.)administration of EPA inhibited the LTP significantly,and there was no significant difference between control and 35mg/kg intragastric(i.g.)administration of EPA slices groups.We also performed complete input-output(I-O)curves at a series of increasing stimulation intensities,after 20 min of EPA superfusion additional I-O curves performed at the same stimulation intensities as under control conditions also showed no detectable changes.So,in our studies,we confirmed that intragastric(i.g.)administration of EPA can also impair the tetanus-induced LTP at Schaffer collateral-CA1 synapse in hippocampal slices in adult mice.As hippocampus LTP is a cell model of learning and memory,we next use a animal behavior test to conform the role of EPA fatty acid on learning and memory.Contextual fear conditioning is an animal model to test the learning and memory that related to hippocampus,so we apply it to test whether intragastric(i.g.)administrations of EPA change the behavior test.We found that intragastric(i.g.)administration of EPA mice exhibited a shorter freezing time than their control mice in the box.This gave us a information that the intragastric(i.g.)administration of EPA impaired the learning and memory of mice,which was consistent to the fact that intragastric(i.g.)administration of EPA suppressed the hippocampal LTP.Thirdly,to determine the effect of endogenous EPA on LTP induction,we generated the fat-1 mice.We performed the gas chromatography and analyzed the fatty acid level in their brain regions.We found that the EPA fatty acid level significantly increased in the fat-1 mice hippocampus and prefrontal cortex(PFC)compared with the control littermates.No significant differences were detected in EPA fatty acid level of the striatum between the two groups.We next test whether the tetanus-induced LTP at Schaffer collateral-CA1 synapse in hippocampal slices in the fat-1 mice was also affected.we recorded fEPSP from young adult fat-1 mice hippocampal slices and their control littermsates to examined the effects of increased level of hippocampus EPA fatty acid on Schaffer collateral-CA1 synapes LTP induced by HFS and TBS at Schaffer collateral-CA1 synapse.Consistent with the above results,fat-1 mice also exhibited impaired hippocampal LTP.We used two stimulation methods to induce the hippocampal LTP,HFS-induced hippocampal LTP and TBS-induced hippocampal LTP,both of which can not induce the hippocampal LTP.To examine the effects of fat-1 mice on synaptic transmission and plasticity,we then performed complete input-output(I-O)curves at a series of increasing stimulation intensities,and found no significant differences between control slices and fat-1 mice slices,which meant that fat-1 mice did not change the basal synaptic transmission.To determine whether fat-1 mice might affect the presynaptic locus,we studied paired-pulse function at 20,50,100,150,200 ms interstimulus intervals.No significant changes were detected between control group and fat-1 mice group,suggesting that the EPA fatty acid do not have presynaptic effects under these experimental conditions.To further understand the effect of EPA fatty acid on learning and memory,we compared fat-1 mice and control mice in three additional behavioral paradigms:novel object recognition(NOR),contextual fear conditioning and morris water maze(MWM).We first performed the NOR test.We found that the fat-1 mice showed no such preference,whereas the control littermates showed a preference toward the novel object during the NOR test.Contextual fear conditioning is an animal model to test the learning and memory that related to hippocampus,so we next apply it to test whether the fat-1 mice also exhibited the impaired contextual fear conditioning.By analyzing the freezing time of the mice in the box,we found that fat-1 mice exhibited a shorter freezing time than their control mice in the box.To further investigate whether the EPA led to functional impairment,we employed the Morris water maze task to examine hippocampus-involved learning and memory.After being trained fourth per day for four consecutive sessions,control littermates were able to reach the hidden platform in a shorter time during the training.Howeve,the learning and memory abilities of fat-1 mice were significantly impaired compared with the control group.There was no significant difference in average swim speed among the groups.These results clearly indicate that EPA significantly severely impaired spatial cognitive performance.In the probe trial of the Morris water maze test,EPA had a significant effect on the time and distance in target quadrant compared with the control group.We found that the fat-1 mice spent less time in the target quadrant and spent less distance in the target quadrant.As the intimate relationship between the 5-HT receptor and the learning and memory,to investigate which 5-HT receptor that EPA was bound to,we performed the Radio-ligand Receptor Binding Assay.We used this method to screen the 5-HT1A,5-HT1B,5-HT1D,5-HT2A,5-HT2C,5-HT5A,5-HT6 receptors.By calculating the percent inhibition,we found that the EPA was selectively bound to the 5-HT6 receptor,with less binding to other receptors.And we also tested another n-3 fatty acid DHA and found its binding to the 5-HT2C receptor.So from this result we can draw a conclusion that the role of impaired LTP by EPA may via 5-HT6 receptor.However,further work will be must to verify their relationship-We then applied the 5-HT6 receptor antagonist SB-399885 and found that this antagonist can rescue the impaired role of EPA.In conclusion,our present study indicates the EPA fatty acid mediated suppression of LTP induction that arises from activating the 5-HT6 receptor in adult hippocampal slices.