Glial ATP,a Key Player In Depression And Epilepsy Co-morbidity

Epilepsy,a brain disorder characterized by recurrent seizures,is a devastating neurological disease with a worldwide prevalence of about 1-2%[1-3].Unfortunately,seizures in 30%of patients with temporal lobe epilepsy are not controlled by anticonvulsant agents,drugs that are directed to neuronal targets.In this case,patients may undergo resective epilepsy surgery to become seizure-free.The key to preventing and curing epilepsy lies in understanding epileptogenesis,the process by which a healthy brain becomes epileptic.Epileptogenesis is triggered by insults,such as trauma,stroke and infection,resulting in progressive changes in brain structure and function,as well as in the balance between excitatory and inhibitory pathways,and then leading to neuronal hyper-excitability and recurrent seizures[5-7].Major depressive disorder(MDD)is a common and disabling illness affecting a rising percentage of the world’s population.At present,nearly all of the medications available for the treatment of MDD have been developed out of the monoaminergic deficit hypothesis of depression that arose in the mid 1960s[8].Although this approach has led to great advances in our ability to treat MDD,the limitations of our current armamentarium of antidepressant drugs are becoming increasingly evident[9].This realization presents a strong impetus to search beyond the monoaminergic systems for a better understanding of the pathophysiological mechanisms underlying MDD,and to consider these newly identified mechanisms in the search for novel antidepressant medications.A wealth of clinical data has established a strong co-morbidity between epilepsy and depression[10-12].This co-morbidity is bi-directional,that is,patients with epilepsy are three to five times more likely to develop depression,and patients with active depression,a history of depression,or a family history of depression nearly twice as likely to develop epilepsy,a risk which rises to 4.2-fold if they have a history of suicide attempt[13].Depression has a more profound impact on the quality of life of individuals with epilepsy than seizure frequency or severity.Importantly,this co-morbidity is also highly detrimental to overall prognosis and outcome,as patients with both disorders exhibit higher rates of re-hospitalization and decrease success with treatment.Indeed,several antiepileptic medications,such as gabapentin,lamotrigine,oxcarbazepine,tiagabine,and valproate,can cause depressed mood,whereas some antidepressants increase seizure risk,particularly in overdose situations[14-15].Several reasons for this co-morbidity have been posited,including shared neurobiological pathways,neuroanatomical regions,and common genetic mechanisms[16].On one hand,growing evidence indicates that glial elements are involved in the neuropathology of several neuropsychiatric illnesses including MDD[17].Post-mortem studies of tissues from patients with MDD describe a reduced number and an altered morphology of glial cells in several brain regions,in particular the prefrontal cortex(PFC)[18-21].Other post-mortem studies demonstrating altered expression of glial fibrillary associated protein(GFAP),glial-specific excitatory amino-acid transporters(EAATs)and glutamine synthetase(GS)in tissue from individuals with MDD[22-23]suggest that the glial cell abnormalities include changes in astrocytic cell function.Recent studies provide evidence that stress exposure may be related to some of the reported glial cell pathology by demonstrating that animals exposed to chronic stress have a decreased glial density in the hippocampus[24]and a reduced production of glial cells in the adult hippocampus and PFC[25-27].On the other hand,emerging evidence suggests a critical role for the glial cells,astrocytes,in epilepsy[28-29].Astrocytes play an important role in maintaining central nervous system function by releasing gliotransmitters,including glutamate,D-serine,and ATP[30].Although a strong co-morbidity exists clinically between epilepsy and depression,the cause of this co-morbidity remains unknown,and a valid animal model is crucial for the identification of underlying mechanisms and the development of a screening tool for novel therapies.Our recent study indicates the deficiency in astrocytic ATP release as a biological mechanism of major depressive disorder.Our goal in this study was to explore whether depression and epilepsy co-morbidity share a common pathological mechanism,insufficiency in astrocytic ATP.First,we need to establish an appropriate animal model to study the pathogenesis of depression and epilepsy co-morbidity.This co-morbidity is bi-directional.Whether chronic epilepsy in mice with depressive-like behavior?Chronic depression can increase seizure susceptibility in mice?We found that in KA intro-amygdala kindling model,mice did not exhibit depression-like behavior in forced swimming test.However,mice exhibited increased seizure susceptibility in chronic social defeat stress model(CSDS)and chronic mild stress model(CMS).Compared with CMS model,CSDS model CSDS is the depression model accompanying with obviously higher seizure susceptibility.Therefore,we use CSDS model to study the mechanism depression and epilepsy co-morbidity.Central nervous system includes neurons and glia.Our previous study found astrocytes involved in the regulation of depression-like behavior in mice.GFAP--glial fibrillary acidic protein is the astrocytic activation marker.We found that in chronic social defeat stress model(CSDS)(accompanied by increased susceptibility to epilepsy),GFAP protein expression decreased in the hippocampus and cortex.It reminds us that astrocytes may participate in depression and epilepsy co-morbidity.Astrocytes are nonexcitable cells,activation of which is carried out by the increased concentration of intracellular calcium ions via G protein-coupled receptors-phosphatidylinositol signaling pathway.IP3R2(Itpr2)is the only unctional IP3 receptors of astrocytes.Therefore,we knocked out IP3R2(Itpr2)to silence astrocyte calcium signaling.We found Itpr2-/-mice exhibit depression-like behavior in the forced swim test.Meanwhile in different animal models of epilepsy,epilepsy susceptibility increased in this mice strain.Therefore,By silencing astrocytic calsium Itpr2-/-mice is an animal tool to study depression and epilepsy co-morbidity.Our previous study found that,decreased release of astrocytic ATP causes depression-like behavior.We performed microdialysis to measure ATP release in cortical and hippocampal CA1 region of the brain from wild-type and Itpr2-/-mice.We found ATP release was at a reduced level in the two brain region of Itpr2-/-mice.T To clarify the relative contribution of glia cells and neurons to the reduction of ATP release in Itpr2-/-mice,we examined ATP release in cultured cortical and hippocampal glia cells or neurons.Microdialysis revealed that ATP release was reduced in glia cells,but not neurons,of Itpr2-/-mice.In addition,we employed microdialysis to determine the ATP level in the cortex and hippocampus of CSDS model mice.Similarly to Itpr2-/-mice,CSDS-exposed mice exhibited a lower ATP level in both cortex and hippocampus.These results prompted that the decreased ATP levels might play a key role in the pathogenesis of depression and epilepsy co-morbidity.To test this possibility,we used dnSNARE mice that were deficient in astrocytic ATP release by means of transgenetic blockage of vesicular gliotransmission.First we confirmed in our experiment the deficiency of ATP release in cortex and hippocampus of dnSNARE mice after deprivation of tetracycline(DOX)for 5 days.Then we checked behavioral consequence relating to depression and epilepsy.It was able to be observed that DOX deprivation mice exhibited depression-like behavior in the forced swimming test and high seizure susceptibility in PTZ epilepsy model,indicative of an important role of ATP insufficiency in the pathogenesis of depression and epilepsy co-morbidity.Next,in order to locate the dominant brain regions mediating the action ATP,we analyzed EEG spectrum.In the resting state,EEG spectrum displayed delta,delta-a,1 Hz,3Hz waves increased,while alpha wave declined in the CA1 region of Itpr2-/-mice,compared to wild type mice.No change in EEG spectrum was detected in cortex,suggesting that hippocampal CA1 mediates behavioral outcomes on depression and epilepsy of ATP-deficient mice.Both pyramidal neuron and interneuron in CA1 region express ATP receptors which consist of P2X and P2Y types.Each receptor type includes several subtypes.P2Y1 receptors are mainly expressed in the interneuron and activate its inhibitory loop.Lower ATP levels decrease the tone inhibitory loop,and thus increase excitability,leading to increased seizure susceptibility.To identify which neuronal type,pyramidal neuron or interneuron,is primarily responsible for the effects of lowed ATP in Itpr2-/-mice,we conducted electrophysiological recordings in CA1 pyramidal neurons.We showed that spontaneous firing rate of pyramidal neurons increased in Itpr2-/-mice,compared to wild type mice.The increased firing rate of pyramidal neurons is due to decreased GABAergic inhibition,as GABAergic input decreased while glutamatergic input was intact.These data indicate that interneuronal P2Y1 receptors manifest the high seizure susceptibility in Itpr2-/-mice.Microinjection of ATP/ATPrS/MRS2365 to CA1 region can rescue high seizure susceptibility in Itpr2-/-mice.Microinjection of MRS2179 to CA1 region causes high seizure susceptibility in WT mice rather than Itpr2-/-mice.Injection of shRNA-mP2Y1 to CA1 region change seizure susceptibility of WT rather than Itpr2-/-mice.P2Y1 receptors of hippocampus CA1 region mediate the antiepileptic effects of ATP.Competitive P2Y1 purinoceptor antagonist change sIPSCs of WT rather than Itpr2-/-mice.Microinjection of MRS2365 can rescue high seizure susceptibility in mice with depressive-like behaviors.ConclusionThe mice that were susceptible to chronic social defeat showed high seizure susceptibility and low GFAP expression and ATP abundance in hippocampus.Similarly,both a lack of inositol 1,4,5-trisphosphate receptor type 2 and transgenic blockage of vesicular gliotransmission-induced deficiencies in astrocytic ATP release,cause depressive-like behaviors and high seizure susceptibility.The high seizure susceptibility of Itpr2-/-mice could be rescued by administration of ATP,which was mediated by the P2Y1 receptor in hippocampal CA1 region.Furthermore,microinjection of P2Y1 receptor agonist to hippocampal CA1 region also prevented the high seizure susceptibility in depressed mice subjected to chronic social defeat stress.