br Materials and methods br Results

Materials and methods
Results
Discussion In the CNS, GLP-1R is associated with G-protein signal transduction pathways, which can activate adenylyl cyclase and protein kinase A by cyclic adenosine monophosphate, and mediates neuronal activities implicated in various pathophysiological changes, including synaptic plasticity, neuronal apoptosis and neuronal excitability [21]. Therefore, an increasing number of researchers believe that GLP-1 or GLP-1R agonistic drugs might be applied to the treatment of many neurological diseases, for instance AD, PD, and stroke, but few studies have examined its effects on epilepsy. Through immunofluorescence staining, our study confirmed that GLP-1R is mainly located in neurons. Moreover, the GLP-1R levels were reduced in the PTZ-treated rats as well as TLE patients. These data indicated that GLP-1R might be involved in epileptic seizure. To further identify the association between epilepsy and GLP-1R, we administered liraglutide and ex9-39 to PTZ-treated rats and observed their behaviors. The results revealed that liraglutide effectively reduced the severity of seizures, whereas ex9-39 aggravated seizure severity and weakened the antiepileptic effects of liraglutide. Our findings suggest that GLP-1R might exert antiepileptic effects, which is supported by previous studies. For example, Abbas et al. discovered that GLP-1 knock-out in mice clearly decreased the seizure threshold and damaged synaptic remodeling and cognitive dysfunction [22]. GLP-1R exerts protective effects on substantia nigra dopaminergic neurons. Activated GLP-1R can increase the number of dopamine receptors in the amygdala region and then activate dopamine D2 receptors, which exert antiepileptic effects [23,24]. Moreover, GLP-1R can increase the PF-670462 synthesis levels of noradrenaline and 5-hydroxytryptamine (5-HT); GLP-1R can also adjust neuronal excitability and reduce the seizure severity [25], indicating that activated GLP-1R has a great probability of reducing the possibility of epilepsy deterioration by mediating the secretion of neurotransmitters [26]. GABA and glutamate neurotransmitters can exert great influence on excitatory and inhibitory currents of neurons. In addition, GABA and glutamate neurotransmitters are the most basic and important neurotransmitters for epilepsy. Moreover, GABA also contributes to inhibiting the excitability of the nervous system. GABARs include three subtypes, GABAAR, GABABR and GABACR, and the lower expression of the former subtype (GABAAR) in epileptic patients and epileptic models has been confirmed [27,28]. A variety of antiepileptic drugs exert their effects by upregulating the level of GABA in brain tissues through different mechanisms [29]. In this study, the expression of GABAAR2/3 (a GABAR subtype) in the rat hippocampi was increased after treatment with liraglutide, and decreased after treatment with ex9-39. Ex9-39 treatment also undermined the effects of liraglutide on GABAAR2/3 expression. Additionally, immunoprecipitation confirmed that GLP-1R interacted with GABAARβ2/3. These results indicate that GLP-1R up-regulation is highly probable to increase the expression of GABAARβ2/3. Farkas et al. found that administration with exendin-4, one of the GLP-1R agonist, activated GLP-1R and facilitated GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) [30]. Korol et al. also confirmed that the GLP-1R regulation during hippocampal functions was consistent with that obtained using GLP-1R agonists, which enhanced GABA signaling through pre- and postsynaptic mechanisms [31]. The above evidence shows that the GLP-1R up-regulation can increase the frequency and amplitude of mIPSCs. Due to the decreased expression of GABA/GABARs, we hypothesized that the frequency and amplitude of GABA/GABAR-mediated mIPSCs were decreased, leading to reduced inhibition of neuronal excitability and triggering epilepsy. In contrast, the up-regulation of GLP-1R reversed the expression of GABAR in seizure-induced rats, leading to the increase in the inhibitory effects on neuronal excitability and thereby alleviating seizures.