Effects Of Melatonin On Levels Of Glutamate, Gaba And Their Receptors In Hippocampus Of Rats With Seizures Induced By Glutamate

Epilepsy is a common and frequently encountered disease of the center nerve system (CNS). It's induced by the repeated paroxysm of excessive bursting activity of the neuron in the brain. People at present to think that their was the intimate connection between the epilepsy and the maladjustment of the neurology-immunology-endocrinology network, the neurology gave priority to the epilepsy, the immunology and endocrinology respectively adjusted it at the level of the receptor, messenger and gene. Thereinto, the primary neurology mechanism was the unbalance of the excitatory system and the inhibitory system in epilepsy patient brain. Glutamate (Glu) is a kind of excitatory amino acids nerve transmitters in CNS. It is widely distributed in cerebral cortex, cerebellum, and nucleus basalis of Meynert in CNS. Many researches discovered Glu participated the epileptiform activity and Glu also was used to make the epilepsy model. The mechanism of Glu promoted the paroxysm of the epilepsy may be via Glu combined with it's receptor in neuron, mediated the G protein coupled ion-channel, or combined with excitatory amino receptor (NMDA) , to lead to large numbers of Ca2+ and Na+ inflow, K+ and Mg2+ efflux, to produce depolarization and induce the epileptiform activity. Glu receptor included ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors (mGluRs). Thereinto, iGluRs included NMDA receptor, AMPA receptor and KA receptor. GluR2 is one kind of the four subtype of AMPA receptor (GluR1-4). The major character of AMPA receptor lies on GluR2. The decreasing of the expression of GluR2 was thought a molecular switch to control the permeability of the AMPA receptor for Ca2+ or reduce the exitotoxicity of Glu, thereby to affect the epilepsy seizure.γ-aminobutyric-acid (GABA) is the most important inhibition amino acids nerve transmitters in CNS. Epileptic animals empirical studies indicate that the contents of GABA was decreased in epileptic animal cerebral cortex and the decreased degree accorded with the epilepsy developed degree. The seizures can be inhibited by injecting the GABA in cistern. Intraperitoneal injection or intravenous injection can antagonize the animal's medico-seizures. The content of GABA in CSF is positive correlation to seizures threshold. The seizures threshold reduced and kindled easily if the content of GABA in CSF decreased. GABA is via it's ionotropic receptor ( GABRA and GABRC) and metabotropic receptor (GABRB) educes inhibitory effect. GABRA is chemical gating ion channel receptor and distributed mainly in the postsynaptic membrane of center neuron or periphery sympathetic neuron. When GABA or other excitomotor combines with GABRA, the chloride channel is opened in postsynaptic membrane, large numbers of Cl- inflows, induces cell membrane hyperpolarization and forms inhibitory postsynaptic potential (IPSP), to mediate the effect of the postsynaptic inhibition of GABA. Melatonin (MT) is an indolamine neuroendocrine hormone synthesized and secreted by the pineal gland, and N-acetyl-5-methoxytryptamine is it's chemical name. It's secretion shows obviously circadian rhythm. Researches indicate that MT has widely physiological function, includes to maintain the circadian rhythm, to promote sleep, to reinforcement the immunological function of the body, and to inhibit the growth of the tumor cell. In addition, MT can also regulate the growth, development, sexual function and lots of other organ function. In recent years, the other functions of MT were discovered in succession, including clearing free radical, antioxidation, neuroprotection, anticonvulsion and inhibiting seizures. But till now, the mechanism of the melatonin on epilepsy is not very clear. Some studies indicate that the anticonvulsion and inhibiting seizures function of MT is implemented via its special receptors in cell membrane. The melatonin receptor antagonist luzindole can attenuated the anticonvulsion and inhibiting seizures functions of MT. The present study is to observe the influence of MT on the behavior of rats induced by L-glutamate and the influence on the expression of Glu and its receptor GluR2, GABA and its receptor GABRA1 in the hippocampus, so as to investigate the mechanism of MT on epilepsy.Our study is composed of two parts. Firstly, 40 healthy male SD rats were divided into A, B, C, D 4 groups at random (10 for each group). Group A: control group of normal saline; Group B: MT+Glu group; Group C: L-glutamate group; Group D: Luz+MT+Glu group. We observed and recorded the behavior change and detected the Glu and its receptor GluR2 in hippocampus by immunohistochemistry staining method. Results showed that Group C and Group D displayed serious epileptic convulsion and discharge, which Group A didn't occur, and Group B showed slightly outbreak. The immunohistochemistry staining method showed that the Glu and GluR2 expressed in pyramidal cell layer of hippocampus CA1-CA3 and granular cell layer of dentate gyrus in each group rats hippocampus. The endochylema of Gluergic neuron were stained. The immunological reaction of Group C and D were much fiercer than that of Group A in each area, the positive cell of Group B were more decreased than that of Group C and D in each area, staining is much lighter, the distinction had statistical significance (P<0.05). Their was no obvious difference between Group A and Group B; the cell membrane of GluR2ergic neuron were stained much stronger. The immunological reaction of Group C and D were much weaker than that of Group A in each area, the positive cell of Group B were more increased than that of Group C and D in each area, the distinction had statistical significance (P<0.05). Their was no obvious difference between Group A and Group B. These research results revealed that MT inhibits the convulsion and discharge in L-glutamate induced rats and one of the mechanisms is that MT potentiates the function of decreasing the expression of Glu, reducing the Glu activity and increasing the expression of GluR2 and then exerts anti-seizures effect.Secondly, 40 healthy male SD rats were divided into A, B, C, D 4 groups at random (10 for each group). Group A: control group of normal saline; Group B: MT+Glu group; Group C: L-glutamate group; Group D: Luz+MT+Glu group. We observed and recorded the behavior change and detected the GABA and its receptor GABRA1 in hippocampus by immunohistochemistry staining method. Results showed that Group C and Group D displayed serious epileptic convulsion and discharge, which Group A didn't occur, and Group B showed slightly outbreak. The immunohistochemistry staining method showed that the GABA and GABRA1 expressed in pyramidal cell layer of CA1-CA3 and granular cell layer of dentate gyrus in each group rats hippocampus. The cell membrane and endochylema of the GABAergic neuron were all stained, the cell membrane of GABRA1ergic neuron were stained much stronger. The immunological reactions of Group C and D were much weaker than that of Group A in each area, the positive cell decreased obviously, the staining was much lighter. The immunological reactions of Group B were much fiercer than that of Group C and D in each area, the positive cell increased obviously, the staining was much stronger. The distinction had statistical significance (P<0.05). Their was no obvious difference between Group A and Group B. These results indicate that MT inhibits the convulsion and discharge in L-glutamate induced rats and one of the mechanisms is that MT reinforces the activity and expression of the inhibitory neurotransmitter GABA and its receptor GABRA1 in hippocampus via its special membrane receptors and then exerts anti-seizures effect.From these data above, we drew a conclusion that MT may play an inhibitory effect in epileptogenesis by reducing the activity and expression of Glu and up-reguluation the expression of GluR2, reinforcing the expression and inhibitory activity of GABA and GABRA1 via its special membrane receptors.