Lactate Enhance The Rundown Of GABA_A Receptor Current

In brain slices and acute dissociated neurons, the rundown of GABAAR current could be inhibited by Mg-ATP and orthovanadate. It indicates that the function of GAB AAR could be affected by the metabolism status of the neuron and the phosphorylation process of GAB AAR. In 2004, Laschet et al found the key glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is the kinase of GABAAR al subunit. The rundown of GABAAR current was significantly reduced by the addition of GAPDH and nearly abolished by the addition of its substrate glyceraldehyde-3-phosphate (G3P). The results of their experiment indicate a direct involvement of glycolysis in the function of GABAAR. In the neurons of surgical resected brain tissue from epilepsy patient, the GABAAR current showed a enhanced rundown compared with "control". In 2007, Pumain et al found G-3-P could inhibit the enhanced rundown of GABAAR current in epilepsy patients, thus discovered a relationship between hypometabolism and the deficiency of GABAAR in epilepsy patients.The researches on "astrocyte-neuron lactate shuttle hypothesis" in the last decade have found that lactate may play an important role in the metabolism of neuron. Furthermore, the inhibition of glycolysis by lactate had been proved by many research groups, and the inhibition is considered to be caused by the competition to NAD between lactate dehydrogenase(LDH) and GAPDH.Thus, we make the hypothesis that lactate might inhibit the function of GABAAR through the inhibition of glycolysis and glycolysis-dependent phosphorylation of GABAAR al subunit.Object:1. To study the influence of lactate on the function of GABAAR。2. To study the relationship between glycolysis-dependent phosphorylation of GABAAR al subunit and the mechanism underlying the influence of lactate on the function of GABAAR.Method:Adult Sprague Dawley rats (21-28 d of age) were anesthetized and killed by decapitation. The brains were removed quickly and placed in cold artificial CSF The brain was cut into 300-400μm thick brain slices in 15min after the decapitation. Then the brain slices were transferred into a ACSF containing(in mmol) 125 NaCl,2.5 KCl,2 CaC12,1 MgC12,1.25 NaH2PO4,10 D-glucose, pH 7.35. After 30-60mins incubation, the brain slice was transferred into the recording chamber and incubated with addition of TTX(lμmol),CNQX (101μmol) and AP-5(50μmol) to the ACSF. Pyramidal neurons in the cortex of the frontal lobe were recorded using borosilicate pipettes (2-5MΩ) filled with a solution containing (in mmol) 140 CsCl,0.5 CaCl2,4 NaCl,10 Hepes,1 EGTA, 4 Mg-ATP,0.3 Na-GTP, pH 7.3. Different additions were made to this standard pipette milieu according to different groups:lactate (500μmol), orthovanadate (100μmol), lactate (500μmol)+orthovanadate (100μmol). Whole cell peak currents induced by GABA(100μM; fast applications,20 sec pulse every 2 min, 5 times) were measured for each application and normalized to I1st. Neurons were held at-70 mV such that GABA evoked inward currents (Cl- equilibrium potential close to 0 mV).The statistical significance was assessed using a one-way ANO VA.Results:1. The GABAAR currents of control group showed a rundown to 79.15%±1.96% (n=16) at the end of rundown protocol.2. The addition of lactate(500μmol) into the pipette milieu significantly enhanced the rundown of GABAAR current (59.78%±3.76%,n=16; ANOVO, p<0.01)3. The addition of orthovanadate (100μmol) into the pipette milieu significantly inhibit the rundown of GABAAR current (96.60%±2.29%,n=16; ANOVO, p<0.01)4. The rundown of GABAAR current in the orthovanadate (100μmol)+ lactate(500μmol) group (92.45%±5.40%,n=12) is significantly lower than the lactate(500μmol) group(59.78%±3.76%,n=16; ANOVO, p<0.01).5. No significant difference were found between the group adding lactate (500μmol)+orthovanadate (100μmol) (92.45%±5.40%,n=12) and the group only adding orthovanadate (100μmol) (ANOVO, p>0.05)Conclusion:Lactate significantly enhanced the rundown of GABAAR current. Orthovanadate almost completely abolished enhancement of the rundown of GABAAR current induced by lactate.Laschet et al has demonstrated that the inhibition of the glycolytic function of GAPDH by iodoacetamide could significantly enhance the rundown of GABAAR current, and vanadate almost completely abolishes the effect of iodoacetamide. We have found the effect of lactate on GABAAR is similar to the effect of iodoacetamide. We conclude two points from our experiment:1.Lactate could inhibit the function of GABAAR; 2. Lactate might inhibit the function of GABAAR through the inhibition of glycolysis and glycolysis-dependent phosphorylation of GABAARαl subunit.