Abstracts

Rationale: Energy metabolism and excitability are coupled, but their precise relationships are not known and, in fact, may seem paradoxical: most inherited energy metabolism defects cause epilepsy rather than hypoexcitability, just as most epileptogenic lesions are associated with diminished glucose metabolism. We set out to investigate epileptogenesis in glucose transporter type I (Glut1) deficiency, an epileptic encephalopathy that arises from impaired glycolysis resulting from decreased blood brain barrier and astrocyte glucose flux. Glut1 is the dominant glucose transporter of the cerebral endothelium and astroglia. Patients afflicted by Glut1 deficiency (OMIM 606777), commonly suffer from generalized spike and wave discharges in the context of anticonvulsant-refractory epilepsy but benefit from a ketogenic diet. Methods: Glut1-deficient mouse brains generated by a transgenic antisense strategy were coronal sliced (350-μm) and acutely maintained in carbogen perfusate containing (in mM): 126 NaCl, 3 KCl, 1.25 NaH₂PO₄, 26 NaHCO₃, 2 MgS0₄, 2 CaCl₂, 20 Glucose (pH 7.3, mOsm 303). Whole-cell patch recordings were obtained from large layer V pyramidal cells located in the sensory barrel cortex. Patch pipettes made of borosilicate glass (R=3-5 MΩ) were filled with an internal solution of (in mM): 110 K-Glu, 20 KCl, 10 NaCl, 4 Mg-ATP, 0.3 GTP, 0.6 EGTA, 5 QX314, 10 HEPES-K (pH 7.3, mOsm 303). Cells were clamped at -67 mV, sampled at 20 kHz with a 5 kHz low pass filter, and all post-synaptic current events detected offline by a template method (Clampfit 10.1). All recordings were obtained after the cell had been voltage-clamped for at least five minutes with stable access resistances (< 20 MΩ). A minimum of 100 events from each cell which had smooth rise and decay phases were obtained for analysis of post-synaptic event properties. Results: At P28, the developmental stage when glycolysis is stimulated in the brain, the amplitude of spontaneous synaptic events was diminished in Glut1-deficient neurons relative to normal littermate cells (median peak amplitude 24.3 versus 28.0 pA, p<0.001, Kolmogorov-Smirnov test), without detectable changes in event half-width or rise time. Cell membrane potential, input resistance and synaptic event frequency were similar to normal brain and constant during the experiment in the mutant slices. Conclusions: Impaired neural glucose flux is associated with diminished neuronal spontaneous events probably reflecting a selective reduction in synaptic cortical input in the presence of normal passive neuronal properties and, possibly, of synapse numbers. Circumventing the blood brain barrier by virtue of slice incubation in glucose-containing medium results in neuronal excitability abnormalities that speak against endothelial-astrocyte barrier dysfunction as the sole pathological mechanism involved in Glut1 deficiency. Therefore, our present efforts focus on investigating alterations in glutamatergic synaptic function as a possible pathogenic mechanism postulated by the extension of the astrocyte-neuron lactate shuttle hypothesis (ANLSH) to the Glut1 deficient state.