Abstracts

Inhibition of glycolysis with 2-Deoxy-D-Glucose (2-DG) produces anti-seizure effects in in vitro brain slices and in vivo animal models, yet the mechanisms remain elusive. We hypothesize that 2-DG may achieve its anti-seizure effect by reducing glycolytic ATP production and thus regulating the functions of two glycolysis-derived ATP-associated machinery: vacuole ATP pump (V-ATPase) and ATP-sensitive K⁺ channel (K_ATP).

Hippocampal slices were prepared from rat pups aged 8-16 days. Spontaneous epileptiform bursts in CA3 were induced by perfusing slices with 0 Mg²⁺ artificial cerebrospinal fluid (aCSF) with 4-aminopyridine (4-AP), and registered using simultaneous whole-cell and field-potential recordings. Evoked excitatory post-synaptic currents (EPSCs) were recorded from CA3 pyramidal cells in voltage-clamp mode at -70 mV and in the presence of a GABA_A receptor antagoist gabazine. Synaptic stimulation was delivered by a concentric bipolar electrode placed in the CA3 stratum radiatum. For paired-pulse stimulation, the inter-pulse interval was 50 ms, and stimuli were delivered at 0.1 Hz. High frequency repetitive stimulation was given at 20 Hz for 1 - 2.5 s (20 to 50 pulses). Tetanic stimulation was delivered at 100 - 200 Hz for 1 s. For K_ATP induction, gramicidin perforated-patch recording was used in order to keep intracellular ATP level intact.

Interictal-like epileptiform bursts consistently occurred in CA3 in 0 Mg²⁺ and 4-AP aCSF. 2-DG consistently abolished epileptiform bursts in the presence of pyruvate (to sustain tricarboxylic acid cycle for oxidative ATP production) at 30-33^oC but not at room temperature (22^oC). Under physiological conditions, 2-DG did not reduce the amplitude of evoked EPSCs or the paired-pulse ratio in CA3 neurons. During repetitive high-frequency (20 Hz, 20-50 pulses) stimulation, 2-DG did not accelerate the decline of EPSCs (i.e., depletion of transmitter release), even when slices were pre-incubated with 8 mM K⁺ to enhance activity-dependent uptake of 2-DG. In addition, in 2-DG, tetanic stimulation (100-200 Hz, 1 s) dramatically increased rather than diminished the occurrence of spontaneous EPSCs immediately post stimulation (i.e., no transmitter depletion). Moreover, a V-ATPase blocker (concanamycin) failed to block epileptiform bursts that were subsequently abolished by 2-DG. Furthermore, 2-DG did not induce detectable K_ATP current in hippocampal neurons. Finally, epileptiform bursts were not affected by either a K_ATP opener (diazoxide) or a K_ATP blocker (glibenclamide) but were blocked by 2-DG in the same slices.

Our data suggest that 2-DG’s anti-seizure action is both glycolysis- and temperature-dependent, but not mediated by V-ATPase or K_ATP. These results provide new insights to understand 2-DG’s cellular mechanisms of action, and more broadly, neuronal metabolism and excitability.

  Supported by Paine Foundation to CES