Abstracts

Rationale: Modulation of metabolic activity alters cellular excitability and manipulation of metabolic pathways (e.g., ketogenic diet, glycolytic inhibition) represents a novel strategy for treatment of drug-refractory seizures. The glycolytic intermediate, fructose-1,6-bisphosphate (F1,6BP) has been previously shown to have an anticonvulsant effect in several models of acute seizures (Lian et al., J Neurosci 27:12007-11, 2007), and was proposed to act through an intracellular metabolic pathway (i.e., pentose-phosphate pathway, PPP). How F1,6BP reduces neuronal excitability and seizure activity is unknown. Here, we investigated mechanisms of action of F1,6BP in hippocampal CA3 neurons and neuronal networks. Methods: Hippocampal slices were prepared from P7-15 rats. Spontaneous (or evoked) epileptiform bursts in hippocampal CA3 were induced in Mg2+-free medium containing 20-50 uM 4-aminopyridine (4-AP), and registered with extracellular field-potential recording or simultaneous whole-cell/field potential recordings. Neuronal and synaptic activity was recorded using whole-cell current/voltage-clamp recordings with K-gluconate-based intracellular solution. Voltage-activated calcium currents (ICa) were recorded with CsMeSO3-based internal solution supplemented with QX-314 (5 mM), and in the presence of DNQX (10 uM), AP-5 (50 uM) and gabazine (10 uM). ICa was activated by depolarizing voltage steps and leak-subtracted online.  Results: Epileptiform bursts consistently occurred in hippocampal CA3 in 0 Mg2+/4-AP medium. Bath application of F1,6BP (2.5-5 mM) completely blocked spontaneous bursts in most slices (6 of 9). Similar blockade of synaptically-evoked bursts was observed in the same medium (n=4). Surprisingly, the blockade effect of F1,6BP occurred rapidly (usually 2-3 min), suggesting that it may act through an extracellular mechanism prior to any intracellular action. F1,6BP did not stop spontaneous intrinsic firing of CA3 neurons in normal aCSF or Mg2+-free/4-AP containing medium (n=5), nor did it eliminate spontaneous or evoked excitatory synaptic currents (s- or eEPSCs, n=6). Unexpectedly, F1,6BP readily blocked ICa by ~40% without altering channel activation/inactivation kinetics (n=8). Consistent with these results, the Ca2+ channel blocker, CdCl2 (100 uM), quickly blocked epileptiform activity in Mg2+-free/4-AP medium (n=3).  Conclusions: These preliminary in vitro data support previous in vivo studies that F1,6BP blocks epileptiform activity. This effect is likely mediated, at least in part, via an acute extracellular action on Ca2+ channels. It remains to be determined whether F1,6BP exerts additional effects on intracellular metabolic pathways. Funding: This work was supported in part by generous gifts from the Mathias Koch Memorial Fund of the Community Foundation of Southern Wisconsin and the Sandra and Malcolm Berman Foundation.